Dangerous prey recognition in avian predators

Transkript

Jihočeská univerzita v Českých Budějovicích
Přírodovědecká fakulta
Rozlišování nevhodné kořisti ptačími
predátory
Mgr. Petr Veselý
Školitel: RNDr. Roman Fuchs, CSc.
CSc.
2010
Veselý, P. 2010:
2010 Rozlišování nevhodné kořisti ptačími predátory. Doktorská disertace,
anglicky s českým úvodem. 88 stran, Přírodovědecká fakulta, Jihočeská univerzita
v Českých Budějovicích, Česká Republika
Veselý, P. 2010:
2010 Dangerous prey recognition in avian predators. Ph.D. Thesis, in English
with Czech introduction. 88 pp., Faculty of Science, University of South Bohemia, České
Budějovice, Czech Republic.
Anotace
Prezentovaná disertační práce shrnuje čtyři publikováné články a dva manuskripty týkající
se významu jednotlivých komponent výstražného signálu v ochraně hmyzí kořisti před
ptačími predátory.
Annotation
The present PhD thesis comprises four published research papers and two manuscripts
in preparation dealing with importace of particular parts of the warning signal in
protection of insect prey against avian predators.
Finanční
Finanční podpora
Tato disertační práce vznikla za finanční podpory Grantové agentury Jihočeské Univerzity
(35/2007/P-PřF), Grantové agentury České Republiky (206/03/H034, 206/08/H044),
Grantové agentury Akademie věd (IAA601410803, A6141102) a Ministerstva školství,
mládeže a tělovýchovy (MSM6007665801)
Podě
Poděkování
Na tomto místě bych rád poděkoval všem, kteří se jakýmkoli způsobem zasloužili o to, že
tato disertační práce vznikla, především všem spoluautorům, ale i dalším členům naší
pracovní skupiny. Největší dík samozřejmě patří mému školiteli Romanu Fuchsovi. Dále
bych chtěl poděkovat Keithu Edwardsovi, Ph.D. a Christopheru Steerovi, B.A. za kontrolu
anglických manuskriptů, jakož i všem anonymním recenzentům, za plodné a užitečné
připomínky k manuskriptům. V neposlední řadě bych chtěl poděkovat své rodině za
podporu.
Prohlašuji, že svoji disertační práci jsem vypracoval samostatně pouze s použitím
pramenů a literatury uvedených v seznamu citované literatury.
Prohlašuji, že v souladu s § 47b zákona č. 111/1998 Sb. v platném znění souhlasím se
zveřejněním své disertační práce, a to v úpravě vzniklé vypuštěním vyznačených částí
archivovaných Přírodovědeckou fakultou; elektronickou cestou ve veřejně přístupné části
databáze STAG provozované Jihočeskou univerzitou v Českých Budějovicích na jejích
internetových stránkách, a to se zachováním mého autorského práva k odevzdanému
textu této kvalifikační práce. Souhlasím dále s tím, aby toutéž elektronickou cestou byly v
souladu s uvedeným ustanovením zákona č. 111/1998 Sb. zveřejněny posudky školitele
a oponentů práce i záznam o průběhu a výsledku obhajoby kvalifikační práce. Rovněž
souhlasím s porovnáním textu mé kvalifikační práce s databází kvalifikačních prací
Theses.cz provozovanou Národním registrem vysokoškolských kvalifikačních prací a
systémem na odhalování plagiátů.
V Českých Budějovicích 19. 7. 2010
……………………………………………………………………………
Obsah
Úvod……………………………………………………………………………………………………………………………1
Aposematismus a formy výstražné signalizace………………………………………………………………1
Mimikry…………………………………………………………………………………………………………….4
Evoluce výstražné signalizace a její studium……………………………………………………….6
Smyslové, kognitivní a mentální schopnosti ptačích predátorů……………………………8
Metodoligické přístupy ke studiu výstražné signalizace…………………………………….11
Cíle disertační práce……………………………………………………………………………………….13
Literatura citovaná v úvodu……………………………………………………………………………..16
Paper 1
VESELÝ, P., VESELÁ, S., FUCHS, R. AND J. ZRZAVÝ (2006) Are gregarious red-black
shieldbugs, Graphosoma lineatum (Hemiptera: Pentatomidae), really aposematic? An
experimental approach. Evolutionary Ecology Research, 8: 881–890
…………………………………………………………………………………………………………………………………24
Paper 2
DOLENSKÁ, M., NEDVĚD, O., VESELÝ, P., TESAŘOVÁ, M. and R. FUCHS (2009) What
constitutes optical warning signals of ladybirds (Coleoptera: Coccinellidae) towards bird
predators: colour, pattern or general look?. Biological Journal of the Linnean Society, 98:
234–242
…………………………………………………………………………………………………………………………………35
Paper 3
PROKOPOVÁ, M., VESELÝ, P., FUCHS, R. and J. ZRZAVÝ (2010) The role of size and colour
pattern in protection of developmental stages of the red firebug (Pyrrhocoris apterus)
against avian predators. Biological Journal of the Linnean Society, 100:
100 890-898
…………………………………………………………………………………………………………………………………45
Paper 4
VESELÝ, P., VESELÁ, S. and R. FUCHS (in prep) Visual anti predatory signal of the red
cotton bug: colour or pattern? Submitted to Ethology
…………………………………………………………………………………………………………………………………55
Paper 5
CIBULKOVÁ, A., VESELÝ, P., POLÁKOVÁ, S. and R. FUCHS (in prep) Efficiency of individual
colours in insect warning displays: experiments with avian predators. Submitted to
Ethology
…………………………………………………………………………………………………………………………………65
Paper 6
VESELÝ, P. and R. FUCHS (2009) Newly emerged Batesian mimicry protects only
unfamiliar prey. Evolutionary Ecology, 23:
23 919-929
…………………………………………………………………………………………………………………………………76
Úvod
Nepřeberná rozmanitost strategií, jimiž se organismy brání predaci je jedno z hlavních
témat behaviorálně ekologických studií již po několik desítek let. Během této doby byl
hlavní zájem věnován používání výstražných signálů nebo naopak kamufláže a
nejrozmanitějším aspektům týkajících se evoluce a udržení těchto znaků (Komárek
2003). V průběhu posledního století a půl fascinovala vědce variabilita barevných signálů
chránící se kořisti, stejně jako rozmanitost adaptací predátora na tyto signály. Teprve
během posledních několika let však byla tato problematika studována detailněji, byly více
vzaty v potaz kognitivní schopnosti predátorů a díky tomu byly lépe poznány a vysvětleny
vztahy panující mezi predátorem a výstražně signalizující kořistí. Navíc nové
experimentální přístupy umožnily poodhalit více z iniciální evoluce aposematismu
(Lindström 1999).
Aposematismus
Aposematismus a formy výstražné
výstražné signalizace
Aposematismus (z řeckého apo – pryč, stranou, sémeion – znamení; signál) je jev
popsaný E. B. Poultonem v roce 1890, popisující situaci, kdy organismus dává najevo, že
je coby potrava nevhodný. Již před touto definicí se Alfred R. Wallace (1878) zamýšlel nad
adaptivním významem barevného zbarvení mnoha hmyzích druhů. Dospěl k názoru, že
Darwinovy myšlenky navrhující pohlavní výběr coby hlavní selekční sílu formující barevnou
variabilitu hmyzu (Darwin 1859), nemusí být vždy dostatečné, neboť i stadia hmyzu
neúčastnící se rozmnožování, vykazují výstražné zbarvení.
Základní princip fungování aposematismu je založen na představě, že organismus mající
nějakou vlastnost, která z něj činí nepoživatelnou kořist, se snaží tuto svou vlastnost dát
najevo a vyhnout se tak napadení ze strany predátora (Guilford 1988). Tento signál by
měl být co možná nejjednodušší a umožnit tak predátorovi snadné a dlouhodobé
zapamatování. Pro tento účel se tedy nejčastěji používají jasné a kontrastní barvy, které
upoutají predátorovu pozornost a vryjí se na dlouho do jeho paměti (Guilford 1986).
Obranné mechanismy, kterými aposematický organismus disponuje mohou být
realizovány nejrůznějším způsobem. Mohou být chemické (nechutné, toxické, nebo
páchnoucí látky vylučované v případě ohrožení z rozličných žláz a žihadel hmyzu,
pavouků, kůže žab a jedových zubů hadů), či mechanické (ostny, chlupy tuhá,
inkrustovaná kutikula, štítky, žihadla jako taková) (Cott 1940, Edmunds 1974, Allen a
Cooper 1994, Ruxton et al. 2004). Obecně se nejedná o kvality pro predátora letální,
neboť z takové obrany neplyne žádné poučení a následná ochrana obdobně zbarvených
příbuzných (Beckers et al. 1996).
Tyto kvality mohou být dávány najevo v několika smyslových kanálech – jsou tedy
multimodální (Rowe a Guilford 1999). Optické signály jsou nejčastější a jsou obyčejně
tvořeny výraznými barevnými odstíny kombinovanými do charakteristických vzorů.
Nejčastější barva užívaná ve výstražné signalizaci je červená, neboť je kontrastní vůči
většině přirozených podkladů a zároveň je většinou opticky se orientujících predátorů
dobře vnímána (Cott 1940). Její výstražná funkce pro ptačí predátory byla prokázána jak
na přirozené (např. Silén-Tullberg et al. 1982), tak umělé kořisti (např. Mastrota a
Mensch 1995) a dokonce i na obarvené vodě (např. Roper a Marples 1997). Nicméně,
výstražnost červené barvy je podmíněna druhem predátora. Bylo prokázáno, že například
plodožraví, nebo zrnožraví ptáci vůči červené barvě nevykazují averzi (Honkavaara et al.
2004, Exnerová et al. 2003). Žlutá barva je také velmi běžná ve výstražné signalizaci
(Schuler a Hesse 1985), i když některé studie naznačují, že její efektivita nemusí být vždy
obdobná (Ham et al. 2006). Poněkud problematická, co se týče signalizace nevýhodnosti,
je bílá barva, vyskytující se zvláště u motýlů. Collins a Watson (1983) zkoumali v tropech
bílé motýli čeledi přástevníkovitých (Lepidotera, Arctiidae), kde byla bílá barva prokázána
jako signál nepoživatelnosti pro řadu obratlovců. Obdobně plodožraví ptáci vykazovali vůči
bílé barvě averzi (Honkavaara et al. 2004). Naopak u čeledi běláskovitých (Lepidoptera,
Pieridae) se nechutnost pojící se s bílým zbarvením prokázat nepodařilo (Lyytinen et al.
1999). Stejně tak bílá mutace ruměnice pospolné (Pyrrhocoris apterus) nebyla chráněna
před ptačím predátorem (Exnerová et al. 2006). Za universálně nevýstražné barvy je
považována hnědá, šedá a zelená (Gamberale-Stille a Silén-Tullberg 1999, Exnerová et
al. 2003) nicméně problematické bývají fialová a modrá, které se obecně v přírodě
vyskytují vzácněji (Mastrota a Mensch 1995, ale Pank 1976).
Nicméně, optická signalizace většiny aposematické kořisti sestává z kombinace barev,
nejčastěji se jedná o některou z výše uvedených spojenou s černým vzorem (Cott 1940).
Kombinace dvou barev zjevně slouží k usnadnění identifikace daného typu kořisti a
konkretizaci jejího signálu (Cott 1940). Každopádně, vzor naplňuje výstražnou funkci
lépe, je-li symetrický a tvořen výraznějšími (většími) komponenty (Forsman a Herrström
2004, Forsman a Merilaita 1999, 2003). Poměrně běžný je vzor tvořící dvě okrouhlé
skvrny umístěné symetricky na těle kořisti – tzv. oční skvrny motýlů (Lyytinen et al. 2003),
nebo skvrny na polokrovkách některých ploštic (Exnerová et al. 2008). Nicméně, bylo
prokázáno, že název oční skvrny není v případě hmyzu úplně na místě neboť nemají
imitovat oči, ať už za účelem vystrašit upřeným pohledem, nebo odvést útok predátora na
méně citlivou část, jak to bylo popsáno např. u ryb a žab (Meadows 1993, Lenzi-Mattos et
al. 2005). Stevens et al. (2008) prokázali, že skvrny na hmyzí kořisti mají spíše za úkol
zvýšit nápadnost a tím i výstražnost kořisti, spíše než imitovat oči.
Otázka vzorů výstražného zbarvení je úzce spjatá s kontrastem s pozadím. Aposematická
kořist by měla být nápadná a snadno identifikovatelná a zapamatovatelná (Guilford
1986). Je tedy předpokládáno, že je kontrastní vůči svému pozadí, proto volí ke své
obraně barvy, v přírodě se běžně nevyskytující (Aronsson a Gamberale-Stille 2009).
Nicméně, experimentálně bylo prokázáno, že barvy na kontrastním pozadí působící
výstražně mohou na pozadí s podobným odstínem fungovat krypticky (Endler a Mielke
2005, Tullberg et al. 2005, 2008). Nicméně, i pro kryptickou kořist platí překvapivě
pevná pravidla, jak má být zbarvena – vzor, symetrie (Cuthill et al. 2006). A naopak, i
některé na první pohled kryptické vzory mohou na vhodném pozadí působit na predátory
výstražně (Niskanen a Mappes 2005).
Kořist samozřejmě poskytuje predátorovi více optických vjemů a mnohé z nich lze také
využít ve výstražné signalizaci. Například větší velikost kořisti může umocnit výstražnost
signálu (Forsman a Merilaita 1999, Mand et al. 2007), což je také jeden z důvodů.proč
aposematická kořist často tvoří agregace a tím zvyšuje celkové množství vysílaného
signálu (Gamberale-Stille & Sillén-Tullberg 1996, 1998; Mappes & Alatalo 1997a). Navíc
agregace
kořisti
způsobuje
snazší
zapamatování
výstražného
signálu
naivním
predátorem, který je v krátkém čase opakovaně konfrontován se stejnou kořistí (Tullberg
et al. 2000, Ruxton a Sherratt 2004).
Nicméně, pokud se jedná o rozpoznávání jednotlivých druhů kořisti, nabízí se i další
optické vlastnosti, u některých skupin hmyzu (Hymenoptera, Vespidae a Formicidae)
například, tvar a postoj těla, tvar a délka končetin a tykadel nebo způsob pohybu (Ito et
al., 2004; Nelson et al., 2006, Kauppinen a Mappes, 2003) a samozřejmě, tím, že
predátor podle těchto vlastností identifikuje daný druh aposematické kořisti, mohou tyto
signály být chápány také jako výstražné.
Další významná složka aposematického signálu je zprostředkována chemicky. Původně
se předpokládalo, že chemický signál je určen pro další příslušníky druhu a způsobuje
rozprchnutí (Gibson 1980, Evans a Schmidt 1990). Nicméně, bylo experimentálně
prokázáno, že chemický signál je určen predátorovi (Roper a Marples 1997), a navíc, že
podporuje snazší učení barevné výstražné signalizaci (Skelhorn a Rowe 2006 a, b).
Nicméně se ukazuje, že , alespoň v případě hmyzu, je chemický signál primárně většinou
adresován spíše srovnatelně velkému predátorovi, který se orientuje převážně čichem
(mravenec, pavouk) a není schopen v plné míře vnímat barevný signál kořisti (Skelhorn a
Ruxton 2008).
V poslední době byl experimentálně prokázán přenos varovných signálů i na akustickém
kanálu (Hauglund et al. 2006). Jednak se jedná o různé cvrčivé a klikavé zvuky vydávané
podrážděnými housenkami (Brown et al. 2007, Bura et al. 2009), větší pozornost je však
věnována zvukovým interakcím netopýrů a různých druhů můr (Hristov a Conner 2005).
Bylo prokázáno, že některé můry z čeledi přástevníkovitých (Arctiidae), nejen že dokáží
zachytit a následně rušit echolokační hvizdy netopýrů (Barber a Conner 2006), ale dokáží
také konkrétním zvukem signalizovat svou nepoživatelnost (Barber et al. 2009). Bylo
navíc
prokázáno,
že
jejich
výstražné
signály
jsou
mezi
jednotlivými
druhy
přástevníkovitých velmi podobné a proto zde zjevně fungují akustické mimikry (Barber a
Conner 2007). Podobně například pestřenky (Diptera, Syrphidae) se snaží bzučet všechny
podobně, což by naznačovalo, že se také jedná o mimikry, nicméně experimentálně
nebylo prokázáno, že by se akusticky snažily napodobovat své modely – blanokřídlé
(Rashed et al. 2009).
Mimikry
Již jsem se zmínil o pojmu mimikry, což je jeden z nejoblíbenějších předmětů zájmu studií
řešících výstražné zbarvení. Pojem mimikry (mimeze) byl původně používán pro
napodobování rostlinných částí živočichy za účelem zneviditelnění pro predátory (Kirby a
Spence 1828). Později byl tento pojem rozšířen na obecné napodobování mezi organismy
(Fischer 1930). Od počátku definice bylo v rámci výstražné signalizace rozlišováno
několik typů mimikry lišící se výhodností vztahu pro jednotlivé účastníky.
Primárně se jedná o vztah symbiotický, altruistický, kdy výstražně zbarvené organismy se
snaží připodobňovat svůj vzhled navzájem – Müllerovské mimikry (Müller 1878). Tento
jev je zapříčiněn snahou neklást na predátora přílišné požadavky a učit ho pouze
omezené množství signálů, za kterými se skrývá nějaká nevhodnost až nebezpečnost
dané kořisti (Huheey 1980). Důsledkem tedy je, že výstražné signály jsou poměrně
uniformní, používá se v nich několik málo barev a vzorů (viz výše) a i v rámci nepříbuzných
skupin si jsou druhy podobné (Mallet a Gilbert 1995).
Nicméně, vždy jsou jednotliví mimici v něčem odlišní a nabízí se tedy otázka, jak moc
dokonalou kopií musí být aby byly mimikry funkční (Ditrich et al. 1993). Různí predátoři
jsou schopni generalizovat signál modelu na různě podobné mimiky a proto fungují i
poměrně nedokonalé mimikry, často postačuje pouhá prezence červené barvy nebo
podobného jasného znaku (Lindström et al. 1997, Exnerová et al. 2008). Nicméně se
ukazuje, že záleží na tom, jaké spektrum modelů a mimiků má predátor k dispozici a
podle toho se rozhoduje, jak odlišného mimika je ochoten zařadit do daného
mimetického okruhu (Lindström et al. 2004).
Druhým základním typem mimeze jsou Batesovské mimikry (Bates 1863). Jedná se o
situaci, kdy některé druhy parasitují na Müllerovských mimikry, napodobují chráněný druh
(model, vzor), ač sami žádnou ochranu nemají. Predátor znalý výstražného signálu, tedy
Batesovského mimika odmítá také, aniž by se přesvědčil o jeho poživatelnosti (Huheey
1980). Nicméně, tento parasitický vztah má podmínku, aby mimici byly v populaci
vzácnější než modely, a tedy aby se predátor častěji setkával s modely a spojil si daný
signál s ochranou, nikoliv s mimikem (Emlen 1968). Batesovské mimikry byly popsány u
mnoha skupin organismů od obojživelníků (Brodie a Howard 1973, Saporito et al. 2007)
přes plže (Field 1974, Edmunds 1991) po pestřenky (Maier 1978, Ditrich et al. 1993,
Edmunds 2000). Nejčastěji studovanou skupinou však jsou zřejmě motýli. V jejich
případě bylo popsáno několik skupin nepříbuzných druhů (mimicry rings), se stejnou
barevnou signalizací a odlišnou mírou ochrany (Clarke 1982, Mallet a Gilbert 1995).
Nicméně, podrobnější studie prokázaly, že se často nedá mluvit o pravých Batesovských
mimikry neboť všechny zúčastněné druhy disponují určitou ochranou, pouze se jedná o
ochranu různě silnou (Ritland a Brower 1991). Na základě těchto poznatků byl navržen
koncept tzv. quasi-Batesovských mimikry nebo také Batesovsko-Müllerovského kontinua
(Mallet a Joron 1999, Speed 1999a, Turner a Speed 1999). Pro quasi-Batesovského
mimika je alespoň částečná ochrana pojistkou pro případ, že se setká s naivním
predátorem neznalým modelu a tím je částečně chráněn před zabitím. Nicméně, stále
nevynakládá na svou ochranu stejnou energii jako model a jeho vztah k němu je stále
parasitický.
Kromě těchto základních dvou typů mimikry jsou rozlišovány další typy, většinou se lišící
okolnostmi užití. Automimikry je termín užívaný pro dva odlišné jevy (Brower 1968). Za
prvé se jedná o situaci, kdy pochází model i mimik z jednoho druhu, často se jedná o
samce a samici (Gibson 1984), nebo o populace živící se odlišně toxickou potravou
(Ritland 1994). Druhý význam tohoto jevu popisuje situaci, kdy je jedinec chráněn částí
svého těla, většinou tím, že napodobuje nějakou strukturu (hlavu, oko – viz výše; LevYadun 2003).
Dalším případem je když jsou mimikry užívány ne za účelem obrany (nenesou tedy
výstražný signál), ale přilákání a pozření kořisti – tzv. agresivní nebo Peckhamovské
mimikry (Sazima 2002, Randall 2005, Marshall a Hill 2009). Dále se mimikry užívají za
účelem přenosu parasita (hnízdního – Soler 2009, myrmekofilního – Geiselhardt et al.
2007 nebo krevního – Davies 1997) nebo rozmnožení (opylení – Schiestl 2005, přilákání
partnera – Kelley 2008 nebo udržení sociálního statutu – Muller a Wrangham 2002).
Evoluce výstražné signalizace a její studium
Nejproblematičtějším bodem studia aposematismu je představa o jeho iniciální evoluci.
Jak se mohl gen pro výrazné zbarvení rozšířit v jinak krypticky zbarvené populaci, když je
jeho nositel okamžitě napaden a zabit kvůli své výraznosti (Guilford 1990)? Pro řešení
tohoto dilematu bylo potřeba experimentálně testovat reakci predátora na nový výstražný
signál. Nicméně predátoři jsou v dnešní době zatíženi přinejmenším evoluční poučeností
o výstražných signálech (když ne individuální). Nelze tedy odfiltrovat nakolik je jejich
reakce zatížena vrozenou nebo získanou averzí vůči aposematické kořisti (Harvey a
Paxton 1981).
Řešení tohoto experimentálního problému bylo nalezeno ve dvou nových přístupech,
které se během posledních desítek let používají na studium iniciální evoluce
aposematismu (Lindström 1999). První z nich používá robotického predátora,
nasimulovaného pomocí počítačového software (Speed 1999b). Takový predátor (a
samozřejmě i kořist) může mít libovolné schopnosti a vlastnosti (např. populační
dynamiku a rozpoznávací schopnosti) a navíc lze sledovat přežívání kořisti a populační
trendy v dlouhém časovém období (tisíce generací; Sherratt a Beatty 2003). Nicméně,
takovýto přístup je velmi citlivý vůči zadaným parametrům. Drobné odchylky ve
vlastnostech predátora nebo kořisti mohou zapříčinit úplně odlišný výsledek celé
simulace. S tímto přístupem, lze tedy nadefinovat, za jakých podmínek může k evoluci a
udržení aposematismu dojít. Nicméně, je otázkou, nakolik lze závěry těchto prací přenést
na reálné predátory a kořist.
Z tohoto důvodu byl vyvinut nový experimentální přístup, který užívá reálné predátory,
uvádí je ale do úplně nových podmínek a konfrontuje je s naprosto neznámou umělou
kořistí. Tento přístup se souhrnně označuje jako Novel World experimenty (Alatalo a
Mappes 1996). V takovém pokusu lze použít reálného predátora (např. sýkoru koňadru –
Parus major), třeba i odchyceného v přírodě a majícího tedy určité vrozené i získané
zkušenosti. Predátor je přesunut do laboratorní místnosti, v níž je na podlaze umístěno
několik jedinců kořisti. Kořist je většinou plátek mandlového jádra, nebo podobná umělá
potrava, zakrytá přilepeným papírkem se symbolem (Mappes a Alatalo 1997a). Tyto
symboly jsou většinou černobílé a schematizované (čtverec, kříž). S ohledem na vzor,
který je nakreslen na podlaze je kořist výstražná nebo kryptická (odlišná nebo stejná jako
podlaha). Dále je kořist buďto jedlá, nebo nejedlá (napuštěná nechutným roztokem).
