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Oviposicin en la planta hospedera, Passiflora biflora (Passifloraceae), por heliconiine, en la presencia del imita huevos
Oviposition on the host plant, Passiflora biflora (Passifloraceae), by heliconiine, in the presence of egg mimics
There is a co-evolutionary arms race between butterflies from the subfamily Heliconiine and their host plants from the family Passifloraceae. Egg mimicry is one adaptation that Passifloraceae has evolved specifically to inhibit heliconiine ovipositions. The effectiveness of egg mimicry was tested in Passiflora biflora by modifying the natural egg mimic glands on the leaves, which are dull green in coloration, with two different colors which are closer in likeness to an actual heliconiine egg. The teal leaf treatment represents an early transitional phase of a Passiflora on its evolutionary pathway to creating a more realistic egg mimic, while the yellow leaf treatment mimics the coloration almost perfectly. Both yellow and teal enhancements to egg mimic glands on these leaves effectively deterred ovipositions on the new growth vegetation, yet the yellow treatments were more effective. This indicated that changes to the P. biflora vegetation could affect heliconiine oviposition even if the changes do not appear exactly like a heliconiine egg, but the closer in appearance an alteration is, the stronger the deterrent is.
Passiflora spp produce glucsidos de cianuro para prevenir la herbivora. La subfamilia de mariposas Heliconiinae (Nymphalidae) ha traspasado esta defensa con la habilidad de ingerir compuestos con cianuro. Una carrera armamentista co-evolucionara de adaptaciones y contra adaptaciones seguidas, en la cual se cree que Passiflora spp evoluciona una serie de defensas adaptativas, como mimetismo de huevos, forma de la hoja, y nectarios extraflorales, para combatir especficamente a estas mariposas.
Text in English.
Butterflies--Costa Rica--Puntarenas--Monteverde Zone
Mariposas--Costa Rica--Puntarenas--Zona de Monteverde
Tropical Ecology 2008
Ecologa Tropical 2008
t Monteverde Institute : Tropical Ecology
1Preferential oviposition by Heliconiinae (Nymphalidae) butterflies on Passiflora biflora (Passifloraceae) leaves with higher cyanide concentrations Phillip Burkholder Department of Chemistry and Biochemistry, Universit y of Tulsa ABSTRACT Passiflora spp produces cyanogenic glycosides to prevent herbivo ry. The butterfly subfamily Heliconiinae (Nymphalidae) has broken through this defense with the ability to ingest the cyanogenic compounds. A coevolutionary arms race of adaptations and counter -adaptations followed, in which it is believed that Passiflora spp. evolved a series of counter-adaptive defenses, lik e egg-mimics, leaf shape, and extrafloral nectarines, to specifically combat heliconiines. W hile sometimes overcoming these adaptations, heliconiines still consider them for oviposition. Additionally, the role of cyanide may also have an effect on oviposition. It has been suggested that while detrimental to larvae, cyanide provides protection that promotes oviposition. There are also numerous stud ies