Tímto způsobem lze simulovat ne/chráněnou, ne/výstražnou kořist a sledovat její
přežívání pod predačním tlakem různě (ale známě) zkušených predátorů a tím odhadnout
jaké principy se podílely na udržení nově vzniklých výstražných signálů.
Před osvojením těchto experimentálních přístupů vzniklo několik teorií, které vysvětlovali
paradox evoluce prvního aposematika. První úvaha se opírala o fakt, že prvotní
aposematik není sám. Pokud se v jedné generaci kořisti, vyskytne najednou několik
výstražných jedinců (sourozenců), měla by kin selekce způsobit přežití alespoň některého
z nich, a ostatní se obětují aby poučili případné predátory (Harvey et al. 1982, Guilford
1985, Mallet a Singer 1987). Tato teorie zároveň předpokládala, že sourozenci se
vyskytují agregovaně a predátor je tedy konfrontován s jednotlivými výstražnými jedinci
v krátkém časovém intervalu (viz výše).
Alternativní teorie se opírala naopak o
individuální selekci a tvrdila, že nově vzniklý výstražný jedinec by mohl útok predátora
přežít, poučit ho a zároveň přenést své geny do další generace (Silén-Tullberg a Bryant
1983, Engen et al. 1986). U aposematického hmyzu se navíc předpokládá větší
mechanická odolnost umožňující právě toto ochutnávání (Evans 1987, Evans a Schmidt
1990). Tyto názory byly experimentálně prokázány s použitím larev motýlů (Wiklund a
Järvi 1982).
Právě nové studie používající reálné v přírodě odchycené predátory v novel world designu
(Alatalo a Mappes 2000) prokázaly, že obě tyto selekční mechanismy mohou hrát roli;
nicméně, hlavní princip zajišťující přežívání aposematické kořisti tkví v kognitivních a
mentálních schopnostech predátora (Lindström et al. 2001a,b). Tyto parametry již
samozřejmě byly brány v potaz i dříve (Guilford a Dawkins 1991, Rowe 1999), nicméně
vzhledem k omezené znalosti mentálních schopností většiny potenciálních predátorů,
nebylo možné si utvořit konkrétní představu o významu těchto parametrů v evoluci
aposematického signálu. Teprve s bližším ohledáním jednotlivých schopností predátorů
(zejména ptačích) mohly být zahrnuty do studia aposematismu a usnadnily např.
modelování virtuálních predátorů (Speed 2001).
Smyslové, kognitivní a mentální schopnosti ptačích predátorů
Nejvýznamnějším smyslem ptáků je stejně jako u člověka zrak. Tato vlastnost se také
významně odrazila na faktu, že ptáci jsou nejpoužívanějšími predátory při studiu
aposematismu (Lindström 1999). Nicméně rozdíly ve vidění ptáků a člověka jsou víc než
významné a je tedy otázkou, nakolik lze zobecňovat lidské vnímání na ptačí. Především
ptáci mají v čípcích 4 nebo 5 typů fotopigmentů (na rozdíl od našich 3), dále mají systém
olejových kapének, které určitým způsobem filtrují přicházející světlo (Epsmark et al.
2000) a jejich zrakový aparát je schopný vnímat rozsáhlejší část spektra přesahující bílé
světlo do UV části (Cuthill et al. 2000, Epsmark et al. 2000, Hastad a Ödeen 2008).
Schopnost kategorizovat a rozlišovat jednotlivé barvy je do jisté míry obdobná jako u lidí,
ale lidské a ptačí vnímání kontrastu a barev není totožné (Jones et al. 2001).
Oblíbeným tématem mnoha prací je především schopnost vnímání UV části spektra.
Bennett et al. (1996 ex Epsmark 2000) zkoumal zebřičky (Taeniopygia guttata), na
kterých prokázal vztah vnímání UV záření a pohlavních preferencí. Podobné výsledky
přinesla i studie na špačcích obecných (Sturnus vulgaris - Bennett et al. 1997 ex
Epsmark 2000). Vliv schopnosti vidět v UV spektru na vyhledávání a identifikaci potravy
byl poprvé popsán u poštolek obecných (Falco tinnunculus- Viitala et al. 1995). Na
sýkorách modřinkách (Cyanistes caeruleus) byla provedena studie zabývající se
využíváním schopnosti vidět hmyzí kořist (různé druhy housenek) v UV spektru (Church et
al. 1998).
V recentních pracích se již počítá s odchylkami ptačích a lidských zrakových schopností.
Například studie Gamberale-Stille & Sillén-Tullberg (1999) nebo Svádové et al. (2009)
obsahuje i naměřená odrazová spektra druhů ploštic, které byly v experimentu použity.
Lze předpokládat, že druhy pro lidské oko nevýstražné mohou odrážet v UV spektru
signály, ptáky chápané jako výstražné (Cuthill et al. 2000). Nicméně, výstražnost UV
signálů není rozhodně obecně zřejmá. Lyytinen et al. (2001) odhalili, že sýkory koňadry
(Parus major) nevykazovaly jasnou preferenci, nebo averzi vůči aposematické, nebo
kryptické kořisti odrážející UV záření. Naopak byly spíše pozorovány sklony ptáků spojit si
UV záření s chutnou kořistí
Další smysly ovlivňující přístup ptačího predátora jsou chuť a čich. V tomto ohledu již ptáci
nejsou srovnatelní s člověkem, jejich čichové a chuťové schopnosti jsou výrazně horší.
Člověk má v dutině ústní 9000 chuťových pohárků, kdežto kuře (Gallus gallus f.
domestica) jich má pouze 24 (Sturkie a Whittow 2000). Co se týče vnímání a preferencí
pro jednotlivé chutě a vůně, existují významné rozdíly mezi jednotlivými druhy ptáků.
Sladkou chuť preferují převážně nektarivorní ptáci (Nectarinidae), zatímco například
špačci (Sturnus vulgaris), odmítají roztok sacharózy a preferují čistou vodu (Sturkie a
Whittow 2000). Vnímání hořké chuti je také nejednoznačné; některé, pro člověka hořké
sloučeniny ptáci odmítají, zatímco jiné jsou ptáky normálně přijímány ( Sturkie a Whittow
2000).
Podobně jako v případě chuťi, ptačí čichové schopnosti jsou relativně potlačeny na úkor
zraku a sluchu (Roper 1997). Pokusy s kuřaty (Gallus gallus f. domestica) odhalily, že
ptáci byli schopni zachytit a reagovat na pachy běžně produkované aposematickým
hmyzem. Pachy, jako například vanilka, které v přírodě nejsou produkovány k odpuzování,
byla kuřata také schopna zachytit a nevyvolávaly u nich žádnou reakci (Marples a Roper
1996).
Vjemy přijaté smyslovými orgány jsou vstupní informace, na nichž se zakládá rozhodování
o případném pozření dané kořisti; nicméně, mohou jej ovlivnit i vnitřní mentální pochody
predátora. Během studia chování ptáků vůči nevhodné kořisti bylo prokázáno, že jejich
celkový přístup k ní je výrazně ovlivněn nejen potravní ekologií ale i mentálními
schopnostmi.
Jedním z hlavních procesů ovlivňující přístup k nevhodné kořisti je neofobie. Jedná se o
situaci, ve které je predátorův útok zastaven při střetu s neznámou novou kořistí.
Neofobie neboli strach z nového, byla zaznamenána již Coppingerem (1969, 1970), který
pozoroval, že naivní predátoři se nové výstražné kořisti vyhýbali a dokonce se u nich
objevovala útěková reakce. Marples a Kelly (1999) poukázali na rozdíl mezi neofobií a
potravním konservatismem, který je způsoben hlavně trváním doby, po kterou je nová
kořist odmítána. Neofobie je považována za krátkodobý stav, trvající pouze několik minut
a má za úkol donutit predátora ohledat blíže kořist a ochránit ho před zbrklým jednáním.
Naopak potravní konzervatizmus způsobuje dlouhodobé odmítání dané kořisti, je
geneticky dán a charakterizuje daného jedince (druh) po celý život (Marples a Kelly
1999). Každý pták se rodí s určitou představou o vhodné a nevhodné kořisti, která je
během života upřesňována ať už vlastní aktivitou nebo s pomocí rodičů (Turner 1964).
Nicméně, variabilita mezi jednotlivými druhy v tomto konzervatizmu a v míře jeho
vrozenosti je velmi výrazná. Např. Exnerová et al. (2007) prokázali, že averze vůči
aposematické ruměnici pospolné (Pyrrhocoris apterus) je vrozená u sýkory modřinky
(Cyanistes caruleus) a uhelníčka (Periparus ater), zatímco sýkora koňadra (Parus major) a
parukářka (Lophophanes cristatus) se jí musí učit.
Další mentální schopnosti, které silně ovlivňují reakci ptačího predátora na
aposematickou kořist jsou schopnost učit se a pamatovat si. Efektivita a udržitelnost
výstražného signálu závisí na schopnosti predátora naučit a zapamatovat si jej, proto jsou
výstražné signály designovány tak aby byly prokazatelně snáze zapamatovatelné než
kryptické (Roper a Wistow 1986 a Roper a Redston 1987). Nicméně, existuje teorie,
která tvrdí, že výstražné signály jsou zapamatovány, ne díky své nápadnosti, ale díky větší
frekvenci útoků na ní provedených (Gittleman et al. 1980).
V poslední době bylo prokázáno, že mnoho druhů organismů, včetně ptáků, vykazuje
individuální variabilitu v reakcích na různé podněty, nejen potravní, ale i sociální (Carere
2003). Tento fenomén je popisován jako personalita a obecně se rozlišují dva základní
typy osobnostních charakteristik (mezi- i vnitrodruhově). První z nich se v anglicky psané
literatuře označuje jako „shy“, „slow“ nebo „proactive“; jedná se o jedince, kteří na nové
podněty reagují opatrně, pomalu, ale důkladně je prozkoumávají, obyčejně se jedná o
jedince sociálně níže postavené (Carere 2003, Drent et al. 2003, Dingemanse a Goede
2004). Druhý typ, popisovaný jako „bold“, „fast“ nebo „reactive“, se vyznačuje povrchním
a rychlým ohledáním nových podnětů a spíše dominantnějším chováním. Osobnostní
charakteristiky mají přirozeně vliv na mnoho aspektů ptačího chování, reakci na
aposematickou kořist nevyjímaje. Exnerová et al. (2010) pozorovala vliv personality
sýkory koňadry (Parus major) na reakci vůči ruměnici pospolné (Pyrrhocoris apterus).
„Slow“ ptáci se chovali opatrněji než „fast“ ptáci, avšak kořisti se naučili vyhýbat rychleji.
Ve výsledku se ale nakonec oba typy ptáků naučily odmítat nechutnou aposematickou
kořist.
Dalším jevem, který ovlivňuje reakci ptáka na aposematickou kořist je tzv. search image.
Jedná se o situaci, kdy predátor má konkrétní představu o hledané potravě a upozadí
ostatní vjemy, i když jsou relevantní, aby se mohl soustředit na vjemy poskytované
hledanou kořistí (Tinbergen 1963, Dukas a Ellner 1993, Blough a Blough 1997). Tato
vlastnost se projevuje především při hledání kryptické kořisti, neboť výstražné signály
obyčejně upoutají pozornost i přes působení search image (Bond a Kamil 2002).
Nicméně, je-li kryptická kořist žádána, search image může způsobit, že z kryptického
zbarvení neplyne taková výhoda a kryptická i aposematické kořist jsou napadány
s obdobnou frekvencí (Dukas a Kamil 2001; Dukas 2002, 2004).
Další mentální schopnost predátora ovlivňující fungování výstražné signalizace, obzvláště
mimikry je schopnost generalizace. Je-li predátor schopen naučit se jednomu
výstražnému signálu, je pro něj výhodnější rozšířit jeho charakteristiky i na podobné
signály, než se učit každý jednotlivý signál zvlášť (Leimar et al. 1986). Tato schopnost je
obyčejně usnadněna Müllerovskými mimikry, které vyvíjí tlak na co největší podobnost
jednotlivých aposematických druhů (Huheey 1980). Nicméně, některé studie prokazují
překvapivě velkou schopnost generalizovat averzi na i velmi odlišné mimiky (Gamberale a
Tullberg 1996, Svádová et al. 2009).
Metodoligické přístupy ke studiu výstražné signalizace
Nicméně rozmanité kognitivní schopnosti predátorů nejsou jediný parametr ovlivňující
výsledek jejich střetu s kořistí a tím i evoluci výstražného signálu. Metodologické přístupy
dobře použitelné pro simulaci evoluce výstražných signálů nemusí vždy přinášet výsledky
plně aplikovatelné pro reálné procesy probíhající v přírodě. Jak již bylo řečeno
aposematický signál je obyčejně složen z mnoha jednotlivých znaků, přenášených na
různých smyslových kanálech (Rowe a Guilford 1999). Práce testující evoluci se obyčejně
zaměřují na jeden konkrétní signál, což je pro potřeby simulace evoluce ideální, nicméně
reálná např. hmyzí kořist (i ta uplně první výstražně zbarvená) poskytuje predátorovi více
znaků, podle kterých je možné ji rozpoznat (Kauppinen a Mappes 2003).
Je tedy nezbytné testovat efektivitu výstražných signálů také na reálné, živé kořisti. Tento
přístup byl samozřejmě používán v počátcích, kdy se testoval základní význam barevných
signálů pomocí experimentů s ptačími predátory (Brower a Brower 1962), obzvláště se
zájmem o mimikry. Poté se studie zaměřovaly na jednotlivé znaky, ať už optické
(Coppinger 1970) nebo chemické (Brower et al. 1968) a tím vznikala potřeba použití
umělé kořisti. Nejdříve se jednalo o prosté pozorování jak přežívají různě zbarvené kusy
potravy pod predačním tlakem divokých, anonymních ptáků (Pank 1976, Jeffords et al.
1979), později se již jednalo o experimenty klecové, kde jsou známy vlastnosti predátora
(Gittleman et al. 1980, Smith 1980). Pokusy s umělou kořistí vyvrcholily v tzv. Novel world
experimentech (viz výše, Alatalo a Mappes 1996, Lindström 1999). V poslední době se
stále více objevují práce popisující reakci přírodních predátorů na potenciální kořist
vybavenou nějakým varovným signálem (např. Hagen et al. 2003, Talianchich et al. 2003,
Meredith et al. 2007, Lindstedt et al. 2008). Poměrně vhodné jsou práce kombinující oba
přístupy, kdy se jedná o kořist přirozenou, nicméně v některém ohledu pozměněnou
(většinou nabarvenou – Schuler a Hesse 1985, Roper 1990, Gamberale-Stille 2001,
Exnerová et al. 2003). Taková kořist poskytuje predátorovi všechny signály stejně jako
přírodní, ale zároveň testuje význam modifikovaného znaku.
Abychom testovali efektivitu reálných výstražných signálů, je třeba používat nejen reálnou
kořist, ale i vhodné predátory. Mnoho prací studujících výstražné signály používala coby
predátory domácí kuřata (Gallus gallus f. domestica; Shettleworth 1972, Gamberale a
Tullberg 1996, Lindström et al. 2001c, Hauglund et al. 2006). Problém s takovým
predátorem tkví především v tom, že je to druh již po generace chovaný v zajetí a za tuto
dobu mohly být jeho potravní návyky posunuty selekcí ze strany člověka na přijímání co
nejširšího spektra potravy (Marples a Kelly 1999). Jeho výhoda spočívá v tom, že se jedná
o druh nekrmivý, lze jej tedy pokusovat prakticky hned po vylíhnutí a lze tedy dobře
manipulovat jeho individuální zkušenost (Schuler a Hesse 1985).
Práce v nichž jsou používány přirozené (nechované) druhy predátorů jsou také poměrně
časté, nicméně omezují se také na úzké spektrum predátorů. Nejčastěji používaným
ptačím druhem užívaným jako predátor aposematické kořisti je kromě kuřete sýkora
koňadra (Parus major), a to jak v přírodě odchycení dospělci (Mappes a Alatalo 1997b,
Exnerová et al. 2003, Hagen et al. 2003) tak ručně odchovaná mláďata (Exnerová et al.
2007, Svádová et al. 2009). Koňadry jsou zároveň predátoři pravidelně používaní v Novel
worldovských pracích (Lindström et al. 1999a,b, 2001b; Riipi et al. 2001; Rowe et al.
2004; Ihalainen et al. 2008). Sýkory mají obecně averzi vůči výstražným signálům
(optickým i chemickým), nicméně alespoň v některých případech není tato averze vrozená
(Exnerová et al. 2007). Zároveň vykazují sýkory velkou přizpůsobivost, a to jak
k laboratorním podmínkám (Merilaita a Lind 2006), tak i nové potravě (Lindström et al.
2004). Nicméně, sýkory, i přes svou ekologickou plasticitu (Royama 1970), nejsou ve
všech případech optimálním predátorem. Práce srovnávající více druhů ptačích predátorů
(Wiklund a Järvi 1982, Exnerová et al. 2003, Pinheiro 2003) ukazují, že různé potravní
specializace výrazně ovlivňují jejich přístup k výstražně signalizující kořisti, a že jejich
averze
nemusí
tedy
být
způsobena
výstražným
signálem,
nýbrž
potravním
konservatismem (Greenberg 1984, Marples a Kelly 1999, Webster a Lefebvre 2000).
Cíle disertační práce
Předkládaná disertační práce je součástí projektu používající jednotnou metodiku, za
účelem testování výstražného významu jednotlivých signálů hmyzí kořisti vůči ptačím
predátorům. V mnou prezentovaných pracích, byly dvěma druhům sýkor (P. major a C.
caeruleus) předkládány v laboratorních podmínkách různé druhy a umělé modifikace
aposematické kořisti a testována jejich potravní reakce. Testované hmyzí druhy byly
voleny tak, aby umožňovaly poměrně dobře odfiltrovat jednotlivé části jejich výstražného
signálu při zachování ostatních.
První skupina prací pouze konfrontuje v přírodě se vyskytující a/nebo uměle
modifikovanou hmyzí kořist (nabarvenou, zbavenou krovek) s testovanými ptáky, za
účelem odhalení významu jednotlivých složek výstražného signálu daného druhu. Kněžice
páskovaná (Graphosoma lineatum), předkládaná v první práci oběma druhům sýkor, se
kromě barevného vzoru a poměrně výrazné chemické signalizace vyznačuje poměrně
charakteristickým tvarem těla. Efekt těchto znaků je tedy testován, společně s efektivitou
celkového výstražného signálu na různě kontrastních prostředích a tím je zároveň
testována hypotéza, že černo-červené pruhované zbarvení kněžice páskované může na
vhodném pozadí (uschlé souplodí mrkvovitých rostlin) působit nejen výstražně, ale i
krypticky.
Výsledky
ukazují,
že
kněžice
je
velmi
dobře
chráněna
původním
nemodifikovaným vzorem, nicméně nabarvení na hnědo tuto ochranu nesnižuje tak, jak
bychom čekali na základě např. výsledků na ruměnici pospolné. Lze tedy předpokládat,
že ptáci jsou schopni rozeznat kněžici na základě jiných něž barevných signálů, např.
tvaru těla.
Několik druhů slunéček (Coleoptera, Coccinellidae), předkládaných sýkorám koňadrám
v druhé studii, bylo vybráno s ohledem na testování různé barevné kombinace (tmavé
skvrny na světlejším pozadí a naopak) výstražného zbarvení (černo-červená, rezavotmavohnědá) ale i prezence vzoru (tečky ano/ne). Dále byly na slunéčkách provedeny
modifikace testující vliv přítomnosti barevného vzoru (nabarvení nahnědo) a celkového
vzhledu (odstranění krovek). Výsledky prokázali především silnou averzi vůči všem
druhům a formám slunéček, neboť žádná z testovaných sýkor nezabila ani nesežrala ani
jedno z nabídnutých slunéček. Dále jsme prokázali význam tečkovaného vzoru v ochraně
slunéček před napadením a to bez ohledu na barevné provedení (dokonce i poměrně
nekontrastní tečkovaný vzor poskytuje dostatečnou ochranu). Naopak slunéčka bez teček
jsou chráněna trochu méně. Nejméně chráněnou formou slunéčka byla ta, jež byla
zbavena krovek a tím byl silně pozměněn celkový vzhled zvířete a sýkory zjevně nebyly
schopny rozpoznat v takové kořisti slunéčko a napadly je.
Pokusy, v nichž byly oběma druhům sýkor (různě velkým) nabízeny larvální instary
ruměnice pospolné (Pyrrhocoris apterus), se pokoušely odfiltrovat vliv a) různé chemické
ochrany (množství i kvalita), b) různého barevného vzoru a c) různé velikosti larev a
dospělců. Byla prokázána překvapivě nízká ochranná síla zbarvení larev ruměnice a také
částečný vliv velikosti (nejvíce byla napadána nejmenší kořist, navíc především největšími
predátory).
Ploštice bavlníková (Dysdercus cingulatus) umožnila testovat vliv různých barevných
signálů (odstínů i vzorů), jako i chemické ochrany na reakce obou druhů sýkor. Navíc,
vzhledem k tomu, že se jedná o Jihoasijskou ploštici, byla zároveň testována schopnost
Evropských ptáků generalizovat známé signály Evropského hmyzu na tyto znaky. Výsledky
ukázaly překvapivě nízkou schopnost generalizovat barevný vzor evropských ploštic na
testovaný druh. Ploštice bavlníkové byly běžně napadány i požírány i přes značnou
podobnost vzorů i celkového vzhledu.
Dvě poslední práce používaly odlišnou metodiku. Ptákům nebyla předkládána v přírodě se
vyskytující kořist, nýbrž kořist částečně uměle vytvořená. Jednalo se o jedlý druh švába,
švába Argentinského (Blaptica dubia), bez jakýchkoliv výstražných signálů a kvalit, který
nesl na své svrchní straně připevněný papírový samolepící štítek s barevným vzorem. Díky
tomuto metodickému přístupu lze odfiltrovat případný vliv chemických signálů a testovat
pouze signály optické.
V první práci nesl štítky s pozměněným vzorem ruměnice pospolné (Pyrrhocoris apterus).
Jednalo se vždy o kombinaci nezměněného černého vzoru ruměnice a různých
podkladových barev., testující vliv barvy a vzoru ve výstražné signalizaci ruměnice
pospolné. Byla prokázána výstražná funkce červené a oranžové barvy, nicméně žlutá,
bílé, modrá a fialová vyvolávali averzi jen u části ptáků. Zelená barva byla napadána se
stejnou frekvencí, jako kontrolní hnědá (bez černého ruměnícího vzoru), což naznačuje,
že černý vzor sám o sobě ochranu kořisti neposkytuje.
Poslední práce použila stejnou metodiku za účelem „výroby“ Batesovského mimika. Jedlý
šváb Argentinský nesoucí štítek s barevným vzorem ruměnice pospolné (mimik) byl
předkládám sýkorám společně se stejně vybavenou (oštítkovanou) chráněnou ruměnicí
(model). Ptáci oba druhy kořisti napadali, Batesovské mimikry tedy fungovaly správně.
Nicméně, druhá část ptáků, která měla předchozí zkušenost se švábem bez jakéhokoliv
štítku byla schopná Batesovského mimika odhalit a napadal jej. Lze tedy předpokládat,
že Batesovský mimik by měl vzniknout pouze v populaci hmyzu, s nímž je predátor pokuď
možno málo obeznámen.
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Evolutionary Ecology Research, 2006, 8: 881–890
Are gregarious red-black shieldbugs,
Graphosoma lineatum (Hemiptera: Pentatomidae),
really aposematic? An experimental approach
Petr Veselý,* Silvie Veselá, Roman Fuchs and Jan Zrzavý
Department of Zoology, Faculty of Biological Sciences, University of South Bohemia,
Branišovská 31, 370 05 České Budějovice, Czech Republic
ABSTRACT
Hypothesis: The coloration of the red-black shieldbug has a warning function. This quality
can be lowered when the shieldbug is presented on a fragmented background.
Organism: We offered wild-coloured and artificially deaposematized (painted brown)
red-black shieldbugs (Graphosoma lineatum) to avian predators (Parus major, Parus caeruleus).
Site of experiments: The experiments were conducted in a cage (0.7 m × 0.7 m × 0.7 m) fitted
with a one-way mirror.
Methods: In succession, we offered five shieldbugs to each bird. We presented the shieldbugs
on contrasting (white) and matching (imitating the shieldbug’s habitat and imitating the striated
shieldbug pattern) backgrounds.
Results: The blue tits avoided all shieldbugs offered to them regardless of their coloration.
The great tits attacked both colour forms, but the brown one more frequently. The wild-coloured
shieldbugs were significantly better protected against repeated attacks. Shieldbugs presented
on any of the matching backgrounds were attacked less frequently than when presented on the
white background.
Keywords: disruptive coloration, Parus caeruleus, Parus major, warning coloration.
INTRODUCTION
Warning (aposematic) colouration, which provides protection to a defended species against
visually oriented predators (usually birds), is a controversial topic in evolutionary ecology.
The initial evolution of aposematic anti-predator signalling is expected to increase
predation risk before reaching a stage when local predators are able to learn to avoid the
unpalatable prey (see Lindström et al., 2001; Marples et al., 2005). Fisher (1958) presented the idea
of aggregation benefit through the survival of related individuals. However, Riipi et al.
(2001) showed that grouping would have been highly beneficial for aposematic prey
individuals, surrounded by naive predators, without requiring any kin selection. They
proposed four possible non-kin selection mechanisms: non-linear growth of detectability
* Author to whom all correspondence should be addressed. e-mail: [email protected]
Consult the copyright statement on the inside front cover for non-commercial copying policies.
© 2006 Petr Veselý
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risk/group size; low additional detectability costs due to conspicuous signals in the groupliving prey; a ‘dilution effect’; and rapid avoidance learning of the warning signal in groups.
In contrast, Beatty et al. (2005) suggest that the common association between aggregation and
distastefulness may primarily arise because of the significant vulnerability of aggregated
palatable prey. However, they investigated responses of human ‘predators’ in computer
‘novel world’ experiments, not a realistic predator–prey system. The true influence of prey
gregarious habit on aposematism efficiency has rarely been analysed experimentally [but see
Reader and Hochuli (2003), who studied a caterpillar prey vs. hemipteran predator system; see
also Nilsson and Forsman (2003) for application of phylogeny-based comparative analyses of
evolutionary relationships of colouration, body size, and gregarious habit in the Lepidoptera].
The Palearctic red-black shieldbug, Graphosoma lineatum L. (Hemiptera: Pentatomidae),
is a textbook example of the aposematic insect (for a review, see Komárek, 2000). Its contrasting
colours form several longitudinal stripes on the dorsal side and a spotted pattern on the
ventral side of its body (Fig. 1). As G. lineatum is a gregarious insect, it would be easy for an
avian predator to learn its unpalatability because the bird is able to meet another prey item
soon after encountering the first (Sillén-Tullberg and Leimar, 1988).
The shieldbugs in general (Pentatomoidea) are known to be extremely distasteful for
predators (e.g. Krall et al., 1999) because they are able, when touched, to release a repellent
secretion, predominantly from their thoracic scent glands. Though the pentatomoids
usually do not combine their chemical defence with warning colouration, there are some
obviously aposematic species, for example Murgantia and Eurydema spp. (Aldrich et al., 1996;
Aliabadi et al., 2002), which are mostly black with yellow or red spots (or vice versa). The
G. lineatum-like longitudinal striation is relatively common in pentatomoids (Tietz and Zrzavý,
1996), but their stripes have usually much less contrasting, presumably cryptic colours
Fig. 1. The shieldbug (Graphosoma lineatum).
Are gregarious red-black shieldbugs really aposematic?
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(mostly brownish and yellowish). The combination of longitudinal striation with contrasting colouration is an evident evolutionary novelty of G. lineatum and a few closely related
species, which suggests that G. lineatum is a suitable model species for studies of the evolution
of warning colouration (Zrzavý, 1994). However, the functional aposematism of G. lineatum
is not evident, although Schlee (1986) showed that G. lineatum is attacked less frequently than
other hemipteran species. The possible warning pattern in G. lineatum is limited to adult
bugs, since the enlarged longitudinally striated shield (mesoscutellum), covering the whole
abdomen dorsally, is not present in the juvenile stages. Only the longitudinal striation on the
head and pronotum (a trapezoid shield just behind the head) is sequentially developed in the
juvenile stages (Tietz and Zrzavý, 1996). The juveniles are yellowish to brownish and hence not
aposematic. This ontogenetic change in the colouration display is interesting for an understanding of possible shieldbug aposematism, because shieldbugs are hemimetabolous
insects with juveniles morphologically and ecologically similar to the adults (they live on the
same plants, later juvenile and adult instars are of comparable sizes and shapes). As there is
no sexual dichroism in G. lineatum, infraspecific colour signalling is unlikely.
Naturally, the striped pattern of G. lineatum could have a different anti-predatory
function. Stripes are often used by animals to disrupt their body outline or to mask vulnerable or important parts of their bodies (e.g. eyes). The shieldbugs feed on umbelliferous
plants (Apiaceae) (Popov, 1971), whose inflorescences (umbels) consist of numerous short twigs
with very small white flowers. Against the background of brown or brown-red dried umbel
twigs, the colouration of the adult shieldbug could be rather cryptic (breaking up the
outline of its body), just like the colouration of juveniles. As most European pentatomoids
(including G. lineatum) hibernate as adults, they are often encountered on dried and rotten
plant remains. The ‘aposematic’ red-black shieldbugs are indeed surprisingly difficult to find
in nature unless they are sitting on a spread of white umbels (Musolin and Saulich, 1995).
In the present study, we examined the warning function of shieldbug colouration experimentally, using two species of wild-caught passerine predators. We examined whether
the colouration of a shieldbug presented on a white background acts as a warning signal,
by comparing the attack rates against wild-coloured and artificially ‘non-aposematic’
(brown-painted) shieldbugs (for methodological details, see Exnerová et al., 2003a, 2006). Furthermore,
we examined whether the colouration of a shieldbug presented on variously patterned
backgrounds still acts as a warning, by comparing the attack rates against the wild-coloured
form presented on white and patterned backgrounds. In each experiment, several trials were
performed with several shieldbugs, thus simulating the gregarious habit of the species.
Instead of presenting a group of shieldbugs to an individual predator, we presented them
sequentially to simulate natural circumstances more closely. In addition, the sequential
design of the experiments had the aims of minimizing a predator’s neophobia and
determining the stability of a bird’s foraging motivation.
MATERIALS AND METHODS
Experimental prey
The experimental shieldbug individuals were collected around České Budějovice (South
Bohemia, Czech Republic) during spring 2002 and 2003. Groups of about 50 individuals
were kept in the laboratory in transparent plastic boxes (16 × 13 × 7 cm). Dry seeds of
carrot (Daucus carota sativa), cow parsley (Anthriscus sylvestris), and wild angelica
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(Angelica sylvestris) together with water were supplied ad libitum. The insects were reared at
258C and in long-day (18 h light, 6 h dark) conditions.
We eliminated the aposematic colouration of bugs using brown watercolour (burned
sienna) spread all over its dorsal side (pronotum and mesoscutellum). The dye is odourless
and non-toxic and the treatment did not affect the locomotion or secretion of the bugs
(see Exnerová et al. 2003a, 2006).
Experimental predators
Adult birds of two tit species (blue tit Parus caeruleus L. and great tit Parus major L), caught
with mist nets in the vicinity of České Budějovice, were used as experimental predators.
Captures were conducted from 2002 to 2003 except in the breeding seasons (May to July).
We have licenses to catch and ring birds (Bird Ringing Centre Praha #975 and #1004) and
conduct experiments with animals (Czech Animal Welfare Commission #489/01). Birds
were kept in standard bird cages at a lowered indoor temperature and under outdoor
photoperiod conditions. Birds were acclimated to the laboratory conditions and food
(sunflower seeds and mealworms) for 1–3 days before the experiments. They were ringed
and released immediately after the end of the trials.
Experimental apparatus
The experimental cages were made from wooden frames (0.7 × 0.7 × 0.7 m) covered with
2
wire mesh (2 mm ) with the front wall incorporating a one-way mirror (for details, see Exnerová
et al., 2003a). The cages were equipped with one perch, a bowl with water, and a rotating circular
feeding tray, containing six small cups (a single cup contained a prey item during a trial). The
distance between the perch and the tray was approximately 25 cm. The colour of the bottom
of the cups was either white or patterned. The UMBEL patterned background was a brown
(burned sienna) pattern consisting of crossed lines in the shape of a schematized umbel seen
from above (Fig. 2). The STRIPES patterned background consisted of a shieldbug-like
pattern (Fig. 3), the colouration and size of which were obtained by scanning the shieldbug.
Standard illumination was generated by a light source (Lumilux Combi 18 W, Osram) that
simulates the full daylight spectrum.
Trials
The 40 blue tits were divided into two groups of 20 individuals. The first group was offered
the wild-coloured form of the shieldbug, the second group the brown one, in both cases on
the white background. The 80 great tits were divided into four groups of 20 individuals. As
with the blue tits, the first group was offered the wild-coloured shieldbugs, the second group
the brown ones, in both cases on the white background. The third and fourth groups,
however, were offered the wild-coloured shieldbugs presented on two different backgrounds
(UMBEL and STRIPES, see above). To avoid pseudoreplication, each individual bird was
used for a single series of trials only.
Each bird was placed into the experimental cage before the experiment to allow it to
adapt to the new conditions, and was provided with food (mealworms) and water. The bird
was deprived of food for 1.5–2.5 h before the experiment. The bird was assumed to be ready
for the experiment as soon as it attacked the mealworm immediately after being offered it.
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Fig. 2. The UMBEL background.
Fig. 3. The STRIPES background.
Each experiment with an individual bird consisted of a series of 10 successive trials, in
which five mealworms and five test bugs were offered alternately, starting with a mealworm.
Repetition of several trials within a single experiment was used to eliminate the effects of
individual differences in predators’ neophobia. The trials with mealworms were used to
check a bird’s motivation to forage, and they ended after the mealworm had been eaten. The
trials with shieldbugs always lasted 5 min.
A continuous description of a bird’s behaviour was recorded in the program Observer
version 3 (1989–1992, ©Noldus). We distinguished three possible results of each trial: (1)
the bug was neither handled nor killed during the 5-min trial; (2) the bug was handled
(touched, pecked or taken by the bird’s bill) but not killed; or (3) the bug was killed.
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Statistical analysis
We used two sets of data for the statistical analyses (all tests were performed in Statistica
5.5, 1984–1999, © StatSoft, Inc.):
1. The numbers of birds in each experimental group that handled/killed at least one of the
five bugs offered to it. Fisher’s exact test was used for this analysis.
2. The numbers of bugs handled/killed by individual birds in each experimental group. The
distribution of these data was not different from a Poisson distribution (KolmogorovSmirnov test, P > 0.05). They were normalized using square root transformation. An
analysis of variance (F-test) with post hoc comparison among particular groups (Tukey
HSD test) was used to analyse these data.
RESULTS
Wild-coloured (‘aposematic’) vs. brown-painted (‘non-aposematic’) form of the
shieldbug on a white background (great tits and blue tits as predators)
The great tits tended to avoid the wild-coloured form of the shieldbug slightly more than the
brown-painted one; however, the difference was not significant. There was a trend for handling (P = 0.111) and killing (P = 0.092) to occur more readily if brown-painted shieldbugs
were offered (Tables 1, 3). In contrast, the numbers of wild-coloured and brown-painted
shieldbugs that were handled/killed by a single bird during the series of trials differed
significantly (Fig. 4; Tables 2, 3). Even the birds that were ready to handle a red-black
shieldbug usually did not re-attack (on average, they handled 1.33 of 5 shieldbugs). The
attacks on the brown-painted shieldbugs were repeated more frequently (on average, the
birds handled 2.69 of 5 shieldbugs). The blue tits did not distinguish the two colour forms
of the shieldbugs and avoided both (the wild form strictly so; see Tables 1, 2).
Wild-coloured (‘aposematic’) shieldbug on patterned
vs. white backgrounds (great tits as predators)
Great tits handled and killed wild-coloured shieldbugs on both patterned backgrounds
slightly more willingly than the same shieldbugs on a white background, but the differences
were not significant (Fig. 4; Tables 1, 2, 3).
DISCUSSION
The anti-predatory function of the red and black colouration of various bugs (Lygaeidae,
Rhopalidae, Pyrrhocoridae) has been proved experimentally (Gamberale-Stille and Sillén-Tullberg,
1999; Aliabadi et al., 2002; Exnerová et al., 2003a, 2006). However, the ecological function of the conspicuous longitudinal pattern has rarely been analysed, in contrast to transversally striped
prey (Järvi et al., 1981; Evans and Waldbauer, 1982; Wiklund and Järvi, 1982; Howarth and Edmunds, 2000; Kauppinen
and Mappes, 2003). Schlee (1986) offered blackbirds (Turdus merula) adult shieldbugs that were
reared in captivity as well as wild-caught ones, and showed that G. lineatum is attacked less
frequently than three non-aposematic pentatomoid species. However, this approach was
unable to separate effects of colouration per se from other biological characteristics
of the species in question (e.g. body size and shape, amount and composition of the
defensive gland secretions, behaviour).
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Table 1. Reaction of the great tit (Parus major) and the blue tit (Parus caeruleus) to different forms of
the shieldbug (Graphosoma lineatum)
Tit species
Parus major (n = 20)
Parus caeruleus (n = 20)
Parus caeruleus (n = 20)
Shieldbug
colour
Background
Numbers of
birds that
handled at least
one shieldbug
wild
brown painted
wild
wild
wild
brown painted
white
white
UMBEL
STRIPES
white
white
6
12
8
8
1
3
Numbers of
birds that killed
at least one
shieldbug
1
6
4
4
0
1
Table 2. Median, mean, minimum, and maximum numbers of shieldbugs handled and killed by
individual birds (Parus major and Parus caeruleus)
Handled
Tit species
Parus major
(n = 20)
Parus major
(n = 20)
Parus major
(n = 20)
Parus major
(n = 20)
Parus caeruleus
(n = 20)
Parus caeruleus
(n = 20)
Shieldbug
colour
Background min–max
Killed
median
mean*
min–max median mean*
wild
white
0–2
0
1.33
0–1
0
1.0
brown
white
0–5
1
2.69
0–5
0
3.71
wild
UMBEL
0–2
0
1.5
0–2
0
1.5
wild
STRIPES
0–5
0
2.5
0–4
0
3.25
wild
white
0–1
0
1.0
0–0
0
0
brown
white
0–2
0
1.67
0–1
0
1.0
* The mean was computed only from data for birds that handled or killed shieldbugs.
Fig. 4. Numbers of bugs handled by individual great tits (Parus major). Each point is an individual
bird.
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Veselý et al.
Table 3. Results of statistical analyses for Parus major
Test result (P-value)
Comparison
WT on Wh vs. B on Wh
WT on U vs. WT on Wh
WT on S vs. WT on Wh
Data type
Handling
Killing
1
2
1
2
1
2
0.111
< 0.05
0.741
0.945
0.507
0.606
0.092
< 0.05
1.0
0.990
0.152
0.407
Note: Multiple comparisons in ANOVA: handling, F76,80 = 3.22, P = 0.027;
killing, F76,80 = 3.51, P = 0.02.
Abbreviations: WT, wild-type bug; B, brown-painted bug; Wh, white background; U, UMBEL background; S, STRIPES background.
Data type: 1, Number of birds that handled/killed at least one bug offered to
them (Fisher exact test). 2, Number of bugs handled/killed by individual birds
(Tukey HSD test of ANOVA).
Although there is no significant difference in the great tits’ readiness to attack any colour
form of a shieldbug, the birds usually do not repeat their attacks on the aposematic wildcoloured shieldbugs. The great tits seem to have to learn that this species is unsuitable as a
prey, and they are able to learn it more accurately if the negative stimulus is associated with
conspicuous colouration. We used wild-caught birds of unknown individual histories, but
they were most probably not familiar with G. lineatum in the field, as great tits do not forage
frequently in the shieldbugs’ microhabitat (Harrap and Quinn, 1996). Exnerová et al. (2003a) studied
the reaction of several passerine species (including the blue and great tits) to another
aposematic bug, the red firebug (Pyrrhocoris apterus L.), using the same experimental
equipment and ‘artificially non-aposematic’ insects as in the present paper. Both great and
blue tits were shown to reject the wild-coloured form of the firebug. This is in line with
the reaction of the blue tits to shieldbugs, but the great tits were more ready to attack wildcoloured shieldbugs (30% of tested birds) than wild-coloured firebugs (16% of tested birds).
In contrast, the mean numbers of the insects handled by an individual bird in our single
trial series was 1.33 for shieldbugs versus 1.80 for firebugs in the experiments of Exnerová
et al. (2003a). These results might suggest that shieldbug colouration is ecologically
important, especially in cases of the repeated encounter of an individual predator with
G. lineatum (see Sillén-Tullberg and Leimar, 1988). Hence, the gregarious habit could be beneficial
for the aposematic insect.
The profound difference between the two tested species of tits is quite surprising,
although Exnerová et al. (2003a) also showed that blue tits were more careful than great tits
(especially to the brown painted form). In our experiments, the blue tits did not handle
shieldbugs at all, regardless of their colour form. The shieldbug-like insects (body size,
solidity) are probably more suitable for more robust predators, as are the great tits. However,
in the parallel study on two juvenile and adult instars of Pyrrhocoris apterus, the predator/
prey body size ratio does not explain the different behaviour of blue and great tits to
the firebug developmental stages (M. Prokopová, R. Fuchs and J. Zrzavý, in preparation). Another possible
explanation is the different foraging ecology and behaviour of both species. The great
tits seem to be more experimental predators, while the blue tits show a higher degree of
889
neophobia. Neophobia was shown to be an important factor in the reaction of bird
predators to aposematic prey (for details, see Marples and Kelly, 1999).
Presenting shieldbugs on variously patterned backgrounds appears to slightly decrease
the strength of the aposematic signal, although not significantly so. The behaviour of a
predator towards aposematic and non-aposematic prey on contrasting and matching backgrounds was examined by Sillén-Tullberg (1985). She examined the reactions of naive zebra
finches (Poephilla guttata) to wild-type red individuals and grey mutants of Lygaeus
equestris (Heteroptera: Lygaeidae) on red and grey backgrounds and showed that the
warning signal of the red form was not weakened on the matching background.
Although the morphology and chemical defence of the pentatomoids do not prevent
attacks by birds in general [17 pentatomoid species were present in the diet of 14 Central
European bird species (Exnerová et al., 2003b)], G. lineatum has never been found in the birds’
diet (Creutz 1953; Exnerová et al., 2003b). The aposematism of G. lineatum appears to be supported by a combination of conspicuous colouration, defensive chemicals, gregarious habit,
living and feeding on the upper parts of herbs (where they are visible to possible avian
predators), and their absence in the bird diet. Nonetheless, the warning signal of the shieldbug colouration does not work unequivocally. It does not prevent the attack but, after
handling the first prey item, the bird learns its unpalatability and does not repeat its attack.
Moreover, it is possible that the shieldbug colouration, when presented on patterned
backgrounds, loses its warning power and becomes more or less disruptive. The effect of
the aposematic signalling of G. lineatum may thus be context-dependent and function in
accordance with other defensive devices.
ACKNOWLEDGEMENTS
We are grateful to M. Šlachta for help in breeding shieldbugs and to V. Novotný for helpful comments
on the manuscript. We thank the Czech Academy of Sciences (A6141102) and the Czech Grant
Agency (206/03/H034) for financial support.
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Biological Journal of the Linnean Society, 2009, 98, 234–242. With 4 figures
What constitutes optical warning signals of ladybirds
(Coleoptera: Coccinellidae) towards bird predators:
colour, pattern or general look?
MICHAELA DOLENSKÁ, OLDŘICH NEDVĚD, PETR VESELÝ*, MONIKA TESAŘOVÁ
and ROMAN FUCHS
Faculty of Science, University of South Bohemia, Branišovská 31, 370 05 České Budějovice,
Czech Republic
Received 21 February 2009; accepted for publication 23 February 2009
bij_1277
234..242
Most ladybirds (Coleoptera: Coccinellidae) possess chemical protection against predators and signal its presence by
less or more conspicuous coloration, which can be considered as a warning. Most ladybirds possess a dotted pattern,
althougn the number, shape, and size of the spots, as well as their colour, varies considerably. Almost all ladybirds
have a characteristic general appearance (body shape). We considered these traits to be used in ladybird recognition
by avian predators. In the present study, we compared the reactions of avian predators (Parus major) caught in
the wild, to four differently coloured ladybird beetles (Coccinella septempunctata, Exochomus quadripustulatus,
Subcoccinella vigintiquatuorpunctata, and Cynegetis impunctata) and two artificial modifications of C. septempunctata; the first was deprived of their elytral spotted pattern by painting it brown, the other had their elytra
removed (i.e. altering their general ladybird appearance). Ladybirds with a spotted pattern were attacked less
frequently than unspotted ones. Ladybirds with removed elytra were attacked much more often than any ladybird
with a preserved general appearance. The results obtained in the present study suggest the high importance of the
spotted pattern as well as general appearance in the ladybird recognition process. Additional experiments with
naïve birds (hand-reared P. major) demonstrated the innateness of the aversion to two differently spotted ladybird
species (C. septempunctata and Scymnus frontalis). © 2009 The Linnean Society of London, Biological Journal of
the Linnean Society, 2009, 98, 234–242.
ADDITIONAL KEYWORDS: body shape – chemical signal – prey recognition – warning coloration.
INTRODUCTION
Warning coloration has been considered as a very
important antipredatory signal subsequent to early
studies dealing with aposematic animals (Komárek,
2003). This signal is addressed to optically orienting
predators (Edmunds, 1974). In the case of terrestrial
invertebrate prey, birds are the most common visual
predators (Smith, 1980; Evans & Schmidt, 1990;
Schuler & Roper, 1992; Roper & Marples, 1997;
Exnerová et al., 2006). The most common colours used
by insects to discourage predators from attacking
them are bright red, orange, and yellow (Cott, 1940).
These colours are distinctive so that predators quickly
determine the connection between coloration and the
*Corresponding author. E-mail: [email protected]
234
toxicity of the prey (Coppinger, 1969; Gittleman,
Harvey & Greenwood, 1980; Harvey & Paxton, 1981;
Guilford, 1986). Nonetheless, other colours, such as
white, may be utilized as antipredatory signals (Lyytinen et al., 1999). The importance of colour per se has
been demonstrated several times using optically orienting predators (Sillén-Tullberg, 1985; Marples, van
Veelen & Brakefield, 1994; Ham et al., 2006).
Colour is not the only aspect of optical warning
signals. Bright colours usually form contrasting patterns (often in combination with black or white),
which should enhance the warning signal (Endler,
1978). The importance of patterns has been experimentally demonstrated using avian predators
(Schuler & Hesse, 1985; Osorio, Miklosi & Gonda,
1999; Endler & Mielke, 2005). Particular parameters
of colour pattern, such as symmetry and size, have
© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98, 234–242
LADYBIRD WARNING SIGNALS
been proposed to be of significance (Forsman &
Merilaita, 1999, 2003; Forsman & Herrström, 2004;
Cuthill et al., 2006). Some studies revealed the possibility that even dull colours may provide a warning
signal. For example, Wuster et al. (2004) and Niskanen & Mappes (2005) demonstrated that the
brownish colour in vipers was sensed as a warning
when forming a zig-zag pattern.
There are additional optical signals possessed by an
insect that may be used by predators to evaluate how
dangerous they are. These traits, such as body shape,
body posture, and shape of legs and antennae, are
often neglected in studies testing optical antipredatory signals. Nonetheless, there are several studies
showing the antipredatory importance of these traits
in ants (Ito et al., 2004; Nelson et al., 2006) and other
hymenopterans (Kauppinen & Mappes, 2003).
The ladybird beetle (Coleoptera: Coccinellidae) is
another example of an animal with a characteristic
general appearance, and this may be important in
their recognition and warning signalization. The
ladybirds comprise a high portion of conspicuously
coloured species (Majerus, 1994). There is amazing
variability in the colour patterning of ladybirds, but
the antipredatory efficiency of many of them has
never been tested. However, it is believed that the
colour patterning is used for signalling unpalatability
to predators (Majerus, 1994). The coloration can
range from a reddish or yellowish background with
dark spots (Adalia, Coccinella, Hippodamia and
many others) to black with light (red, yellow) spots
(Adalia, Scymnus, Exochomus) or brown with light
spots (Adalia, Calvia). Moreover, there are several
inconspicuous species with fragmented brownish patterns (Aphidecta, Rhizobius, Subcoccinella, Harmonia
quadripunctata) that can be considered as a warning,
as well as cryptic, according to background circumstances (Majerus, 1994). Another trait that affects the
conspicuousness of ladybirds is the type of surface on
the elytra and pronotum, which can be smooth and
shiny, or covered in hairs, resulting in a matt appearance. Colour variability is quite broad not only among
members of the Coccinellidae, but also within particular species, where several colour forms coexist (e.g.
Adalia bipunctata: Holloway et al., 1995; A. bipunctata and Adalia decempunctata: Honěk, Martínková
& Pekar, 2005, Harmonia axyridis: Kholin, 1990). The
majority of ladybird species possess any type of
spotted pattern, but the number and size of spots
varies markedly (Majerus, 1994). Only a small proportion of ladybirds have plain coloration (unspotted
reddish: Coccidula rufa; brownish: Cynegetis).
Despite the broad range of colours and patterns
present in ladybirds, the shape of the body is very
uniform within this family. Ladybirds have a quite
distinct body contour, which is caused mainly by the
235
shape of the elytra (quite broad and convex) and
broad pronotum. There are few other beetles in
Europe with a similar body shape.
Ladybirds possess chemical protection against
predators. In most ladybird species, noxious and often
poisonous chemical substances have been found and
isolated in their haemolymph; the most common comprising alkaloids, polyazamacrolides, and polyamines
(Laurent, Braekman & Daloze, 2005). Moreover,
pyrazines, which are chemicals known to provide
long-distance (i.e. perceived by smell, not by taste)
antipredatory protection (Guilford et al., 1987), were
found in ladybird haemolymph (Rothschild & Moore
1987). When sensing danger, ladybirds emit poisonous and odious droplets (reflex bleeding). It has been
demonstrated that chemical signals of ladybirds can
prevent attacks by olfactory orienting predators such
as ants (Sloggett, Wood & Majerus, 1998; Pasteels,
2007), spiders (Camarano, Gonzalez & Rossini, 2006),
true bugs (HoughGoldstein, Cox & Armstrong, 1996),
lacewings (Lucas, 2005), and other ladybirds (Agarwala & Dixon, 1992). The presence of toxic chemicals
in ladybirds also represents a potential danger for
optically orienting predators such as birds. Marples
(1993) and Marples, Brakefield & Cowie (1989) demonstrated that some ladybird species are strongly
toxic for birds (e.g. blue tits). Therefore, it is essential for at least some birds to recognize ladybirds
precisely.
The optical signals of ladybirds vary markedly,
however traits used in the recognition of dangerous
prey should (i.e. based on aposematism theory) be as
comprehensible as possible and thus uniform (Guilford, 1990). If the predator was to have to learn every
particular form of ladybird, there would be extreme
demands for predator memory and cognition (Speed,
2001). Furthermore, the presence of antipredatory
chemicals is not correlated with the presence of
warning coloration (Majerus, 1994). Therefore, it may
be dangerous to use a particular colour combination
in ladybird recognition. The general appearance
(especially the shape of the body) or presence of any
spotted pattern appears to be a more credible parameter for the optical recognition of ladybirds.
In the present study, we attempted to reveal the
importance of particular components in the optical
warning signal of ladybirds for recognition by a model
bird predator. We tested the signal efficiency of
several ladybird species (and their artificial modifications) using adult (caught in the wild) and handreared great tits (Parus major) as predators.
In the first experiment, we compared the response
of adult tits to four ladybird species: two presumed to
possess warning colour signals together with a shiny
surface (Coccinella septempunctata: red with black
spots; Exochomus quadripustulatus: black with red
236
M. DOLENSKÁ ET AL.
spots), one species with fragmenting, less conspicuous
coloration (Subcoccinella vigintiquatuorpunctata:
reddish–brown background with many dark spots and
matt surface) and one species with light brown spotless and matt coloration (Cynegetis impunctata).
In the second experiment, we compared the
responses of adult tits with three varieties of C.
septempunctata: unmodified, brown painted (warning
coloration together with spotted pattern and shining
surface removed) and with elytra removed (general
‘ladybird’ appearance altered). Additionally, we tested
the innateness of the aversion to ladybirds in handreared great tits. We compared their response to two
ladybird species possessing differently conspicuous
coloration: C. septempunctata (red with black spots
and shiny surface) and Scymnus frontalis (black with
red spots and matt surface).
MATERIAL AND METHODS
PREDATORS
The great tit (P. major, Linnaeus, 1758) is an insectivorous bird, 14–15 cm long, mass 14–23 g, occurring
throughout Europe and much of Asia. It is commonly
used for testing the efficiency of antipredatory signals
(Lindström, 1999). Its ability to recognize aposematic
prey according to colour signals has been shown in
several studies performed with invertebrate prey
(Sillén-Tullberg, Wiklund, Järvi 1982; Lyytinen et al.,
1999; Exnerová et al., 2003, 2006; Hagen, Leinaas &
Lampe, 2003). Moreover, it has been demonstrated
that great tits are able to learn the connection
between unpalatability and warning signals in the
red firebug (Pyrrhocoris apterus; Exnerová et al.,
2007). Furthermore, Marples et al. (1989) showed
that some ladybird species are highly toxic to their
relatives blue tits. Therefore, we may presume that
recognizing ladybirds is essential for great tits, which
can be considered as potential ladybird predators
(Cramp & Perrins, 1993).
Adult experimental individuals were caught with
mist nets in South Bohemia, Czech Republic, during
the nonbreeding period in 2004–2006. Birds were
kept in standard birdcages (50 ¥ 30 ¥ 30 cm) at
lowered indoor temperature and under an outdoor
photoperiod. Birds were acclimated to the laboratory
conditions for 1–2 days prior to experiments. Sunflower seeds and mealworms (larvae of Tenebrio
molitor) were provided during this acclimation. To
avoid pseudo-replication, each individual was used for
a single series of trials. The birds were ringed and
released immediately after the experiment.
Naïve predators were obtained from nest boxes in
the same forest where the adults were caught. They
were taken from the boxes (four individuals from each
nest; only from broods containing at least eight
chicks) at the age of 12–14 days (approximately 2
days before leaving the nest). Birds were kept in
standard bird cages in groups according to kinship. A
curd cheese mash (curd cheese, grated carrot, grated
boiled eggs, vitamins, and crushed egg shells), commercial feeding for insectivorous birds, and mealworms were provided as diet. When birds were able to
ingest prey by themselves (approximately 4 weeks
after hatching), training in experimental cages
started. When a bird responded to offered prey
(mealworm) in the cage appropriately (i.e. attacked
immediately after offering), experiments were
started. After the series of experiments, birds were
trained to forage (by presenting various live prey) and
fly in an aviary (4 ¥ 4 ¥ 4 m) and, when found ready,
were released.
PREY
SPECIES
Five species of ladybirds were used as potential avian
prey for the experiments (Fig. 1): C. septempunctata
(Linnaeus, 1758), E. quadripustulatus (Linnaeus,
1758), S. vigintiquatuorpunctata (Linnaeus, 1758), C.
impunctata (Linnaeus, 1767), and S. frontalis (Fabricius, 1787). Adult ladybirds were collected during the
autumn months, and were in the stage of reproductive diapause. All ladybirds were stored at 10–15 °C
for several months before the experiments. Only
water in cellulose cotton was provided.
The seven spot ladybird, C. septempunctata, is a
large species (6–8 mm long), and its coloration consists of a shiny red background of the elytra, with
small black spots, and a black and white pronotum
and head. It contains the alkaloids coccinelline
(Tursch et al., 1971) and precoccinelline (Karlsson &
Losman, 1972). The pine ladybird, E. quadripustulatus, is medium sized (3–5 mm) and shiny black with
four red spots. It contains exochomine, a dimeric
alkaloid (Timmermans et al., 1992). The 24-spot
(or alfalfa) ladybird, S. vigintiquatuorpunctata, is
medium sized (3–4 mm) and brownish-red, with many
small dark markings. This species is somewhat variable in the number and size of spots and possesses
grey hairs on the upper surface, giving the beetle
a matt appearance. Its alkaloid, Na-quinaldyl-Larginine·HCl, has a unique molecular structure
among ladybird alkaloids (Wang et al., 1996). The
grass ladybird, C. impunctata, is medium sized
(3–4 mm), pale brown, and is generally found in
central Europe, usually without black markings. The
upper surface is hairy with a matt appearance.
Unlike the other four species, it appears cryptic
rather than conspicuous. Scymnus frontalis is small
(3 mm) and black, with four large reddish–brown
spots on the elytra. The upper surface is hairy with a
237
Figure 1. Experimental ladybird species. A, Cynegetis
impunctata (photo Stanislav Krejčík, meloidae.com); B,
Subcoccinella vigintiquatuorpunctata (photo Malcolm
Storey, http://www.bioimages.org.uk); C, Exochomus
quadripustulatus (photo Stanislav Krejčík, http://
meloidae.com); D, Coccinella septempunctata ı unmodified
(photo Stanislav Krejčík, http://meloidae.com); E, Coccinella septempunctata with removed elytra (photo Oldřich
Nedvěd); F, Coccinella septempunctata brown-painted
(photo Oldřich Nedvěd); G, Scymnus frontalis (photo K. V.
Makarov, http://www.zin.ru). Species are not depicted to
scale.
b
et al., 2003), and causing no obstacle to motion for the
beetles; (2) by excision of the elytra by scalpel; the
ladybirds would then immediately extend the lower
membranous wings, thus the entire ladybird appearance (shape, etc.) was altered, together with
coloration and pattern (Fig. 1E). During these modifications, each ladybird was held on a piece of cellulose cotton that absorbed the released haemolymph
droplets with protective compounds. At least 1 h
elapsed before experimentation with the bird predator to allow volatile protective compounds to disappear from surface of the beetle, and to allow the
ladybird to replenish volatiles in its haemolymph.
EXPERIMENTAL
EQUIPMENT
Experiments were performed in cubic cages
(71 ¥ 71 ¥ 71 cm). All walls except one were covered
with a wire mesh (2 mm). The front wall consisted of
a one-way mirror. The cage included a perch, a bowl
with water, and a revolving circular feeding tray
containing six cups. The bottom of each cup (diameter
5.5 cm, depth 1.5 cm) was white. Each of the cups
contained a single prey item during a trial. The
distance between the perch and the tray was approximately 25 cm. Illumination was generated by a daylight spectrum fluorescent tube (LUMILUX COMBI
18 W; Osram).
TRIALS
matt appearance. The identities of defensive chemicals for the latter two species are unknown.
Two artificial non-aposematic modifications were
made from the seven spot ladybirds (Fig. 1E, F): (1)
by painting the upper surface with brown tempera
colour, which is matt, nontoxic, non-odious (Exnerová
Collectively, 120 great tits were used as predators.
Adult experimental birds were divided into six groups
of 15 individuals. Four groups were presented with
particular unmodified species of ladybirds (C. septempunctata, E. quadripustulatus, S. vigintiquatuorpunctata, and C. impunctata); two groups were presented
with modified C. septempunctata (brown-painted and
elytra-cut). Hand-reared birds were divided into two
groups of 15 individuals: one group was offered C.
septempunctata and the other S. frontalis.
238
M. DOLENSKÁ ET AL.
Two hours before the start of an experiment, a bird
was placed into the experimental cage, with access to
water but no food. Previous experiments showed that
2 h is sufficient to evoke food-searching but not stress.
Each bird was assumed to be ready for the experiment when it attacked a mealworm in the feeding
tray immediately after it was offered. Each experiment with an individual bird consisted of a series of
ten trials. Control prey (mealworm) was offered alternately with experimental prey (any form of ladybird),
and this was repeated five times (sequence mealworm, ladybird, mealworm, ladybird, etc.). The repetition of five successive presentations was used to
reveal a possible effect of neophobia, which was
previously demonstrated to be a short-term event
(Marples & Kelly, 1999). The control prey was used to
check the bird’s motivation to feed, and the trial
ended after the prey had been eaten. Each trial with
experimental prey lasted 5 min. There were three
possible results for a particular trial: the bird
attacked and killed the offered prey; the bird attacked
(handled by bill) offered prey, but the prey managed
to survive; or the bird did not attack the prey. Repetition of attacks occurred only sporadically: 15 birds
out of 120 attacked the prey twice (when confronted
with brown painted or elytra-cut modifications of
C. septempunctata) and two birds attacked the prey
three times (with elytra-cut C. septempunctata).
Therefore, all the results obtained were summarized
for every particular bird and data were used in statistical comparisons in a binomial distribution: 1 = a
certain activity was observed during at least one of
the five experimental prey trials; 0 = the activity was
not registered during any of the five trials with a
particular bird.
STATISTICAL
ANALYSIS
The binomial data were compared using generalized
linear models (binomial distribution, logit link function, analysis of variance (ANOVA), and the F-test
were used as the test criteria) with post-hoc Tukey’s
honestly significant difference (HSD) tests. Fisher’s
exact test was used in pairwise comparisons. The
probability level showed clear significance without
the need for Bonferroni adjustment. All tests were
performed in STATISTICA, version 6 (StatSoft, Inc.).
RESULTS
KILLING
OF PREY
All mealworms offered in the trials were eaten
(killed). In none of the total 600 trials (120 series,
each of five offers) did we record any killing of any
offered ladybird; each ladybird survived 5 min of the
Figure 2. Numbers of great tits attacking particular
ladybird species. A, Cynegetis impunctata; B, Subcoccinella vigintiquatuorpunctata; C, Exochomus quadripustulatus; D, Coccinella septempunctata. The line indicates a
significant difference.
trial duration. Thus, the strongest reaction of the
experimental birds comprised the attack.
COMPARISON
OF FOUR LADYBIRD SPECIES
There was a significant difference in bird reaction
towards the four tested ladybird species (ANOVA:
F60,57 = 5.197; P = 0.003; Fig. 2). The number of birds
attacking the brown coloured C. impunctata was
higher (post-hoc Tukey’s HSD test, P = 0.002) compared to those attacking the other three ladybirds.
COMPARISON
OF MODIFIED
COCCINELLA
SEPTEMPUNCTATA
There was a significant difference in bird reaction
towards the three variations of C. septempunctata
(ANOVA: F45,42 = 9.172; P < 0.001; Fig. 3). The birds
attacked naturally-coloured as well as brown-painted
beetles less often than elytra-cut ones (post-hoc
Tukey’s HSD test; naturally-coloured, P < 0.001;
brown-painted, P = 0.014). The reaction to naturallycoloured ladybirds did not differ from the reaction to
brown-painted ones (P = 0.096).
COMPARISON
OF ADULT AND NAÏVE PREDATORS
Both tested ladybird species (C. septempunctata and
S. frontalis) were attacked by the same proportion of
birds (Fig. 4). Naïve birds attacked naturally-coloured
C. septempunctata more often than adult birds
(Fisher’s exact test, P = 0.021; Fig. 4).
DISCUSSION
KILLING
THE LADYBIRDS
Although every tested ladybird species or artificial
modification was at least sporadically attacked, none
Figure 3. Numbers of great tits attacking particular
modifications of Coccinella septempunctata. D, unmodified;
E, with removed elytra; F, brown-painted. The line indicates a significant difference.
Figure 4. Numbers of adult and hand-reared great tits
attacking particular ladybird prey. D, adults versus
Coccinella septempunctata; G, hand-reared versus
Scymnus frontalis; I, hand-reared versus C. septempunctata. The line indicates a significant difference.
were killed regardless of their optical warning
signals. The ladybirds thus appear to be highly protected against the great tit, that should not be
regarded for other warningly coloured prey. Similar
experiments (Sillén-Tullberg et al., 1982; Exnerová
et al., 2003; Veselý et al., 2006) with this predator
species showed that they are willing to kill and even
eat several species of true bugs (Heteroptera) when
their optical protection is weakened.
We assume that attacking birds were discouraged
from continuing the attack by a chemical signal (i.e.
sensed by smell or taste) when attacked ladybirds
actively released their haemolymph, containing repelling substances (i.e. reflex blood). Thus, we assume
that the chemical protection of tested ladybirds works
239
very well not only against insect predators (see
Introduction), but also against great tit. The strong
efficiency of this protection is not weakened by differences in chemical composition among tested species
(i.e. containing various alkaloids and other defensive
chemicals; see Material and methods).
However, the results obtained from experiments
with the great tits cannot be generalized to all avian
predators. Japanese quails in experiments of Marples
et al. (1994) killed and ate mealworm beetles tainted
by ladybird toxicity. Moreover, other quails fed
directly with ladybirds did not show any discomfort.
By contrast, strong toxicity of some species of ladybirds for blue tits was demonstrated (Marples et al.,
1989; Marples, 1993). We used two ladybird species in
these studies. The seven spot ladybird (C. septempunctata) was shown to be highly toxic for young blue
tits (Marples et al., 1989) and the pine ladybird
(E. quadripustulatus) caused serious discomfort and
inhibited the growth of tit chicks slightly (Marples,
1993). These blue tits were offered pellets made of
smashed ladybird bodies. They were willing to eat
pellets containing extracts of most of the tested ladybird species, including those causing them subsequent digest problems, such that they were not
repelled by the content of alkaloids (which are possibly not detectable by smell or taste). The results
obtained in the present study suggest that living
ladybirds possess additional protective substances
causing the noxiousness. Rothschild & Moore (1987)
revealed the presence of pyrazines in ladybirds’
hemolymph. These chemicals were shown to provide
chemical protection for several insect species (Guilford et al., 1987) and are quite volatile, and thus may
provide olfactory protection.
ATTACKING
THE LADYBIRDS
Although no bird was found to kill any ladybird, the
portion of attacking birds differed among tested ladybird forms. We may assume that this is caused by the
differently effective optical protection of ladybirds. In
experiments with adult birds confronted by four ladybird species, the spotless and brownish coloured C.
impunctata was attacked more often than other
species. All spotted ladybirds were protected very
well; even the brownish and dark spotted S. vigintiquatuorpunctata. If great tits would recognize particular ladybird species, we must presume that all
three tested spotted species represent equal danger
for them, and are more dangerous than the brown,
spotless Cynegetis at the same time. This is in contrast with equally effective chemical protection of all
four species (none was killed). Similarly, Marples
(1993) and Marples et al. (1989) demonstrated the
differing chemical impact of the seven spot ladybird
240
M. DOLENSKÁ ET AL.
and the pine ladybird on blue tits. An a lternative
explanation suggests that great tits avoid all spotted
species, regardless of their coloration, whereas the
spotless ladybirds are put to the test of edibility. The
spotted pattern appears to be the most important part
of the optical warning signal, at least for the ladybird
species tested in the present study. It protects even
those species that have inconspicuous background
colours. The significant importance of spots in the
warning signalization of ladybirds is also in concordance with the variability of coloration in ladybirds.
Although the colour of background as well as of spots
varies considerably in ladybirds, only a small proportion does not possess any type of spotted pattern
(Majerus, 1994).
The theory that Cynegetis is attacked more because
of its absence of spots, and not because of its smaller
chemical protection, is supported by experiments with
brown painted seven spot ladybirds, which were
attacked by approximately by the same proportion of
birds as the brown coloured Cynegetis. By contrast to
experiments conducted in several ladybird species,
the brown painted form had the same chemical protection as well as other traits (e.g. body size) similar
to the unmodified seven spot ladybird.
Nonetheless, even Cynegetis and the brown
painted ladybird are not attacked by one half of
birds, which suggests that they are still recognized
as a ladybird. However, when the elytra of the
seven spot ladybird are removed, it is attacked by
more than 80% of birds (i.e. significantly more than
in the case of the unmodified as well as the brown
painted form). The importance of the general ladybird appearance in the antipredatory signal is thus
indicated. Ladybirds possess a characteristic (oval,
convex) body shape that is common among most
species, regardless of size, colour or pattern
(Majerus, 1994). When this shape was changed
(together with loss of spots), great tits became confused and mostly attacked the ladybirds.
We can conclude that the results obtained in the
present study suggest that the general appearance
and spotted pattern suffice to explain ladybird recognition, at least by some birds. This could mean that
ladybirds may not be forced by strong selective pressure to unified coloration, as are many other groups
of warningly coloured animals (Guilford, 1990). This
relative selective freedom may explain the wide
palette of colours present among ladybirds (Majerus,
1994). The theory of importance of general body shape
in the antipredatory signal of ladybirds is also supported by the high colour uniformity (i.e. red background with seven melanization centres on each
elytron) of members of the subtribe Hipodamiina,
which have a completely different appearance (i.e.
elongated and depressed body).
The importance of optical signals unrelated to coloration in antipredatory signalization has often been
neglected. Chemical signals and colour pattern are
always considered to be the most important cues
in aposematic prey recognition (Guilford, 1990).
However, the warning signal should be clear and
comprehensible (Guilford, 1988); a uniform and distinct general appearance may comply with this. Two
studies have considered the importance of optical
signals unrelated to coloration in antipredatory
optical signalization and, interestingly, both dealt
with non-avian predators. Nelson et al. (2006) demonstrated mantises to react to ants similarly to their
reaction to ant-mimicking jumping spiders (i.e. equal
in body shape, not in chemical signals). Kauppinen &
Mappes (2003) showed the particular importance of
the characteristic shape of wasp bodies in optically
repelling dragonflies. Artificially nonstriped wasps
were attacked less often than identically coloured
flies. These results suggest that other optical traits
may play a significant role in the recognition of dangerous animals.
The results obtained in the present study with
respect to ladybirds cannot by generalized to other
optically signalling insects. The role of precise coloration pattern is rather different in coreoid true bugs,
which comprise another well described and studied
group of aposematic insects. Experiments on red firebugs and great tits (Exnerová et al., 2003) revealed
that, in contrast to natural ones, the brown-painted
firebugs were not only attacked, but also frequently
killed. Even mutants of bugs with native black patterning, but differing in background colour, were less
protected than naturally-coloured individuals. SillénTullberg et al., (1982) demonstrated a higher attack
rate to seed bug (Lygaeus equestris) larvae when their
background colour was grey instead of red. Similarly,
Exnerová et al. (2006) showed a lower protective function of yellow and white backgrounds instead of red,
in the firebug (P. apterus). By contrast to ladybirds,
the general appearance (as well as black pattern) has
no importance in coreoid true bug recognition by
great tits, or, from the point of view of cognitive
science, great tits do not posses a general concept
(Shettleworth, 1998) of coreoid true bugs.
In experiments with naïve, hand-reared great tits,
the partial innateness of aversion to ladybirds was
demonstrated. Comparable experiments (Exnerová
et al., 2007), using red firebugs (P. apterus) as prey
and naïve great tits as predators, showed practically
no innate wariness. It is interesting that the firebug
possesses the black–red colour pattern as in many
ladybird species. Although at least some ladybirds are
strongly toxic to some tits (Marples et al., 1989),
eating the firebug may only cause slight discomfort,
such that the penalty is not as high. Therefore, selec-
tion pressure for innate aversion to the ladybird is
stronger than that to the firebug. This type of explanation could also be used to clarify the different role
of general appearance in the recognition of ladybirds
and coreoid true bugs. However, we have insufficient
knowledge of the general danger posed by both of
these insect groups.
ACKNOWLEDGEMENTS
The team of authors is licensed to catch and ring
birds (Bird Ringing Centre Prague, No. 1004) as well
as to treat animals experimentally (Czech Animal
Welfare Commission, No. 489/01). The study was supported by grants of the Academy of Sciences of the
Czech Republic (IAA601410803), the Czech Grant
Agency (206/08/H044), and the Ministry of Education,
Youth, and Sports (MSM6007665801).
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Biological Journal of the Linnean Society, 2010, 100, 890–898. With 4 figures
The role of size and colour pattern in protection
of developmental stages of the red firebug
(Pyrrhocoris apterus) against avian predators
MILENA PROKOPOVÁ, PETR VESELÝ*, ROMAN FUCHS and JAN ZRZAVÝ
Faculty of Science, University of South Bohemia, Branišovská 31, 37005 České Budějovice,
Czech Republic
Received 5 January 2009; revised 19 February 2010; accepted for publication 19 February 2010
bij_1463
890..898
We investigated how predator/prey body-size ratio and prey colour pattern affected efficacy of prey warning signals.
We used great and blue tits (Parus major and Cyanistes caeruleus), comprising closely related and ecologically
similar bird species differing in body size, as experimental predators. Two larval instars and adults of the
unpalatable red firebug (Pyrrhocoris apterus), differing in body size and/or coloration, were used as prey. We showed
that prey body size did not influence whether a predator attacked the prey or not during the first encounter.
However, smaller prey were attacked, killed, and eaten more frequently in repetitive encounters. We assumed that
body size influences the predator through the amount of repellent chemicals better than through the amount of
optical warning signal. The larger predator attacked, killed and ate all forms of firebug more often than the smaller
one. The difference between both predators was more pronounced in less protected forms of firebug (chemically as
well as optically). Colour pattern also substantially affected the willingness of predators to attack the prey. Larval
red–black coloration did not provide a full-value warning signal, although a similarly conspicuous red-black
coloration of the adults reliably protected them. © 2010 The Linnean Society of London, Biological Journal of the
Linnean Society, 2010, 100, 890–898.
ADDITIONAL KEYWORDS: chemical protection – larvae – red firebug – titmice – warning signal.
INTRODUCTION
Prey that is defended chemically, and is therefore
more or less distasteful, often manifests its unpalatability by conspicuous ‘warning’ or ‘aposematic’ coloration (Poulton, 1890). It is difficult, however, to
‘dissect’ the complex aposematic display into individual components (body size, colour pattern, behaviour, etc.) with the aim of understanding the network
of predator–prey interactions.
Exnerová et al. (2003) described different responses
of several Palaearctic, mostly insectivorous, passerine
bird predators to the red firebug, Pyrrhocoris apterus
(L., 1758) (Hemiptera: Pyrrhocoridae). In this study,
great and blue tits (Parus major L., 1758; Cyanistes
caeruleus L., 1758), robins (Erithacus rubecula L.,
*Corresponding author. E-mail: [email protected]
890
1758), and blackcaps (Sylvia atricapilla L., 1758)
generally avoided naturally-coloured (red and black)
firebugs, but readily attacked ‘artificially nonaposematic’ (brown-painted) individuals. By contrast,
these experiments showed no discrimination by blackbirds (Turdus merula L., 1758), which frequently
attacked and consumed both ‘forms’ of firebugs, or
long-tailed tits (Aegithalos caudatus L., 1758), which
strongly avoided both. Interestingly, blackbird and
long-tailed tit were the heaviest and the lightest
tested species, respectively, which suggests that relative predator body size might affect its attitude
towards the prey. Similar results were obtained by
Wiklund & Järvi (1982), who showed a higher willingness of starlings (Sturnus vulgaris L., 1758) and
quails (Coturnix japonica Temminck & Schlegel,
1849) than great and blue tits to attack chemically
protected and warningly coloured prey, in this case
adults and larvae of butterflies (Papilio machaon
APOSEMATISM OF LARVAE OF THE RED FIREBUG
L., 1758; Pieris brassicae L., 1758; Zygaena filipendula L., 1758), red firebugs, and seven-spot ladybirds
(Coccinella septempunctata L., 1758).
These studies suggest that, if the predator/prey
body size ratio is relatively high, the predator may be
less cautious to attack the prey because there is a
lower risk of physiological repercussion after digestion. Thus, the general prediction, that smaller predator species show a higher degree of dietary
conservatism than larger ones (Webster & Lefebvre,
2001), is in accordance with these results. Another
possible approach to test the importance of the
predator/prey body size ratio is by using various sizes
of a single prey species, confronted with one predator
species.
Generally, differently sized prey could affect predator attitude through two principles. Already at the
beginning of studies concerning aposematism, it was
noted that one of the ways how to enhance the
warning signal is to enlarge the prey’s body size (Cott,
1940; Turner, 1984; Guilford, 1986). This has been
tested experimentally by using domestic chicks
(Gallus gallus f. domestica L., 1758) as predators and
artificial paper butterflies as prey (Forsman & Merilaita, 1999). This study, as well as a comparative
analysis of moth larvae (Nilsson & Forsman, 2003)
and poison frogs (Hagman & Forsman, 2003), found a
correlation between body size and efficacy of warning
coloration. Similarly Mand, Tammaru & Mappes
(2007) showed larger conspicuous prey to be less
acceptable for predators in experiments with artificial
larvae. Nevertheless, in experiments conducted with
natural prey, larger larvae survived less, most likely
as a result of higher detectability (Sandre et al. 2007).
Moreover, the higher protection provided by warning
signals in gregarious prey (providing a higher amount
of signal) was also shown experimentally (GamberaleStille & Sillén-Tullberg, 1996, 1998; Mappes &
Alatalo, 1997). In these cases, the visual stimulus is
stronger, although the danger connected with sampling one prey remains the same. (Riipi et al., 2001).
Another effect of body size that enhances the protection of prey is the amount of repellent (toxic)
chemicals in their body. Bigger prey, of course, have
the possibility of possessing a larger volume of defensive substances and thus are better protected (Holloway, Dejong & Ottenheim, 1993). The simplest effect
of body size that provides protection from predators
comes from predator food preferences. Too large or too
small a prey may not fulfil a predator’s image of an
appropriate prey and thus it is not attacked. Consequently, different body size of tested birds may result
in generally different food preferences (Webster &
Lefebvre, 2001).
In the present study, we compared the reaction of
bird predators towards natural insect prey. We used
891
Figure 1. Adults (AD) and larvae (L5, L3) of Pyrrhocoris
apterus (photo: P. Veselý).
third- and fifth- (ultimate) larval instars (‘L3’ and
‘L5’) and adults of the red firebug. The L3 animals
are markedly smaller than L5; on the other hand,
both larval instars have equal red coloration with a
black pattern and, when attacked, their scent glands
emit defensive secretions of equal composition
(Farine et al., 1992), although of course of a different
dose.
Whereas L3 and L5 differ only in size, the adult
body size is almost identical to that of L5, but the
adults have quite a different black-red pattern
(Fig. 1). This allows the possibility of comparing the
relative role of body size and colour pattern with
respect to their aposematic signal. The combination of
colours in a conspicuous geometric pattern has been
demonstrated to significantly affect the efficacy of
warning optical signal (Forsman & Merilaita, 1999;
Endler & Mielke, 2005). One of common patterns in
aposematic prey is a rounded spot (Lyytinen, Brakefield & Mappes, 2003; Stevens, Stubbins & Hardman,
2008). Unfortunately, the adults differ from larvae not
only in their black-red patterns, but also in the chemical composition of the defensive secretions (Farine
et al., 1992). Nevertheless, the effect of chemical
defence per se can be tested easily by removing the
warning optical signal (Exnerová et al., 2003).
Because we compared two predators differing in
body size (great and blue tit), we also decided to test
their willingness to attack and eat large prey, as
represented by the mealworm beetle (Tenebrio molitor
L., 1758), which is significantly bigger than any of
presented forms of the red firebug, but does not
possess any optical or chemical warning signal.
Our study aimed to answer several questions:
1. Is the red firebug better protected against smaller
bird predators?
2. Are the larger forms (L5 and Ad) of the red firebug
better protected than smaller ones (L3)?
3. Is the better protection of larger form (L5 and Ad)
of the firebug caused by the amount of optical
892
M. PROKOPOVÁ ET AL.
signal, the amount of repellent chemicals or a
general aversion to bigger prey?
4. Are adults of the firebug better protected than
larvae (L3 as well L5)? If yes, does this protection
reside in different coloration or in chemical
composition?
MATERIAL AND METHODS
PREDATORS
AND PREY
We used blue tit (C. caeruleus) and great tit (P. major)
as experimental predators. They differ in body size
(great tit, approximately 19 g; blue tit, approximately
11.5 g) but share similar foraging/feeding behaviour
and habitats (Cramp & Perrins, 1993). Both distinguished aposematic and non-aposematic ‘forms’ of P.
apterus (Exnerová et al., 2003). Nevertheless, blue tits
hesitated to taste the attacked prey and usually
released it without injury, whereas great tits tended to
kill the brown firebugs. We used 112 individuals of
great tits and 112 blue tits. Birds were caught in the
vicinity of České Budějovice (Czech Republic) from
July to April (except during the breeding season),
1999–2002. They were kept for 1–10 days before the
experiment in wire cages with free access to sunflower
seeds and water. During this time, we trained them to
accept mealworm larvae in the experimental cage. All
tits were released to the wild after the experiments.
Mealworm beetles (T. molitor) were reared in wheat
groats with vegetables, dried bread and water ad
libitum; 25 °C and under an 18 : 6 h light/dark cycle.
The firebugs were reared under equal conditions, as
in a previous study (Exnerová et al., 2003).
The three firebug categories and mealworm
beetles differed in body size. The mean ± SE
body length of mealworm beetles was 1.42 ±
0.08 cm (range 1.33–1.58 cm); L3s measured
0.55 ± 0.06 cm (range 0.44–0.61 cm); L5s measured
0.91 ± 0.04 cm (range 0.83–0.98 cm); and adult body
length was 1.03 ± 0.03 cm (range 0.96–1.08 cm); so
the general ratio can be counted as 2.6 : 1.0 : 1.7 : 1.9
(beetle : L3 : L5 : Ad). The mean ± SE body weight
of mealworm beetles was 89.8 ± 12.27 mg (range
76.2–111.7 mg); L3 weighed 10.6 ± 2.99 mg (range
5.3–14.5 mg); L5 weighed 45.9 ± 4.05 mg (range 39.2–
51.0 mg); and adults weighed 47.9 ± 5.95 mg (range
38.1–58.1 mg); so the ratio was 8.5 : 1.0 : 4.3 : 4.5
(beetle : L3 : L5 : Ad). Both these measures were
assessed by measuring 20 individuals per category.
The non-aposematic prey ‘forms’ were obtained
using odourless, nontoxic brown watercolour paint
(tempera colour, KOH-I-NOOR, Hardtmuth) applied
to the upper part of the insect body. The painting
treatment did not affect the motion and secretion of
firebugs (Exnerová et al. 2003).
EXPERIMENTAL
EQUIPMENT AND PROCEDURE
We used the same experimental equipment and procedure as described in detail by Exnerová et al.
(2003). Experiments were performed in an experimental cage (70 ¥ 70 ¥ 70 cm) equipped with a rotating circular feeding tray of eight white cups (diameter
5.5 cm), perch, water bowl, and light source
(LUMILUX COMBI 18W, Osram; simulating the daylight spectrum).
Birds of both species were separated into seven
treatment groups of 16 individuals for the seven test
prey categories (L3, L5, and Ad, all either naturallycoloured or brown-painted and mealworm beetle).
Each bird was pre-trained to attack mealworms in the
experimental cage. Offered mealworms were ninth
larval instar of mealworm beetle (T. molitor) reaching
1.82 ± 1.28 cm (range 1.56–2.19 cm) and 68.8 ±
10.72 mg (range 56.2–77.7 cm). Two hours before the
start of an experiment, the bird was deprived of food
to gain motivation for the experiments. After this
period, each bird was tested in ten consecutive trials.
Every trial started when a prey item was offered, and
then continued until the prey was eaten, or 5 min had
elapsed. Offered insects alternated in a sequence: a
mealworm, a tested prey, a mealworm, a tested prey,
etc., so there were five trials with tested prey for each
bird. Every control mealworm was attacked and
immediately eaten during every experiment.
The behavioural events recorded were attacking
(counted in all situations when a bill contacted the
prey body), killing (the bird killed the attacked prey),
and eating (the bird ate at least part of the killed
firebug).
STATISTICAL
ANALYSIS
First, we assessed the willingness of birds to attack
and kill the mealworm beetles (as number of birds
attacking and eating at least one of the five offered
beetles). Numbers of birds of both species (i.e. which
decided to attack or kill at least one of five
offered mealworm beetles) were compared using
2 ¥ 2 tables and two-tailed Fisher’s exact P-value as
the criterion.
In experiments with the red firebug forms, for each
bird, we noted willingness to attack and kill the first
(of five) offered prey (the initial acceptance). These
data had a binomial distribution. Two generalized
linear models (GLM; McCullagh & Nelder, 1989)
were formed [for attacking and for killing; a logit
link function, using factorial analysis of variance
(ANOVA), chi-square test as test criterion; and
Tukey’s honestly significant difference (HSD) test for
post-hoc comparisons]. We assessed the effect of three
independent factors: (1) developmental stage of prey
(adult/L5/L3); (2) prey coloration (nonmodified/brownpainted); and (3) predator species (great/blue tit), and
all possible interactions.
Additionally, we assessed differences in learning
abilities of birds. The presence/absence of attacking/
killing during particular trials (one of five) was used
to compile data (binomial). Particular trials with one
bird individual were grouped using a nested design of
analyses (trial nested in particular birds). Two GLMs
were formed (attacking and killing, binomial data,
logit link function, using nested ANOVA, chi-square
test as test criterion; and Tukey’s HSD test for posthoc comparisons). We assessed the effect of four independent factors: (1) developmental stage of prey
(adult/L5/L3); (2) prey coloration (nonmodified/brownpainted); (3) predator species (great/blue tit); and (4)
trial number (1–5) and all possible interactions.
Finally, we compared palatability of different prey
categories (as number of eaten firebugs per bird,
range 0–5) by forming another GLM (Poisson distribution, log link function, using factorial ANOVA and
chi-square test as test criterion, with post-hoc Tukey’s
HSD test). The tests included only data concerning
individuals of both bird species that attacked at least
one offered firebug, aiming to test only the effect of
edibility, and not that of long-distance signals (optical
or chemical). This analysis included the same independent factors as in the GLMs for initial acceptance:
(1) developmental stage of prey; (2) prey colour;
and (3) predator species, as well as all possible
interactions.
All calculations were performed using R 2.8.1 for
Windows (R Development Core Team, 2009).
RESULTS
REACTION
OF BOTH TESTED BIRD SPECIES TO
MEALWORM BEETLES
We did not find any difference between the great and
blue tits in attacking (Fisher’s exact: P = 1; Fig. 2), as
well as killing the mealworm beetles at the first
encounter (Fisher’s exact: P = 0.79; Fig. 2). Great tits
as well as blue tits attacked and killed mealworm
beetles more often (in absolute numbers) than any
form of firebug (Fig. 2).
INITIAL
ACCEPTANCE OF PARTICULAR FORMS OF THE
FIREBUG (REACTION TO THE FIRST OF FIVE OFFERED
PREY, WHERE ALL MENTIONED
POST-HOC
P-VALUES REFER
TUKEY’s HSD TESTS)
TO
Attacking
The number of birds attacking the first offered
firebug was significantly affected by the colour of the
firebug and species of the bird (Table 1). Brownpainted firebugs were attacked more often than the
893
Figure 2. Numbers of great (Parus major, PM) and blue
tits (Cyanistes caeruleus, CC), that attacked/killed the first
of five offered prey. Prey categories in treatment groups:
Tm, adult beetle of mealworm (Tenebrio molitor); Ad,
adults; L5 and L3, fifth- and third-instar larvae of the red
firebug (Pyrrhocoris apterus). *The tested prey was not
attacked. The total number in each group was 16. White,
striped and grey columns refer to attacking, black to
killing.
unmodified ones (P < 0.001; Fig. 2). Great tits
attacked firebugs more often than the blue tits
(P = 0.003; Fig. 2). The effect of the developmental
stage of the firebug was not significant (Table 1).
Nevertheless, the interaction of the developmental
stage of the firebug and its colour highly significantly affected the numbers of birds attacking the
first firebug (Table 1). The brown-painted firebugs
were attacked more than unmodified ones only when
they were adult. Larvae were attacked without reference to their colour (L3: P = 0.337; L5: P = 0.197;
Fig. 2). Unmodified larvae were attacked more than
unmodified adults (L3: P = 0.022; L5: P = 0.050;
Fig. 2), but less than brown-painted adults (L3:
P = 0.050; L5: P = 0.022; Fig. 2).
Killing
By contrast to attacking, the number of birds killing
the first offered firebug was significantly affected by
its developmental stage (Table 1). L3 firebugs were
killed significantly more often than adults (P = 0.041;
Fig. 2). The species of tested bird and colour of the
firebug were also significant factors (Table 1). Brownpainted firebugs were killed more often than the
unmodified ones (0.002; Fig. 2) and great tits killed
firebugs more often than blue tits (P << 0.001; Fig. 2).
The interaction of bug colour and bird species was
also shown to be important (Table 1). Brown-painted
firebugs were killed more often than the unmodified
ones only by great tits (P < 0.001) and great tits killed
more firebugs than blue tits only in the case of brownpainted ones (P << 0.001).
894
Table 1. The importance of predator and prey parameters in the willingness to attack (ATTACKING), kill (KILLING)
and eat (EATING) firebugs
Type of experiment
data family
Dependent
variable
Initial acceptance
Binomial (0/1)
Attacking
Killing
Learning abilities
Binomial (0/1)
Attacking
Killing
Edibility of prey
Poisson (0–5)
Eating
Independent variable
F
d.f.
P
Prey coloration
Prey developmental stage
Predator species
Stage ¥ Coloration
Prey coloration
Predator species
Predator ¥ Coloration
35.87
1.58
8.97
5.07
10.16
3.13
31.11
5.71
1
2
1
2
1
2
1
1
<< 0.001
0.209
< 0.001
0.007
0.002
0.046
<< 0.001
0.018
Prey coloration
Predator species
Trial number
Stage ¥ Predator
Coloration ¥ Trial
Stage ¥ Coloration ¥ Predator
Prey coloration
Predator species
Trial number
Stage ¥ Predator
Predator ¥ Trial
Stage ¥ Coloration
42.58
8.94
18.39
26.82
10.70
7.82
6.69
3.62
20.27
24.57
74.64
11.65
9.17
7.83
5.68
3.84
1
2
1
4
2
1
4
2
1
2
1
4
2
1
4
2
<< 0.001
<< 0.001
<< 0.001
<< 0.001
<< 0.001
0.005
<< 0.001
0.027
<< 0.001
<< 0.001
<< 0.001
<< 0.001
< 0.001
0.005
< 0.001
0.022
Prey coloration
Predator species
Stage ¥ Predator
1.11
22.58
41.22
2.98
6.18
1
2
1
1
2
0.529
<< 0.001
<< 0.001
0.050
0.010
Generalized linear model (logit link function for both data families), analysis of variance (chi-square test, P). Interactions
of independent variables are shown only when their effect is significant.
LEARNING
ABILITIES OF TESTED BIRDS (PRESENCE OF
ATTACKS/KILLS IN EACH OF FIVE SUCCESSIVE
TRIALS
– TRIAL
NUMBER NESTED IN BIRD NUMBER)
Attacking
All tested factors were proven to affect the presence of
attack in particular trials (Table 1). L3s were
attacked more often than adults (P < 0.001; Fig. 3);
nevertheless, the interaction of developmental stage
of the firebug and bird species was also significant
(Table 1). The L3 firebugs were attacked more often
only by great tits (great tit: P << 0.001; blue tit:
P = 0.984; Fig. 3). The brown-painted firebugs were
attacked more often than the unmodified ones
(P << 0.001; Fig. 3); nevertheless, there was again a
significant interaction of firebug colour and bird
species (Table 1). Blue as well as great tits attacked
brown-painted firebugs more often than the unmodified ones (great tit: P << 0.001; blue tit: P = 0.042;
Fig. 3); however, the great tit attacked the brownpainted firebugs more often than the blue tit
(P < 0.001; Fig. 3). Of course, the great tit attacked
firebugs more often than the blue tit (P < 0.001;
Fig. 3) without regard to any other factors. There was
also a strong effect of trial number (Table 1). Firebugs
were attacked most often during the first trial than in
any other (second: P << 0.001; third: P << 0.001;
fourth: P << 0.001; fifth: P << 0.001). Nevertheless,
the significant interaction between trial number and
firebug colour (Table 1) shows that, in the case of the
unmodified firebugs, the significant difference is only
between the first and the fifth (last) trial (P = 0.030).
Killing
Again, all tested factors were shown to affect the
presence of killing in particular trials (Table 1). The
L3 firebugs were killed more often than adults
Figure 3. Numbers of firebugs attacked/killed by individual great (Parus major, PM) and blue tits (Cyanistes
caeruleus, CC). Prey categories in treatment groups: Ad,
adults; L5 and L3, fifth- and third-instar larvae. The total
number of tested birds in each group was 16. Striped and
grey columns refer to attacking, black to killing. Boxes
refer to 25–75% percentile, whiskers refer to 1–99%
percentiles.
(P << 0.001; Fig. 3) as well as L5 firebugs (P << 0.001;
Fig. 3). Similarly, similar to attacking, there was a
significant interaction between the developmental
stage of the firebug and the bird species (Table 1). The
L3 firebugs were killed more often than adults and
L5s only by the great tits (adults: P << 0.001, L5:
P << 0.001; Fig. 3). By contrast to attacking, there
was a significant interaction between firebug colour
and developmental stage (Table 1). L3 firebugs were
killed more often than L5 and adult only when brownpainted (L5: P < 0.001; adult: P << 0.001; Fig. 3),
whereas the unmodified L3 differed only from the
unmodified adult (P = 0.001; L3). The brown-painted
firebugs were generally killed more often than the
unmodified ones (P << 0.001; Fig. 3), with the same
significant interaction as in attacking (firebug
colour ¥ bird species; Table 1). Brown-painted firebugs were killed more often than the unmodified ones
only by great tits (great tit: P << 0.001; blue tit:
P = 0.624; Fig. 3). As in attacking, firebugs were killed
most often during the first trial (second: P < 0.001;
third: P < 0.001; fourth: P << 0.001; fifth: P << 0.001);
however, there was a significant interaction between
trial number and bird species (Table 1). Firebugs
offered during the first trial were killed more often
only by great tits (blue tits; second: P = 0.939; third:
P = 0.939; fourth: P = 0.939; fifth: P = 0.986).
EDIBILITY
OF FIREBUGS (NUMBERS OF EATEN BUGS
BY PARTICULAR BIRDS)
The number of eaten firebugs was significantly
affected by the developmental stage of the firebug and
895
Figure 4. Numbers of firebugs eaten by individual great
(Parus major, PM) and blue tits (Cyanistes caeruleus, CC).
Prey categories in treatment groups: Ad, adults; L5 and
L3, fifth- and third-instar larvae. The total number of
tested birds in each group was 16. Boxes refer to 25–75%
percentile, whiskers refer to 1–99% percentiles.
species of the bird (Table 1). Birds ate more L3s than
either L5s (P < 0.001; Fig. 4) or adults (P < 0.001;
Fig. 4). The number of eaten L5s and adults did not
differ (P = 0.999; Fig. 4). Great tits ate on average
more of the offered firebugs than blue tits (P << 0.001;
Fig. 4). The difference was so pronounced that the
number of unmodified firebugs eaten by great tits was
slightly higher than the number of brown-painted
ones eaten by blue tits (P = 0.058). The effect of colour
was not significant (Table 1). Birds that attacked at
least one of the offered firebugs ate the same number
of unmodified and brown-painted ones (P = 0.529;
Fig. 4).
DISCUSSION
BODY
SIZE OF THE PREY
The probability of attack at the first encounter of bird
and firebug is not affected by the body size of the
firebug. Smaller prey are just killed more than larger
prey. Moreover, there is no difference between L3s
and L5s, but only between L3s and adults. However,
smaller prey are more often attacked as well as killed
in repeated encounters. The effect is stronger in the
case of killing because, in the present study, we can
find a significant difference between L3s and L5s and
not only between L3s and adults. Finally, L3s were
eaten more often than larger firebugs (adults as well
as L5s).
On the basis of these results, we assume that size
works better through the amount of defensive chemicals than through the amount of optical stimulus.
Firebugs release repellent substances when attacked
(Farine et al., 1992), which may discourage some
896
portion of the attacking birds. L3s, which are significantly smaller than L5s and adults, possess a lesser
amount of defensive substances. At the first encounter, lowered chemical protection just increases the
probability of killing the firebug. In the case of
repeated encounters, birds learn slowly to refuse
worse chemically protected firebugs because their
unpleasant experience is smaller.
The killing rate of unmodified firebugs in repeated
encounters is less affected by prey size than is the
case for brown-painted firebugs. We may thus assume
that conspicuous coloration enhances the learning
process in the case of smaller L3s. If the efficacy of
warning coloration was lower with L3s (as a result of
smaller colour area), this handicap should have been
added to the handicap caused by a lower amount of
repellent substances.
Previous experimental studies showed the importance of colour signals in the effect of body size on
protection of aposematic prey. Experiments performed
by Gamberale-Stille & Sillén-Tullberg (1996, 1998)
demonstrated a higher warning efficacy of aggregation of red seed bugs (Spilostethus pandurus Scopoli,
1763; and Tropidothorax leucopterus Goeze, 1778;
both Hemiptera: Lygaeidae) as a result of a higher
amount of warning optical signal. They compared
solitary and aggregated bugs (up to 27 specimens).
Therefore, the difference between their experimental
modifications was more pronounced than for the
arrangement used in the present study. Artificial prey
(paper ‘butterflies’ or almonds covered by paper) used
in other studies (Forsman & Merilaita, 1999; Lindstrom et al., 1999; Mand et al., 2007) only provides a
warning optical signal. In our experiments, L3 possessed a lower amount of warning optical signal;
nevertheless, it is still a firebug and may provide
additional optical signals (body shape, posture) but
also typical scent. Moreover, the efficacy of warning
optical signal of larvae is in our experiments generally lower than those in adults.
Experiments with firebugs cannot exclude the effect
of general preference of the tested birds for smaller
prey. Nevertheless, adult mealworm beetles (i.e.
bigger than adult firebugs) were attacked and killed
more often than any tested form of firebugs. Therefore, we may rule out the effect of size per se.
BODY
SIZE OF THE PREDATOR
Because both tit species commonly attacked and
killed mealworm beetles, we may exclude the possibility that birds differ in their reaction to variously
sized firebugs as a result of different prey size
preferences.
The larger great tit generally attacked, killed and
ate more firebugs than the smaller blue tit, both at
the first encounter and repeated trials. Differences
between blue tit and great tit are in concordance with
the results obtained in studies using other variously
large avian predators (Wiklund & Järvi, 1982; Exnerová et al., 2003) where the larger ones were always
more willing to attack aposematic prey.
Only the great tit attacks and kills L3s more often
than L5s and adults, or brown-painted firebugs more
often than the unmodified ones. The more careful
approach of the blue tit to our prey is in concordance
with its general feeding behaviour. As observed previously, the blue tit is more deliberate in utilizing new
prey and is not as innovative as the great tit (Fisher
& Hinde, 1949), which is in contrast to the practically
equal dietary requirements (Cowie & Hinsley, 1988,
McGregor & Healy 1999 or Naef-Daenzer & Keller,
1999).
In general, the wariness of the blue tit can be
explained by higher innate neophobia and dietary
conservatism, which is repeatedly described in
smaller species (Greenberg, 1984; Marples & Kelly,
1999), and thus does not allow it to be as experimental as the great tit.
PREY
COLORATION
There was no significant difference in numbers of
birds attacking and killing nonmodified and brownpainted firebugs of both larval instars at the first
encounter. However, unmodified larvae of both tested
instars were better protected against bird attack than
the brown-painted adults. Taken together, red larval
coloration has a certain repellent effect; however, it is
much weaker than in adult coloration. When prey is
presented repeatedly to predators, differences in the
protective function of the coloration of adults and
larvae diminish. Birds learn during the experiment to
refuse unmodified larvae as well as adults.
The fact that unmodified adult firebugs are better
protected against bird attack than brown-painted
ones confirms the results reported in previous studies
(Wiklund & Järvi, 1982; Exnerová et al., 2003). Nevertheless, the low protection of coloration of larvae is
in contrast to the general assumption that contrasting red and black coloration per se is a universal
aposematic signal (Cott, 1940; Edmunds, 1974; Tullberg et al., 2008). Experiments by Sillén-Tullberg,
Wiklund & Järvi (1982) also showed worse colour
protection in larvae of the red seed bug (Lygaeus
equestris L., 1758; Hemiptera: Lygaeidae), another
Palaearctic red and black aposematic bug. This suggests that the role of individual colour pattern elements should be considered in more detail (e.g. in
models simulating evolution of mimetic complexes
with different, but equally perfect, signals; Speed &
Turner, 1999).
Experimental studies with artificial prey suggest
that efficacy of warning coloration increases with
increasing size and symmetry of the individual colour
pattern elements (Forsman & Merilaita, 1999; Lindstrom et al., 1999). Thus, we suggest that coloration of
P. apterus adults may be more efficient because of two
large, rounded, and laterally symmetrical black spots
on their forewings (Fig. 1). Firebugs tend to live in
large aggregations, including larvae of various instars
as well as adults. Advantages gained from the gregarious way of life of larvae and adults were demonstrated by Sillén-Tullberg et al. (1982). L5s of the red
seed bug have been shown experimentally to survive
better when presented to a predator with previous
experience of adult bugs, whereas the survival rate of
adults did not improve after a previous encounter
with larvae.
The results obtained in the present study are in
apparent contradiction to the results reported by
Exnerová et al. (2006) who tested reactions of passerines to colour mutants of the red firebug (orange,
yellow and white). They found that the background
colour of the pattern was highly important in aposematic signals, whereas there was practically no effect
of the black spots because the white-and-black as well
as yellow-and-black mutants were attacked with the
same frequency as the plain brown-painted firebug.
Similar results were produced in experiments with
artificial stimuli and chicks (Aronsson & GamberaleStille, 2008). A possible interpretation is that colour
as well as pattern is necessary for the red firebug to
have fully working protection from tits.
By contrast to attacking and killing, the number of
eaten bugs was not affected by their colour. This
result demonstrates the low importance of optical
signals and therefore the significant importance of
chemical signals with respect to the result of an
already started attack.
EFFECT
OF AVOIDANCE LEARNING
Most attacks as well as kills occurred during the first
encounter between the bird and firebug. The lower
aversion to the first prey shows that there was no
neophobia, which would have an opposite effect
(Marples & Kelly, 1999). During the first experiment,
most birds managed to learn the unprofitability of the
prey and remembered it at least throughout the other
four repetitions (20 min). The higher attack rate
during the first experiment is more significant in the
case of brown-painted firebugs. Nevertheless, this is
not caused by a different learning rate, but instead by
better protection of unmodified firebugs during the
first encounter with a predator.
Exnerová et al. (2007) showed that many more presentations (up to twenty) were necessary for naïve,
897
hand-reared great tits to learn firebug inedibility.
Because we used wild caught birds, they might have
different experiences with various preys, including
other bugs (but also the red firebug in some cases).
During our experiments, birds might have considered
the presented firebugs as suspicious even when they
did not recognize them unambiguously and were able
to generalize them to some familiar prey. Thus, birds
attacked them as a demonstration of this, but they
quickly discovered the inedibility of the bugs. Handreared birds are confronted with a firebug for the first
time and have no experience with any similar prey;
thus, it takes them a longer time to learn. This
difference between naïve and partially experienced
predators might be more general, which would significantly favour even imperfect aposematic species in
nature.
ACKNOWLEDGEMENTS
We thank the Academy of Sciences of the Czech
Republic (IAA601410803), the Czech Science Foundation (206/08/H044) and the Ministry of Education,
Youth and Sport of the Czech Republic (MSM
6007665801) for financial support. We thank Dr Keith
Edwards for language correction. Authors are licences
for manipulation with birds (Bird Ringing Centre,
Czech Animal Welfare Commission no. 489/01).
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The red cotton bug is similar to Middle European true bugs that are considered aposematic by avian
predators. However, it differs in colouration (yellow-orange vs. red) and details of its black pattern as
well. We tested if two titmice species (great and blue tit) refusing Middle European true bugs
generalise their optical signal to the red cotton bug.
Four forms of the cotton bug were tested: unmodified (orange and black pattern); brown-painted (to
eliminate potential warning colouration); with removed hemielytra exposing the red abdomen without
black pattern (similar colour but different pattern from Middle European true bugs); with exposed
white and red striped bottom (similar colouration as some butterfly larvae). To reveal the effect of
chemical signals, we also tested two forms of the red firebug (unmodified and brown-painted): a
chemically protected and familiar true bug species.
The tits were not able to generalise from the firebug to the unmodified cotton bug. Similarly, the
striped bottom of the cotton bug was not considered as a warning. The only cotton bug form perceived
as a warning was the one with removed hemielytra exposing a plain red abdomen. Therefore, the
different (and less conspicuous) colour of the cotton bug was the factor resulting in the failure to be
generalised to Middle European true bugs.
Despite having very similar composition of protective substances, cotton bugs were eaten much more
often than firebugs. This means that even a little change in chemical composition of protective
substances may lead to significant changes in efficacy of protection.
,QWURGXFWLRQ
Aposematic colouration has been a favourite
topic of studies since the theory of
aposematism and mimicry was defined
(Komárek 2003). From its beginning,
multimodality of warning signals has been
proposed (Rowe & Guilford 1999). Signals
perceived by several senses are advantageous
for prey. Nonetheless, even in a single signal
channel, there may be several components with
warning significance. Most “aposematic
studies” use birds as predators, which are
oriented mainly by sight when searching for
food. In this signal channel, four basic
components can be distinguished: size, general
appearance (shape of body, legs and antennae),
colour and colour pattern (Guilford 1990).
Colour is the most studied component of an
optical warning signal. As the aposematic
signal must be easily recognizable and
comprehensive; conspicuous colours (red,
yellow) should be present (Cott 1940). Several
experimental studies showed that birds have a
higher ability to learn the inedibility of
unfamiliar
prey
when
connected
to
conspicuous colours (Marples et al. 1994;
Marples & Roper 1996; Roper & Marples
1997). Studies using natural insect prey noted
the importance of conspicuous colours as well.
Exnerova et al. (2006) showed that mutant
forms of the red firebug (Pyrrhocoris apterus)
are considered aposematic only when their
background colour is red or orange (the black
pattern remained unchanged). Yellow and
white mutations were attacked equally often as
an artificially de-aposematized (brownpainted) form.
The effect of pattern on warning displays is a
less common subject of investigation. Some
patterns were showed to be universally
considered as warnings (e.g. eye spots on
1
wings of butterflies – Brakefield & French
1999; Lyytinen et al. 2003; Stevens et al. 2007,
2008). Nevertheless, the importance of patterns
per se (achromatic patterns) is studied only
rarely. One approach in experimental studies is
to form artificial patterns and present them to
predators (Osorio et al. 1999; Forsman &
Merilaita 1999, 2003; Forsman & Herrstrom
2004). Generally, these studies showed higher
ability of predators to learn the unprofitability
of prey, when connected to larger and
symmetrical patterns. Several studies using
natural insect prey proved a high importance of
pattern regardless of colouration. SillénTullberg et al. (1982) showed that differently
patterned (and equally coloured) larvae of the
red seed bug (Lygaeus equestris) are less
protected than adults. Similarly, Dolenska et
al. (2009) revealed a high importance of
spotted pattern per se in antipredatory optical
signals
of
ladybirds
(Coleoptera,
Coccinellidae).
In the present study, we tried to test the
antipredatory effectiveness of particular
components of optical signals in one species of
a stainer bug: the red cotton bug (Dysdercus
cingulatus, Fabricius, 1777, Heteroptera,
Pyrrhocoridae).
We offered red cotton bugs to two species of
titmice, great tit (Parus major, L., 1758;
Passeriformes, Paridae) and blue tit (Cyanistes
caeruleus, L., 1758; Passeriformes, Paridae).
Blue tit inhabits Europe and adjacent areas of
northern Africa and western Asia, while the
great tit is distributed throughout the whole of
Eurasia to the Sunda Islands (Cramp & Perrins
1993). The red cotton bug is an IndoAustralian species, originally inhabiting a
range from eastern India to northern Australia,
but due to cotton cultivation, it was dispersed
to tropical areas of the world. Since the
experimental individuals of titmice were
caught in the Czech Republic in Central
Europe, we may assume that they had no
individual experience with the cotton bug.
Nevertheless, there is a question if tested
species could have some inherited avoidance to
the red cotton bug.
Naïve chicks of great tits were showed not to
refuse offered red firebugs (Pyrrhocoris
apterus, L., 1758, Heteroptera, Pyrrhocoridae)
and attacked them repeatedly, while adult great
tits usually refused them at the first encounter
(Exnerova et al. 2007). On the contrary, blue
tits possess an innate aversion to red firebugs
(Exnerová et al. l.c.). As the optical signal of
the red cotton bug is to some extent similar to
Middle European species of true bugs, which
are familiar to the tested birds, the cotton bug
may thus be generalized to them.
The red cotton bug, has quite a complex
optical signal (Fig. 1), consisting of yelloworange coloured hemielytra with one black
spot on each, a red head and upper parts of the
abdomen, a yellow-brown coloured pronotum
with a white collar on its forepart, and white
bottom with red and black stripes. The red
cotton bug possesses several conspicuous
colours and patterns which should provide
protection against predators.
The upper part of the cotton bug is quite
similar to most Middle European aposematic
true bugs (Exnerova et al. 2008), although, the
background coloration of the cotton bug is
lighter, rather yellow-orange than red. Several
studies demonstrated that some species of
Middle European true bugs provide a warning
signal to avian predators (e.g. red firebug
Pyrrhocoris apterus – Exnerova et al. 2003;
red seed bug Lygaeus equestris – SillénTullberg et al. 1982). At the same time,
Exnerova et al. (2008) proposed the existence
of a mimicry ring containing Middle European
true bugs and probably some other red and
black insects (ladybirds and froghoppers). This
mimicry ring appears to work in a MüllerianBatesian continuum with well protected model
species (Lygaeus, Spilostethus, Graphosoma),
moderately protected mimics (Pyrrhocoris) and
poorly protected mimics (Eurydema). The
unmodified upper part of the colour signal for
the red cotton bug could be generalised to
these species according to having similar
colour and pattern (most similar to models of
the above mentioned mimicry ring).
The upper part of the abdomen of the red
cotton bug, covered by hemielytra, is
conspicuously red, which is similar to the
colour of most Middle European true bugs.
Nevertheless, there is no melanin pattern on
the abdomen of the red cotton bug. The lower
part of the body of the red cotton bug has quite
a distinct pattern not similar to any European
true bugs; nevertheless, it may be generalized
to the pattern of several lepidopteran larvae,
especially from the families Arctiidae and
Papilionidae, which were proved to possess
signals considered as warnings (Järvi et al.
1981; Bowers & Stamp 1997). Nevertheless,
2
caterpillars differ markedly in general
appearance from true bugs.
In our experiments, we offered these three
modifications of the red cotton bug to two
species of titmice. We presumed that, if the
offered cotton bug is refused, it was
generalized to any of the similar European
aposematic insects (true bugs or caterpillars).
Nevertheless, the refusal of conspicuously
coloured prey need not be caused only by
coloration. To filter out the effect of general
appearance, we covered the colour signal of
the red cotton bug by painting its upper parts
with brown water colour. We may consider the
effect of coloration to be important for a
warning signal only if the reaction to the
unmodified form is weaker than to the brownpainted form.
The red cotton bug also possesses chemical
defence against predators. The chemical
compounds of the defensive glands of this
species have been described by Farine et al.
(1992a) and shown to be nearly identical to
those in its relative, the red firebug (Farine et
al. 1992b, 1993; Tab. 1), Nevertheless, as
showed e.g. by Aldrich et al. (1997), chemical
compounds depend strongly on food, from
which defensive chemicals are sequestered.
That is why we added the red firebug to our
experiments (unmodified as well as brownpainted). Only if the reaction of the tested birds
to brown-painted red cotton bugs and brownpainted red firebugs is equal may we eliminate
the effect of chemical signal.
0DWHULDODQG0HWKRGV
([SHULPHQWDOSUH\
The experimental individuals of both bug
species (D. cingulatus, P. apterus) were
obtained from laboratory stock cultures. They
were reared in groups of circa 50 individuals in
glass jars (15 cm in diameter). Dry seeds of
linden trees (Tilia cordata, T. platyphyllos) and
creole cotton (Gossypium barbadense), and
water were supplied ad libitum. The insects
were reared at 25°C and under long-day (18 h
light, 6 h dark) conditions.
We offered the red cotton bug to the predators
in four forms (Fig. 1): a) an unmodified adult,
b) an adult with the upper parts covered with
brown water colour (burned sienna – proven as
not impacting the secretion or behaviour of the
bug as well as the bird – see Exnerova et al.
2003; brown-painted form), c) an adult with
removed hemielytra (the red abdomen is
visible, elytra-cut form), d) an adult placed on
its back on a sticker background (locked with
exposed bottom; the bottom form). There were
two forms of the red firebug (Fig. 1): a) an
unmodified adult and b) an adult with brown
painted upper parts.
Fig 1: Forms of bugs used in the experiments (from the left): unmodified form of the red cotton bug
(Dysdercus cingulatus), brown-painted form of the red cotton bug (D. cingulatus), the red cotton bug
(D. cingulatus) with removed hemielytra, the red cotton bug (D. cingulatus) with exposed bottom,
unmodified form of the red firebug (Pyrrhocoris apterus), brown-painted form of the red firebug (P.
apterus)
3
Compound
(E)-2hexenal
(E)-2Hexenyl
acetate
(E)-2Octenal
others
Dysdercus
cingulatus
(%), male
Dysdercus
cingulatus
(%), female
Pyrrhocoris
apterus (%),
male
Pyrrhocoris
apterus (%),
female
88.53
86.87
76.09
76.94
3.22
6.26
1.92
1.87
2.86
2.38
9.94
9.64
5.39
4.49
12.05
11.55
Tab 1: Relative percentages of the most common components identified in the metathoracical glands
of adults of the red cotton bug (Dysdercus cingulatus) and the red firebug (Pyrrhocoris apterus), ex
Farine et al. 1993.
([SHULPHQWDOSUHGDWRUV
Adult birds of two tit species (Parus major,
Cyanistes caeruleus), caught in mist nets in the
vicinity of ýeské BudČjovice, were used as
experimental predators. Captures were
conducted from 2002 to 2003, except during
the breeding season (May to July). Birds were
kept in standard birdcages at lowered indoor
temperature and under outdoor photoperiod
conditions. Birds were acclimated to laboratory
conditions and food (sunflower seeds and
mealworms) for 1-3 days prior to the
experiments. The birds were ringed and
released immediately after the trials finished.
([SHULPHQWDOHTXLSPHQW
Experimental cages were made of wooden
cubic frames (70x70x70 cm) covered with wire
mesh (2 x 2 mm) with the front wall made of a
one-way mirror (see Exnerova et al. 2003, for
details). The cages were equipped with one
perch, a bowl with water, and a rotating
circular feeding tray, containing six small cups
(a single cup contained a prey item during a
trial). The distance between the perch and the
tray was approximately 25 cm. Cup bottoms
were white (normal as well as with a sticker).
Standard illumination was generated by a light
source (LUMILUX COMBI 18W, OSRAM)
which simulated the full daylight spectrum.
7ULDOV
There were six experimental groups of
predators. Each group was offered one of the
forms of bug as described above. There were
20 great tits and 20 blue tits in each
experimental group. No individual bird was
tested with more than one form of bug in order
to avoid pseudo-replication. Each bird was
placed into the experimental cage before the
experiment to become adapted to the new
conditions, and was provided with food
(mealworms) and water. The bird was deprived
of food for 2 hours before the experiment; this
motivates the bird to forage but does not cause
stress. The bird was assumed to be ready for
the experiment as soon as it attacked the
mealworm immediately after offering. Each
experiment with an individual bird consisted of
a series of 10 immediately succeeding trials, in
which 5 mealworms and 5 tested bugs were
offered alternately, starting with a mealworm.
Repetition of several trials within a single
experiment was used to eliminate effects of
individual differences in predators’ neophobia,
for some birds did not attack the first offered
prey. The trials with mealworms were used to
check a bird’s motivation to forage, and they
ended after the mealworm had been eaten. The
trials with bugs lasted 5 minutes.
A continuous description of bird behaviour
was recorded using Observer ver. 3 (1989–
1992, ©Noldus). We distinguished three
possible results of each trial: (1) the bug was
neither handled nor eaten during the 5 minute
trial, (2) the bug was handled (touched, pecked
or taken by the bird’s bill) but no part of it was
eaten, or 3) some part of the bug was eaten.
Taken together, we summarised results of all
five trials with bugs to assess the reaction of a
tested bird, so that data for analyses were in a
binomial form (presence or absence of the
reaction of the bird – handling or eating,
during any of the five sessions with the bug).
For eating, only birds that handled at least one
of the offered bugs were included, so that
portion of birds that decided to eat the bug was
described in order to reveal the edibility of a
particular presented bug form.
6WDWLVWLFDODQDO\VLV
Two General Linear Models (GLM, binomial
distribution, logit link function, using ANOVA
4
and Chi-square tests for deviance analyses and
post-hoc Tukey HSD test; McCullagh &
Nelder 1989), one for the handling data and
one for eating (only handling birds included)
the prey, were formed to test the reactions of
the tested birds. We assessed the effect of two
5HVXOWV
+DQGOLQJ
Variability in the number of birds that handled
at least one of the five offered bugs was best
explainable by the form of the offered bug
(ANOVA, F=14.91, d.f.=5, p<<0.01, 32.7% of
explained variability). Nevertheless, the other
explanatory variable, predator species, was
only
marginally
important
(ANOVA,
F=3.6365, d.f.=1, p=0.058, 1.6% of explained
variability).
The blue tit handled slightly fewer bugs than
the great tit (Tukey HSD, p=0.05778). Post
hoc tests divided tested bugs into two groups.
The unmodified and bottom forms of the
cotton bug, and the brown-painted bugs of both
species were attacked more often than the
elytra-cut cotton bug and unmodified firebug
(Tab. 2, Figs. 2 and 3).
(DWLQJ
Fig 2: Numbers of Great (Parus major) and
Blue tits (Cyanistes caeruleus) handling at
least one of five offered bugs of particular
forms.
independent factors: (1) cotton bug form
(together six forms of two bug species; see
above) and (2) predator species (P.major,
C.caeruleus). All tests were performed in R
2.8.1 for Windows (The R Foundation for
Statistical Computing, 2002-2009).
Variability in the number of birds which ate at
least part of one of the five offered bugs (only
handling birds included) was best explained by
the form of the offered bug (ANOVA, F=5.11,
d.f.=5, p<0.01, 21.8% of explained variability).
The other explanatory variable, predator
species, was also shown to be important
(ANOVA, F=9.97, d.f.=1, p<0.01, 8.5% of
explained variability).
Great tits ate more of the handled bugs than
blue tits (Tukey HSD, p=0.000243). The post
hoc test divided tested bugs into three groups.
All forms of the cotton bug except for the
elytra-cut form, were eaten more than the
brown-painted firebug. Unmodified firebug
and elytra-cut cotton bug did not differ from
any other form. However, while the proportion
of eaten elytra-cut cotton bugs is on average
comparable to both other groups, only three
unmodified firebugs were attacked (and none
was eaten).
Fig 3: Proportion of Great (Parus major) and
Blue tits (Cyanistes caeruleus) eating at least
one of five offered bugs of particular forms.
Numbers above every particular column shows
the total number of handling birds (from which
the proportion was counted).
5
Unmodified
cotton bug
Unmodified cotton bug
Exposed
bottom
cotton bug
Elytra-cut
cotton bug
Brownpainted
cotton bug
Unmodified
firebug
Brownpainted
firebug
0.8415561
0.2902197
0.9241398
0.1420412
0.0004155
0.8495739
0.9999470
0.3712920
0.0166845
0.7677000
0.8005909
0.6588722
0.3230299
0.0097387
Exposed bottom cotton bug
0.9734779
Elytra-cut cotton bug
0.0139875
0.0009261
Brown-painted cotton bug
0.9958294
0.9998547
0.0024302
Unmodified firebug
0.0000001
0.0000001
0.0619063
0.0000001
Brown-painted firebug
0.9998547
0.9121648
0.0304687
0.9734779
0.9959148
0.0000003
Tab 2: Results of post hoc comparisons of differences between particular forms of bugs presented to
both titmice species (Tukey HSD test, p). Handling in the left-low cells and eating (only handling birds
included) in the right-up cells. Bold indicates significant differences, italics indicates marginally
significant differences.
'LVFXVVLRQ
The unmodified upper part of the red cotton
bug did not provide a warning signal to both
tested titmice species. We can thus conclude
that birds were not able to compare and
generalize this signal to any familiar one
proven as a warning (e.g. red firebug
Pyrrhocoris apterus – Exnerova et al. 2003;
red seed bug Lygaeus equestris – SillénTullberg et al. 1982). The red cotton bug
differs from these species by colour of the
background. The aposematic Middle European
true bugs possess red colour while the red
cotton bug is yellow-orange. Nevertheless, the
black pattern present on this colour is quite
similar in both bugs. It thus seems that the red
colour is the key part of the warning optical
signal of aposematic true bugs as proposed by
Exnerova et al. (2008). Results similar to ours
were obtained in a study offering colour
mutants of the red firebug to several passerine
species (Exnerova et al. 2006). This study,
though, showed the warning meaning of the
orange colour as well. This result implies that
the same colour has various effects under
different contexts. In the case of the red
firebug the red and orange mutants differed
only in colour while the pattern remained
unchanged, so the generalization was much
easier than in the case of the cotton bug and
Middle European true bugs.
The key importance of colour is supported also
by experiments with the cotton bug having
removed hemielytra and exposed upper part of
the abdomen. The red coloured abdomen,
contrary to yellow-orange hemielytra, is
considered as a warning by titmice, despite the
lack of any melanisation pattern, which is
singular among Middle European true bugs.
Nevertheless, the protection gained by the
simply red colouration is slightly weaker than
in the case of the unmodified red firebug. This
raises the question of why the best (most
universal) warning signal of the red cotton bug
is covered by the hemielytra. Some insect
species were showed to actively expose their
abdomen (conspicuously coloured) by moving
their wings (elytra, hemielytra) when
confronted by a predator (Evans & Schmidt
1990). Nevertheless, this behaviour has never
been observed in our experiments with the
cotton bug.
The bottom of the cotton bug did not provide
any warning signal to titmice despite the
presence of a red and black pattern similar to
several European caterpillars (Arctiidae,
Papilionidae). We could thus conclude that
birds are not able to generalize these patterns
together. Nevertheless, the caterpillar has a
completely different body shape. This shows
that birds are able to use other stimuli than
colour in generalization processes. We are
lacking knowledge of titmice behaviour to
warningly coloured caterpillars similar to the
cotton bug bottom pattern. Only Järvi et al.
(1981) presented caterpillars of the swallowtail
(Papilio machaon, red, black and green
stripped) to great tits, which refused them
strictly according to optical and chemical
signals.
The effect of chemical signal in antipredatory
protection of the cotton bug is quite an
interesting topic. It probably does not influence
6
the decision to attack the bug, because the
number of birds attacking either the brownpainted cotton bug or brown-painted firebug
did not differ. However, this result can be
explained by both species being equally
protected based on the composition of their
protection chemicals (Farine et al. 1993).
Nevertheless, this conclusion is disproved by
the portion of eaten bugs. The brown-painted
cotton bug was eaten much more often than the
brown-painted firebug. Thus, it seems that
chemical protection of the firebug does not
work over longer distances, and cannot deflect
the attack of the bird, but it significantly
affects the probability of continuation of the
attack (contrary to the cotton bug).
While the chemicals of the cotton bug per se
cannot deflect the attack, the warning
colouration of the abdomen provides a
particular protection even against being eaten,
because the elytra-cut cotton bugs were not
eaten more often than the brown-painted
firebugs (contrary to other forms of the cotton
bug). It is evident that birds take a more
cautious approach to warningly coloured prey
despite other components of their antipredatory
signal. This finding should be important in
evolution and preservation of Batesian
mimicry where the comprehensive signal being
stronger than any other is essential (Mappes &
Alatalo 1997).
As mentioned above, the red cotton bug and
red firebug possess similar chemicals in their
metathoracical gland (Farine et al. 1993; Tab.
1). As our bugs were reared under equal
conditions as in the studies describing their
chemical protection (Farine et al. 1992a,
1993), we can assume our bugs possessed
equal composition of protective chemicals.
Nevertheless, the cotton bugs in our study were
much less protected against titmice than the
firebugs. It is obvious that even a small change
in chemical composition of protective
substances may lead to significant changes in
the efficacy of protection. This parameter
should be factored into comparisons of
chemical protection of many other insects.
In our study, we used two titmice species as
predators to reveal possible differences in the
perception of prospective warning signals of
the red cotton bug. We chose the great tit and
the blue tit, because differences in their
reaction toward aposematic prey were proven
before. The blue tits showed a greater wariness
in reactions to several forms of warningly
coloured true bugs (Exnerova et al. 2003,
2007; Vesely et al. 2006). The difference
between both tested predator species was
shown in their reaction to cotton bugs as well.
Generally, the blue tit was more cautious in
their approach to all forms of the red cotton
and firebug, but the difference was more
significant in eating than handling. It seems
that the blue tit has a similar degree of
willingness to attack suspicious prey, but they
are more often deflected from finishing the
attack. This indicates a higher sensitivity of
blue tits to chemical protection of the cotton
bug.
$FNQRZOHGJHPHQWV
We are grateful to Markéta Patoþková for help
in breeding the cotton bugs and Keith Edwards
for language corrections. We thank the
Academy of Sciences of the Czech Republic
(IAA601410803),
the
Czech
Science
Foundation (206/08/H044) and the Ministry of
Education, Youth and Sport of the Czech
Republic (MSM 6007665801) for financial
support. Authors were granted with licenses
permitting the catching and ringing of birds
(Bird Ringing Centre Praha No. 975 and 1004)
and experimentation with animals (Czech
Animal Welfare Commission No. 489/01).
5HIHUHQFHVFLWHG
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8
Efficiency of individual colours in insect warning
displays: experiments with avian predators.
Alena Cibulková, Petr Veselý, Simona Poláková and Roman Fuchs
Abstract
The importance of various colours in warning signals to optically orienting
predators is tested surprisingly seldom. Several colours (red, orange, and yellow) are
considered to be universally warning; nevertheless, the warning meaning of others is rarely
tested and usually with artificial prey. In the presented study we offered edible prey (Guyana
spotted roach – Blaptica dubia), equipped with colour displays, to avian predators (great tit –
Parus major). Colour was modified using paper stickers placed on the insect’s back. These
stickers were of eight various colours and usually (except for the brown control) supported by
a black pattern equal to that seen in the red firebug (Pyrrhocoris apterus), experimentally
proved to induce avoidance in great tits. We confirmed red and orange to provide the best
protection; nevertheless, yellow elicited significantly weaker avoidance. White, blue and
violet preys were attacked similarly to the yellow one, differing in time amount necessary for
recognition by the predator. White and blue preys were evidently novel for tested birds, so
they looked at them from a distance for a long time until they decided to approach and
possibly attack it (more often in the case of blue). The green prey was the only one attacked
equally as often as the brown control. The often attacked green prey suggested a low
importance of the black pattern in red firebug protection. Red, orange, yellow and white
cockroaches were attacked practically equally as much as mutant forms of the red firebug in
previous studies, so the efficiency of this methodology (using paper stickers to make Batesian
mimic) was certified.
Keywords
Colour, pattern, generalization, titmice, firebug, cockroach, warning signal
Contact
P. Veselý, Department of Zoology, Faculty of Biological Sciences, University of South
Bohemia, Branišovská 31, 370 05 ýeské BudČjovice, Czech Republic
e-mail: [email protected]
1
Introduction
The efficiency of warning signals is primarily based on colour patterns and chemical signals
(Cott 1940; Brakefield and French 1999; Gamberalle-Stille and Guilford 2003; Skelhorn and
Rowe 2005). Several colours are generally considered to be universal warning signals (red,
orange, yellow), as they contrast to most natural backgrounds and enhance recognition and
learning processes in most optically orienting predators (Poulton 1890; Cott 1940, Roper and
Marples 1997, Ham et al. 2006). Nevertheless, various studies show various efficacies of
particular colours in warning signals.
The warning effect of red, orange and yellow colours is generally confirmed in experimental
studies using natural insect prey (e.g. Sillén-Tullberg et al. 1982; Kauppinen and Mappes
2003), artificial prey (Schuler and Hesse 1985; Mastrota and Mensch 1995 or Rowe and
Skelhorn 2005) or even painted water (Roper and Marples 1997). Nevertheless, the efficacy
of such signal depends on the type of predator attacking the prey. Frugivorous or granivorous
birds do not show significant avoidance of red prey (Honkavaara et al. 2004, Exnerová et al.
2003), as it represents most of their natural diet.
Other colours are not generally used in warning displays; nevertheless, in some species they
do occur. The warning meaning of white in particular has been proven experimentally several
times (Collins and Watson 1983; Honkavaara et al. 2004).
Nevertheless, other studies did not show such an effect (Lyytinen et al. 1999, Exnerová et al.
2006), so we may consider the warning meaning of white to be context dependent. Green,
brown and grey are colours generally considered to be non-warning as they match most
natural backgrounds (Rowe and Guilford 1996; Gamberalle-Stille and Sillén-Tulberg 2001;
Rowe and Skelhorn 2005). Nevertheless, even in these colours a warning effect has been
proven, when forming an appropriate pattern like the dorsal zig-zag pattern of vipers
(Niskanen and Mappes 2005). The colours blue and violet are quite rare in nature and they
contrast to most natural backgrounds (which both is convenient to the demands on aposematic
prey – Cott 1940); they are nevertheless used as warning displays only rarely and usually in a
shiny variant as seen in beetles (Bonacci et al. 2008).
In any event, the warning meaning of any colour is more efficient when combined with a
contrasting (black) pattern, because it enhances the recognition of a particular prey species
(Cott 1940). Nevertheless, such a pattern should meet several criteria to provide a functional
warning signal. It should be symmetrical (Forsman and Herrström 2004), contrasting within
the used background colour (Aronsson and Gamberalle-Stille 2009) and idealy possess some
specific components like rounded spots – so called eyespots (Stevens et al. 2008).
In the presented study we offered edible prey carrying paper sticker shields placed on their
backs with colour patterns derived from the natural colouration of the red firebug
(Pyrrhocoris apterus, L. 1758: Hemiptera, Pyrrhocoridae) to an avian predator. The red
firebug was proven to possess a warning signal eliciting aversion in several passerine species
(Exnerová et al. 2003), consisting of a red background with several melanisation centres on
the pronotum and hemielytra including rounded black spots (Zrzavý 1995). Nevertheless, the
red firebug also possesses chemical protection (Farine 1992) which causes certain avoidance
in birds as well (Exnerová et al. 2003). To test the effect of the optical signal alone, we
decided to use an edible insect as prey with the colour signal of the red firebug printed on a
paper sticker shield placed on the insect’s back. Therefore, we created a Batesian mimic of the
red firebug possessing no protection, but a similar signal (Huheey 1980). This method was
proven to work well, as the mimic was protected against attack by the avian predator equally
to the model (Veselý a Fuchs 2009). Such prey allows us to test the effect of the colour optical
signal per se; nevertheless, other optical signals typical for the edible prey are still present
(legs, antennae, body shape). It has been shown that predators are able to use such traits in
2
prey recognition (Kauppinen and Mappes 2003, Nelsson et al. 2006, Dolenská et al. 2009).
The shields on the edible prey used depicted the colour signal of the red firebug, preserving
the black pattern but altering the colour shade of the background. To test the effect of the
black pattern a plain brown control shield with no black pattern was used as well.
Material and methods
Prey
We used third larval instars (7-11 mm length) of Guyana spotted roach (Blaptica dubia,
Audinet-Serville 1983: Dictyoptera, Blaberidae) as an edible prey carrying the model colour
pattern. It possesses no chemical or other warning signals and protection; it is commonly used
as fodder. It is similar to the model in size and general appearance, nevertheless, it’s way of
moving, body posture and shape of it’s legs and antennae little different. Roaches were bred
in a glass terrarium in large groups (100 individuals) and vegetables (carrot, beet, and
potatoes), cat food, and water supplied to them ad libitum.
The roaches carried paper sticker shields placed on their backs, possessing a modified colour
pattern of the red firebug. There were seven modifications with a preserved black pattern
equal to that of the firebug, differing in the background colour (red, orange, yellow, white,
green, blue, violet) and one modification without the black pattern, plain brown, used as a
control, non-warning signal. See Table 1 for digital specification and characteristics of the
particular colour shades in RGB as well as CMYK channel regime.
Table 1. Digital specification of particular colour shades used in experiments
Colour shade
Red
Orange
Yellow
White
Blue
Violet
Green
Brown
R
255
255
255
255
0
140
0
130
G
0
140
255
255
140
0
255
90
B
0
0
0
255
255
255
0
55
C
0
0
3
0
71
62
54
42
M
95
51
3
0
19
61
0
66
Y
84
87
82
0
0
0
93
87
K
0
0
0
0
0
0
0
3
Predators
We used individuals of the great tit (Parus major, L. 1758: Passeriformes, Paridae), caught in
the wild, as predators. Great tits were shown to refuse unmodified forms as well as some
colour modifications of the red firebug (Exnerová et al. 2006) and respond equally to the
unmodified red firebug and its artificially made Batesian mimic (Veselý and Fuchs 2009).
The birds were caught using mist nets during two winter seasons (2006-2008, September to
March) on winter feeders.The birds were caged in standard bird cages and bred under a
lowered indoor temperature (15 °C) and daytime light regime with sunflower, mealworms and
water supplied ad libitum. The birds were usually kept one to two days prior to the
experiment. All birds were ringed and released at the place of capture after the experiments
were finished. Together we used 160 birds divided into 8 groups. Each bird was tested only
once to avoid pseudoreplication.
Experiment
The experiments were conducted in an observatory cage consisting of a wooden frame (70
x70 x 70 cm) covered with wire mesh (3x3 mm mesh diameter), and with one side formed by
a one-way mirror, behind which the observer. The cage was equipped with a perch and a
3
water bowl and illuminated with a daylight-simulating light source (OSRAM). At the mirror
side a rotating circular tray with six white cups was placed. In these cups the experimental
prey was placed and turned into the cage, with only one cup containing prey during the
experiment.
Every bird was placed in the observatory cage several hours before the experiment and trained
to attack offered prey (mealworm). After this training, the birds were deprived of food for two
hours, to be motivated to attack prey in the experiment. During the experiment ten prey items
were offered successively, alternating five mealworms with five roaches with stickers, starting
with a mealworm. Five repetitions of the presentation were used in order to filter out the
effect of neophobia, which was showed to be quite a short-time event (Marples and Kelly
1999). Every prey was offered for five minutes and the birds’ behaviour was observed by
camera and simultaneous recording into ethological software (Observer ver. 3, 1989 – 1992,
©Noldus).
Data and statistical analyses
We observed three behavioural responses: approaching the prey (latency of the first approach
and total number of particular approaches), attacking the prey (touching the prey with the
beak) and eating the prey (at least part of the body). As there was no difference among five
particular subsequent repetitions of the experiments with cockroaches, their results were
combined. Therefore the data used in analyses were: 1) presence of any attack during all five
roach trials (values 1 or 0 for every particular bird), 2) presence of any eating event during all
five roach trials (values 1 or 0 for every particular bird), 3) total time to the first approach
during all five roach trials (0 to 5x300 seconds) and 4) mean number of approaches in one of
five roach trials (0 to 3). Data for the number of attacking and eating birds were of binomial
distribution. The effect of sticker shield colour on these behavioural response was evaluated
using the generalized linear model (GLM) and differences between particular shield types
were evaluated using the Fisher exact test with Bonferroni adjustment (p level lowered to
0.007). The data for latency of approaching were of normal distribution, so an analysis of
variance (ANOVA) was used to evaluate the effect of shield colour on this type of data. This
test was used as well in the case of a number of approach events, which was however of
lognormal distribution, so transformation of this data was used (ln(A-0.01)). Particular
differences in these two data types were evaluated using post-hoc Tukey HSD tests.
All calculations were done using R 2.8.1 for Windows (R development core team 2009) and
Statistica 8 (1984 - 2009, StatSoft, Inc.).
4
Results
Attacking and eating
The number of birds that decided to attack or eat at least one of five offered roaches was
significantly affected by the colour of the sticker shield being carried by the roach (GLM,
binomial, logit link function, attacking: F = 31.4, df = 7, p > 0.01; eating: F = 35.9, df = 7, p >
0.01). The most attacked and eaten colour of shield was brown (without a pattern) together
with green (Fig. 1). All tested colours with pattern except for the green differed significantly
from the brown control sticker (Tab. 2). Red and orange stickers were attacked as well as
eaten more often than green ones. Moreover, yellow, white and violet were eaten more often
than green (contrary to attacking).The yellow form was attacked marginally more often than
the red form (Tab 2).
20
18
16
14
12
attacking
eating
N 10
8
6
4
2
0
red
orange
yellow
white
blue
violet
green
brown
Figure 1. Numbers of birds attacking and eating particular prey forms.
Table 2. Particular differences between colour forms of prey in numbers of attacking or eating birds. Fisher
exact p values. Significant differences in bold. Bonferroni adjustment applied (p level lowered to 0.007).
Attacking in left-lower and eating in right higher part of table.
eating
attacking
red
orange
yellow
white
blue
violet
green
brown
red
1
0.065
0.127
0.031
0.065
>0.001
>0.001
orange
yellow
white
blue
violet
green
brown
1
0.661
0.661
0.408
0.408
1
0.065
0.065
0.301
0.501
0.408
0.408
1
1
0.501
>0.001
>0.001
0.001
0.004
0.054
0.004
>0.001
>0.001
>0.001
>0.001
>0.001
>0.001
0.182
0.155
0.273
0.082
0.155
>0.001
>0.001
1
1
1
0.026
>0.001
0.748
1
0.010
>0.001
1
0.048
0.001
0.023
>0.001
0.342
5
Approaching
The latency of the first approaching to the offered prey was significantly affected by the
colour of the sticker carried by the prey (ANOVA: F = 3.62, df = 7, p = 0.001). Similarly, the
number of approaches to the prey was affected by its colour (ANOVA: F = 3.13, df = 7, p =
0.004). The white form was approached after the longest period and quite seldom (Fig. 2 a 3,
Tab. 3). The blue form was approached also after a longer period; nevertheless, it was
approached a little more often. Orange and violet were approached rarely; nevertheless, quite
quickly. The most often and earliest approached colours were brown and green (Fig. 2 a 3,
Tab. 3).
3,0
Number of approaches
2,5
2,0
1,5
1,0
0,5
0,0
red
orange
yellow
white
violet
blue
green
brown
Median
25%-75%
Non-Outlier Range
Outliers
Extremes
Figure 2. Mean numbers of approaches to particular prey forms.
1400
1200
latency (s)
1000
800
600
400
200
0
red
orange
yellow
white
violet
blue
green
brown
Median
25%-75%
Non-Outlier Range
Figure 3. Latency to the first approach in particular prey forms.
Table 3. Particular differences between latency and number of approaches to particular colour forms of prey.
Post hoc Tukey HSD test p values. Significant differences in bold. Latency in left-lower and number of
approaches in right higher part of table.
number
latency
red
orange
yellow
white
blue
violet
green
brown
red
1
0,812
0.125
0.211
0.516
0.215
0.114
orange
yellow
white
blue
violet
green
brown
0.567
1
0.567
0.554
0.814
0.714
1
0.567
1
0.614
0.251
1
0.251
0.848
0.251
1
0.567
1
0.614
1
0.251
0.182
0.002
0.173
0.017
0.182
0.001
0.171
0.268
0.179
0.251
0.654
0.211
0.118
0.094
0.120
0.950
0.685
0.256
0.486
0.241
0.026
0.002
0.154
0.094
0.061
0.627
0.411
0.993
6
Discussion
The least attacked as well as eaten colour forms of presented prey were orange and red,
which is in concordance with our general understanding of warning colours (Cott 1940, Ham
et al. 2006; Exnerová et al. 2006; Svádová et al. 2009). These forms were also approached
quite quickly and scarcely, so we may conclude that the birds had no problem with identifying
them and assigning them quickly to the signal of natural red firebug. The colour orange was
probably not differentiated from the natural coloration of red firebugs, as their coloration has
a certain natural variability (Socha and NČmec 1992).
The yellow and the white form of the prey used in our study were protected a little less
against the titmice attack than the red and orange ones. This result is in concordance with
results using mutant forms of the red firebug (Exnerová et al. 2006). Yellow and white
mutants were attacked as well as killed more often by great tits than the red natural form. In
the case of the colour yellow it is quite an interesting result, as this colour is usually
considered as universaly warning, especially when combined with black (Schuler and Hesse
1985; Kauppinen and Mappes 2003, Hauglund et al. 2006) but even a plain yellow prey was
attacked equally seldom as the red one (Rowe and Skelhorn 2005). We may consider that
great tits were not able to generalize the signal of the natural red firebug to this yellow form
as showed by Svádová et al. (2009). Nevertheless, yellow is still provided a sufficient
protection to the cockroachs in our study (better than in the case of the brown control shield).
Moreover, yellow forms were approached very often and not often repeatedly, so birds did not
have any problem with evaluating its meaning.
The case of white is more complicated as several studies confirm its warning sense (Collins
and Watson 1983; Honkavaara et al. 2004) while others do not (Lyytinen et al. 1999,
Exnerová et al. 2006). Moreover, the birds in our study tended to observe it for a very long
time before they decided to approach it; moreover they approached it only seldomly. It seems
that birds were not able to generalize the natural coloration of the red firebug to this form and
moreover, they had problems with the generalization of any familiar signal to it, as white
insects with black pattern are quite scarce in the Middle-European fauna (except for
butterflies which possess a completely different appearance; Lyytinen et al. 1999).
Nevertheless, after this period, birds mostly evaluated this prey as unacceptable and avoided it
(though a little less than orange and red).
The violet and the blue prey were attacked as well as eaten at a similar rate to yellow and
white ones. Nevertheless, they differed in the time which birds needed to evaluate their
dangerousness. The blue form was approached by birds very slowly; birds needed more time
to decide if to approach it or not, while the violet form was approached quite quickly.
Moreover, the violet form was approached very scarcely while the blue one a little more
often. It seems that birds were not able to recognize and categorize the blue form and compare
it to any familiar prey, while in the case of violet this problem was not that important. This is
quite surprising, because the inner contrast of violet and black in the pattern was the lowest
among all tested colours, so the black pattern was not clearly visible to the human eye.
Patterns are supposed to enhance the recognition process in warningly signalling prey
(Stevens et al. 2008; Aronsson and Gamberalle-Stille 2009), so we expected the latency to
approach the violet prey to be longer. The short time necessary for violet prey recognition
suggests that great tits were able to sense the contrast between these two colours. The higher
ability to perceive contrast between two colours is generally supposed in birds (Jones et al.
2001).
Neither of these colours is at all common in nature. Several insect species possess them, but
usually in a shiny, metal gloss form. The rates of attack and eating events show, that after the
evaluating process, the birds usually decided to avoid these forms. Most metal shining insects,
usually beetles (Coleoptera) possess certain protection (Bonacci et al. 2008), and if titmice
7
tested in our study generalized their signals to the presented roaches, it was more profitable to
avoid them. Nevertheless, the blue prey were attacked, but above all eaten, at practically the
same rate as the green ones, which suggests, that its warning meaning is a little lower than in
the case of violet prey. Mastrota and Mensch (1995) showed northern bobwhites (Colinus
virginianus) to attack blue prey at an equal rate as green ones and much less than red and
yellow. We may assume that blue is considered to be unfamiliar, and may elicit certain
avoidance; nevertheless, it is not suitable for carrying a warning signal.
Green prey were attacked as well as eaten by most of the tested birds and with the same rate
as the brown control and significantly more often than the red and orange form. Moreover, it
was approached most quickly (from all forms possessing the black pattern) but it was not
approached as often as the brown one. These results imply two concerns. That green is not
considered as a warning, it is quickly recognized and birds do not need to observe it
repeatedly before the attack, which is in concordance to the general opinion (Rowe and
Guilford 1996; Gamberalle-Stille and Sillén-Tulberg 2001; Rowe and Skelhorn 2005).
The other result is thus quite surprising. In the green prey the black pattern was retained equal
as in natural red firebugs. Nevertheless, this pattern did not provide sufficient protection when
the background colour of the insect was green. Most warning displays are formed by colour
patterns, usually in combination with black to enhance the signal and facilitate the recognition
and learning process of the predator (Cott 1940). The pattern of the red firebug complies with
most of criteria assumed as important in the efficacy of warning signals: it is symmetrical
(Forsman and Herrström 2004), contrasting (Aronsson and Gamberalle-Stille 2009) and
possesses rounded spots, proved to enhance the warning display (Stevens et al. 2008).
Moreover, it has been shown, that even inconspicuous colour may elicit avoidance in
predators when formed into proper patterns – e.g. the zig-zag pattern of snakes (Niskanen and
Mappes 2005). The explanation of our result may reside in the low ability of titmice to
generalize the normal red and black pattern of the red firebug to the green one. Svádová et al.
(2009) showed that great tits (hand-reared) were not able to generalize the signal of the
natural red and black firebugs to white mutants (which were considered to be non-warning by
adult birds), even if they possessed the equal black pattern. We may therefore assume that the
black pattern has a certain effect on the efficacy of the protection of the red firebug;
nevertheless, it has to be supported by a warning background colour.
In our study the prey was represented by edible cockroaches carrying paper stickers on their
backs. These stickers possessed modified colour patterns of an inedible red firebug, so we
may consider such prey as a Batesian mimic (Huheey 1980). In our previous study (Veselý
and Fuchs 2009) we proved that the cockroach carrying paper sticker with the red and black
pattern of the red firebug to be as equally protected as the model (red firebug). As the attack
rates for various colour modifications of our cockroaches (red, orange, yellow and white)
were practically equal to attack rates (of the great tits) for coloured mutants of the red firebug
in the study of Exnerová et al. (2006) we may consider tested birds not to be able to
recognise/ see through the fakery of such mimics. The Guyana spotted roach carrying a paper
sticker still provides several traits to the predator which might enable its recognition (smell,
but also shape of legs and antennae, or way of moving). According to our result, we may
consider the effect of such traits as negligible in prey recognition, tested titmice were not able
to distinguish the cockroach from the firebug and their reactions were affected most by the
colour pattern on the paper sticker shield.
Acknowledgements
We thank B. A. Christopher Mark Steer for the language improvement. We thank the
Academy of Sciences of the Czech Republic (IAA601410803), Ministry of Education, Youth and Sport of the
Czech Republic (MSM 6007665801) and the Czech Science Foundation (206/08/H044) for financial support.
Authors are licensed for catching and ringing birds (Bird Ringing Centre Prague No. 1004) and for animal
experimentation (Czech Animal Welfare Commission No. 489/01).
8
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DOI 10.1007/s10682-008-9281-1
ORIGINAL PAPER
Newly emerged Batesian mimicry protects
only unfamiliar prey
Petr Veselý Æ Roman Fuchs
Received: 14 September 2006 / Accepted: 30 October 2007 / Published online: 14 November 2008
Ó Springer Science+Business Media B.V. 2009
Abstract The evolution of Batesian mimicry was tested experimentally using avian
predators. We investigated the effect of a search image on the protection effectiveness of a
newly emerged Batesian mimic. The two groups of predators (adult great tits, Parus major)
differed in prior experience with prey from which the mimic evolved. The Guyana spotted
roach (Blaptica dubia) was used as a palatable prey from which the mimic emerged, and
red firebug (Pyrrhocoris apterus) was used as a model. Optical signalization of the insect
prey was modified by a paper sticker placed on its back. The cockroaches with the firebug
pattern sticker were significantly better protected against tits with no prior experience with
cockroaches. The protection of the firebug sticker was equally effective on cockroaches as
it was on firebugs. The cockroaches with firebug stickers were not protected against attacks
of tits, which were familiar with unmodified cockroaches better than cockroaches with a
cockroach sticker. We suppose that pre-trained tits acquired the search image of a cockroach, which helped them to reveal the ‘‘fake’’ Batesian mimic. Such a constraint of
Batesian mimicry effectiveness could substantially decrease the probability of evolution of
pure Batesian mimic systems.
Keywords Evolution of Batesian mimicry Warning signalization Neophobia Search image
Introduction
One of the striking phenomena connected with antipredatory signalization is mimicry. Two
basic forms of mimicry have been distinguished (see Fisher 1930): Müllerian, in which two
protected species are mutually protected by similar signalization; and Batesian when one
unprotected species (mimic) parasites on the protected one (model) by imitating its signal.
P. Veselý (&) R. Fuchs
Department of Zoology, Faculty of Biological Sciences, University of South Bohemia, Branišovská 31,
370 05 Èeské Budı̀jovice, Czech Republic
e-mail: [email protected]
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The initial evolution of Batesian (as well as Müllerian) mimicry has always been
considered trouble-free. The basic dilemma of evolving the warning colouration (predators
unfamiliar with a novel conspicuous prey kill all emerged individuals and, thus, such a
form cannot spread in the population; see e.g., Guilford 1990; Alatalo and Mappes 1996) is
solved in this case, as the predator is familiar with a warning signal of the model species.
Nonetheless, the low amount of evidence of pure Batesian systems in nature (see e.g.,
Turner and Speed 1999) indicates that there are some constraints in the evolution of
Batesian mimicry.
First and foremost, the population of the model species must be bigger than the population of the mimic species (Fisher 1930) so that the predator has a higher probability to
encounter the model and learn (or recall) the connection between signal and quality
(unpalatability) than the connection between the signal and mimic (without quality).
Experimental evidence for this precondition was provided by Lindström et al. (1997).
Experimental predators (great tits) were confronted with models (warning signal and
unpalatability) as well as mimics (warning signal without unpalatability) in a novel world
design. This experimental approach uses artificial prey, which can be considered as being
completely novel to the predators even in terms of evolutionary history (see Lindström
1999 for details). The experiments of Lindström et al. (1997) show that a higher proportion
of mimics in the prey population reduced the protection of the models against predator
attack. Moreover, this study tested the effect of the degree of model unpalatability on the
effectiveness of Batesian mimicry. The more unpalatable is the model the better protected
is the mimic.
Another parameter that could condition the effectiveness of Batesian mimicry is the
accuracy of the mimic signal. The more perfect (similar) is the mimic signal, the higher
should be the probability of its confusion with the model (Hetz and Slobodchikoff 1988;
Johnstone 2002; Sherratt 2002). The novel world experimental design was used to test this
parameter in experiments carried out by Mappes and Alatalo (1997). They showed that
even an imperfect mimic signal may provide some protection. Similarly, Lindström et al.
(2006) did not observe any increase of mimic mortality if their signal was imperfect
(Müllerian mimics were tested in this case). On the contrary, Dittrich et al. (1993) found
different results. Pigeons were trained to assess the similarity of presented items (so called
same/different task). During the experiment, the pigeons were offered less or more perfect
Batesian mimics (several dronefly species) together with their hymenopteran models. The
pigeons were not able to distinguish a perfect mimic from the model, but the imperfect
ones were distinguishable. The difference between this study and the novel world experiments studies can be explained by the different experimental approach. While Dittrich
et al. (1993) were interested in the cognitive abilities of pigeons (they did not perceive the
items as a prey), the novel world experiments observed bird reaction to a prey. The
approach of a predator to any prey (including Batesian mimics) is affected not only by the
quality of the warning signalization, but also by the predator cognitive abilities (Osorio
et al. 1999) or psychology (Speed 2000).
Phobia is one of the perspectives of predator psychology that should be important in the
study of Batesian mimicry; this is supposed to lower the willingness of the predator to
attack encountered prey (Speed 2000). A specific case of phobia is fear of an unknown
prey. This phenomenon was described as neophobia or dietary conservatism (see Marples
and Kelly 1999). Both of these perspectives describe a wariness of new, unknown prey, but
their principles are different. Neophobia is an immediate response that may subside rather
fast, while dietary conservatism is a stable long-term phenomenon (see e.g., Greenberg and
Mettke-Hoffman 2001). There are several experiments with various bird predators (zebra
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921
finch, domestic chick, robins or blackbird), which prove the neophobical reaction towards
unknown warning colours or odours (Marples and Roper 1996; Marples et al. 1998; Kelly
and Marples 2004, respectively). A computer simulation (Speed 2001) as well as an
experimental study (Thomas et al. 2003) proved that neophobia (or/and dietary conservatism) induced the spreading of an unfamiliar conspicuous form in the population. The
review of Marples et al. (2005) also emphasizes the key role of neophobia in the evolution
of warning signalization.
The predator’s reaction to a warning signalling prey could also affect another aspect of
animal cognitive abilities caused by the limited rate of information processing in the
animal brain. This constraint is called limited attention and it affects various components
of animal behaviour (Dukas 2002, 2004). In foraging processes, limited attention results in
selective attention to a specific (e.g., most abundant or most profitable) prey; this is more
effective than spreading the search for various prey types (Bond and Kamil 1999; Dukas
and Kamil 2001). This selective attending to the desired prey is called a search image
(Dukas 2002). The existence and principles of a search image have been experimentally
tested with several avian species (Great tits—Tinbergen 1960; chicks—Dawkins 1971 and
blue jays—Pietrewicz and Kamil 1979; Bond and Kamil 1999; Dukas and Kamil 2001).
Neophobia and search image work together, because the former causes the refusal of an
unfamiliar prey and the latter a preference for a desired (and familiar) prey. Therefore, if a
Batesian mimic arises in a population of unprotected palatable prey; Both the search image
and neophobia of a predator should lower the willingness to attack it. Lindström et al.
(2004) found that predators were more likely to refuse an unfamiliar warning coloured and
unpalatable prey as well as its Batesian mimics if an alternative cryptic and palatable prey
was more available. We suppose that the predators form a search image of the cryptic prey,
resulting in a lack of interest to other potential prey.
Nevertheless, if the predator prefers the ancestor prey and possesses its search image, its
reaction to a newly emerged Batesian mimic could be quite different. Forming a search
image may employ several prey features, such as conspicuousness (Blough 1989a; Dukas
and Ellner 1993), size (Blough 1989b) or shape (Blough 1985; Blough and Franklin 1985).
The colour is undoubtedly the most conspicuous characteristic of a prey, but other features
can be used for prey recognition as well. For example, insects provide several characteristics like body shape, length of legs or antennae, way of movement (e.g., Yamawaki 2000,
2003) or absence of repugnant odour (Roper and Marples 1997; Rowe and Guilford 1999;
Lindström et al. 2001). If a search image includes such features of a prey, a predator can
reveal the signal fake of a Batesian mimic and attack it. The newly emerged warning
coloured form will not spread in the population in such a case.
In the present study, we experimentally simulated the emergence of a warning coloured
Batesian mimic in the population of an edible prey. We used a real insect prey species,
which provides several signals to the predator (Guyana spotted roach—Blaptica dubia). It
was provided with a perfect colour pattern of a well protected model insect species, the red
firebug (Pyrrhocoris apterus; see e.g., Exnerová et al. 2003). Adult great tits (Parus major)
were used. These were caught in the wild as predators supposedly being familiar with the
model from nature (Exnerová et al. 2003).
We tested the following hypotheses:
H1 The cockroach mimicking red firebug is protected against predators unfamiliar with
the cockroach.
H2 The cockroach mimicking red firebug is less protected against predators familiar with
the cockroach (possessing a search image of it) than against unfamiliar ones.
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Materials and methods
Experimental prey
The red firebug, Pyrrhocoris apterus (L., 1758), has a red and black colouration that has been
proved to be a warning signal (Wiklund and Järvi 1982; Exnerová et al. 2003, 2007). It also
possesses a chemical defence based on short-chained alkanes and their derivatives, produced
by the repugnatory metathoracic glands (Farine et al. 1992). Firebugs for the experiments were
collected in the surroundings of České Budějovice (South Bohemia, Czech Republic) during
2004 and 2005. Groups of about 50 individuals were kept in the laboratory in glass jars
(15 cm in diameter). Dry linden tree seeds (Tilia cordata) and water were supplied ad libitum.
The Guyana spotted roach, Blaptica dubia (Audinet-Serville, 1838), is used as a
common feed for insectivorous animals. Similarly as with other cockroach species, it uses
chemical protection when endangered; it empties its stomach and stains the predator with
its contents. Cockroaches in our experiments used this strategy when trying to avoid
attacks of experimental great tits, but as they were fed mostly with carrots, the titmice were
not repelled by these excretions and considered Guyana spotted roaches palatable. In our
experiments, we used the second and third larval instar (8–14 mm in length equalling
firebug length), which are brownish with dark and pale spots. They were kept in a glass
terrarium (40 9 30 9 20 cm) at high densities. Fresh vegetables (carrot, beet root, and
potatoes), dry cat and dog foods and water were supplied ad libitum.
Both insect species were reared at 25°C under long-day (18 h light, 6 h dark) conditions. These two insect species are of comparable appearance, with similar body shapes
and movements. The main differences between the species are in the shape of the antennae
and legs, and in body posture.
The natural colouration of the experimental prey was changed using paper stickers
placed on its back, thus covering the body of the insect from above and partially from the
sides. Stickers are very useful in modifying the colouration of insects and do not influence
the insect’s behaviour. There were four experimental prey types (Fig. 1), as each of the
tested prey species could ‘‘wear’’ two types of stickers, either a cockroach or a firebug
pattern. The sticker patterns were made from printed photographs of both insect species.
Experimental predators
Adult great tits (Parus major L., 1758), caught with mist nets in the vicinity of České
Budějovice (South Bohemia, Czech Republic), were used as experimental predators.
Captures were conducted during 2004–2006, except during the breeding seasons
Fig. 1 Experimental prey types. From the left firebug with firebug sticker, firebug with cockroach sticker,
cockroach with firebug sticker, cockroach with cockroach sticker
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923
(May–July). Birds were kept in standard birdcages at lowered indoor temperature and
under outdoor photoperiod conditions. Birds were acclimatized to laboratory conditions
and food (sunflower seeds and mealworms or cockroaches, see ‘‘Trials’’) for one to 2 days
prior to the experiments. They were ringed and released immediately after the experiments.
Experimental equipment
The experimental cages were made from wooden cubic frames (0.7 m 9 0.7 m 9 0.7 m)
covered with wire mesh (2 9 2 mm) and with a one-way mirror as a front wall (see
Exnerová et al. 2003 for details). The cages were equipped with one perch, a bowl with
water, and a rotating circular feeding tray, containing six small cups (only one cup contained a prey item during each trial). The distance between the perch and the tray was
approximately 25 cm. The bottom of the cups was white. Standard illumination was
generated by a light source (LUMILUX COMBI 18 W, OSRAM) that simulates full
daylight spectrum (including UV radiation).
Trials
The 120 tested great tits were divided into two groups. The first group (80 individuals) was fed
mealworms during acclimatization in the laboratory. The other 40 individuals were fed
cockroaches. The food was supplied ad libitum in both groups, so that several tens of prey items
were eaten during the pre-experimental period. Subsequently, the birds from the first group
were divided into four groups of 20 individuals each. The first group was offered cockroaches
with cockroach pattern stickers. The second group was given cockroaches with firebug pattern
stickers. The third group was offered firebugs with cockroach stickers and the fourth group
was presented with firebugs with firebug stickers. The birds that were fed cockroaches
were divided into two groups of 20 individuals. The first was offered cockroaches with
cockroach stickers and the other group was presented with cockroaches with firebug stickers.
To avoid pseudo-replication, each individual bird was used for a single series of trials only.
Each bird was placed into the experimental cage before the experiment in order to adapt
itself to the new conditions, and was provided with food (mealworms or cockroaches
according to the experimental group) and water. Each bird was deprived of food for
1.5–2.5 h prior to the experiment to enhance its motivation.
The bird was assumed to be ready for the experiment as soon as it attacked the offered
prey immediately after introduction. Each experiment with an individual bird consisted of
a series of four trials, in which two control preys (mealworm or cockroach without any
sticker) and two experimental preys (stickered cockroach or firebug) were offered alternately, starting with the control prey (sequence control/experimental/control/
experimental). The control prey was used to check the bird’s motivation to forage, and the
trial ended after the prey was eaten. The trials with experimental prey always lasted 5 min.
We distinguished three possible results of each trial: (1) the experimental prey was
neither handled nor killed during the 5 min trial, (2) the prey was handled (touched, pecked
or seized) but not killed; (3) the prey was killed.
Statistical analyses
The numbers of birds that handled or killed at least one of the two offered experimental
prey were used to compare the experimental groups of predators (all tests were performed
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Evol Ecol (2009) 23:919–929
Table 1 Fisher exact test statistics comparing the frequencies of handled or killed insects between the first
and second group
First group
Second group
Sticker
pattern
Fisher exact test P
value
Predator search
image
Prey
species
Predator search Prey
image
species
Mealworm
Cockroach Cockroach Mealworm
Cockroach Firebug
0.0003
Mealworm
Cockroach Firebug
Mealworm
Firebug
Firebug
0.4075
0.3416
Mealworm
Firebug
Cockroach Mealworm
Firebug
Firebug
1
1
Mealworm
Cockroach Firebug
Mealworm
Cockroach Cockroach Cockroach
Cockroach
Sticker
pattern
Cockroach Firebug
Cockroach Cockroach
Handling Killing
0.0012
\0.0001 \0.0001
0.2308
0.0471
in Statistica 5.5, 1984–1999, ÓStatSoft, Inc.). A Fisher exact test was used to compare the
proportions of birds handling or killing the offered prey (Fisher 1922). The compared
groups are described in Table 1.
Results
All control mealworms or cockroaches were killed and eaten during the 5 min trials.
Warning function of the firebug pattern placed on unfamiliar palatable prey
(experiments with predators used to being fed mealworms)
Cockroaches were well protected from attacks of predators inexperienced with cockroaches
(as a prey) when disguised by the firebug pattern stickers. A significantly smaller number of
great tits handled and killed cockroaches with firebug stickers than those with cockroach
stickers (Table 1, row 1; Fig. 2). Cockroaches with the firebug stickers were protected
against being handled or killed equally as firebugs with the firebug stickers (Table 1, row 2;
Fig. 2). On the other hand, the cockroach stickers did not lower the protection of firebugs
from predator’s attack. The firebugs with the cockroach stickers were not handled nor killed
more often than the firebugs with the firebug stickers (Table 1, row 3; Fig. 2).
Warning function of the firebug pattern placed on familiar palatable prey (experiments
with predators used to being fed cockroaches)
Firebug pattern did not protect cockroaches from attack of predators experienced with
cockroaches as prey. The Great Tits used in these experiments handled and killed cockroaches more often when familiar with the cockroach (Table 1, row 4; Fig. 2). Since some
great tits inexperienced with cockroaches did not attack cockroaches with the cockroach
sticker, there was a significant difference in the killing, but not on handling, of this prey
between experienced and inexperienced great tits (Table 1, row 5; Fig. 2).
Discussion
Our experiments with predators used to the mealworms proved the protective function of
the firebug pattern placed on palatable as well as unpalatable insect prey. The edible prey
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Evol Ecol (2009) 23:919–929
925
Fig. 2 Number of great tits that handled or killed at least one of two offered prey. Under each column there
is (from above down): a prey species, b sticker type, c experience of predator
was protected equally from predator attack as the inedible one. These results support the
traditional theory of evolution of Batesian mimicry formulated in the nineteenth century
(Bates 1862 and Poulton 1890 ex. Komárek 2003). We have shown that prey protected by
no chemical defence or other defensive mechanism profits from mimicking other protected
species. This result is in concordance with conclusions of most experimental studies testing
the efficacy of Batesian mimics’ signalling using real unmodified prey and real predators
(Prudic et al. 2002; Kauppinen and Mappes 2003; Taniguchi et al. 2005; Nelson et al.
2006a, b or Sherbrooke and Westphal 2006).
On the other hand, our experiments showed that the unpalatable prey was protected
regardless of its colouration; the predators were able to identify the firebug even when
covered with a cockroach sticker. This result suggests that the red firebug’s warning
signalization comprises not only conspicuous coloration. It is possible that birds can detect
the scent of firebugs upon closer inspection, but a significant proportion of the experimental birds avoided attacking a cockroach stickered firebug without even approaching it.
Moreover, firebugs actively release a noxious substance only when disturbed (Socha 1993).
We can presume that other optical firebug signals, such as body posture, shape and length
of antennae and legs or whole body shape, could help the predator to recognize a firebug.
Our finding, that the protection of unpalatable prey remains even when warning colouration is artificially removed, is not unique. Exnerová et al. (2003, 2007) reached similar
conclusions with the red firebug and some passerine predators (e.g., marsh tits). Similarly,
in experiments with dragonflies as predators, wasps (as prey) lost a significant part of their
protection when deprived of their natural warning colouration (Kauppinen and Mappes
2003).
In contrast to great tits fed with mealworms, the birds used to being fed with cockroaches were not repelled by the warning colouration of cockroaches with firebug stickers.
None of the tested birds was misled by the colouration of the sticker.
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Evol Ecol (2009) 23:919–929
It is probable that the compared groups of birds possessed different prey search images
in their minds. The mealworm-diet birds were ready to attack prey of a worm-like
appearance. Therefore, they were not able to distinguish an edible cockroach under the
warning colour sticker. Birds used to the cockroach diet searched for a prey of cockroachlike appearance, since they were able to identify a cockroach under the sticker.
This result is unique in the context of current studies which test Batesian mimic efficacy
experimentally. The cause of this incoherence lies doubtlessly in the different design of our
experiments. In other studies, pre-trained predators had been used, but they were always
familiarised only with model species (Ritland 1991; Taniguchi et al. 2005; Darst and
Cummings 2006), not with the species from which the Batesian mimic evolved.
The most important conclusion of our experiments is the possibility that Batesian
mimics are less protected against predators possessing a search image of the unprotected
prey from which the Batesian mimics evolved. Such a limitation of Batesian mimicry
effectiveness should significantly decrease the profitability (and thereby the probability) of
the emergence of Batesian mimics. Most species have their more or less specialized
predator(s), which possesses its search image at a high probability. Restricting the protection of Batesian mimics could significantly help to clarify the low occurrence of pure
Batesian systems in nature.
Predators form a search image of frequently encountered (and therefore common) prey
(see e.g., Dukas and Kamil 2001). A Batesian mimic originating in a population of rare (in
space as well as in time) prey should be protected better than a mimic that emerges from an
at least locally common population. Fisher (1930) proposed the necessity of a low abundance of mimic species as a basic precondition for the function of Batesian mimicry. Our
results suggest that, for the evolution of a Batesian mimic, a low abundance of ancestral
species per se is necessary, regardless of the abundance of the model species.
Our results show that, in some circumstances, the warning colouration itself is not
sufficient for the protection of Batesian mimics. The search image of a prey is a multimodal phenomenon perceived by different senses (e.g., optical and olfactory—Chiszar
et al. 1985 or Gazit et al. 2005). Evolution should favour Batesian mimics differing from
their unprotected ancestors and resembling the model in as many features as possible.
Some observations from nature, describing behavioural Batesian mimicry (e.g., similar
way of movement in droneflies and hymenopterans—Golding and Edmunds 2000; Golding
et al. 2001; Srygley 2004 or the evidence of compound mimicry in ants and their spider
mimic—Nelson and Jackson 2006), are in concordance with this prediction.
Droneflies (Diptera, Syrphidae) are a group where pure Batesian mimics very often
occur; see Howarth et al. (2004). This can be explained by the theory of search image as
well. These flies are well known for their unusually high flight speed (see e.g., Nachtigall
2003). A predator possessing a search image of a fly (e.g., spotted flycatcher Muscicapa
striata searching for prey using the ‘‘sit and wait’’ foraging technique, see e.g., Davies
1977) then has only a minor opportunity to examine a mimicking dronefly elaborately and
thus confuses it easily with a wasp. However, a predator searching for prey using the
gleaning foraging technique (e.g., great tit in our experiments) could easily reveal a fake
behind the mimicking, because it has enough time for prey observation.
Acknowledgments We thank Dr. Keith Edwards for the language improvement. We thank the Academy
of Sciences of the Czech Republic (IAA601410803), Ministry of Education, Youth and Sport of the Czech
Republic (MSM 6007665801) and the Grant Agency of the Czech Republic (206/08/H044) for financial
support. Authors are licensed for catching and ringing birds (Bird Ringing Centre Prague No. 1004) and for
animal experimentation (Czech Animal Welfare Commission No. 489/01).
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927
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Dangerous prey recognition in avian predators

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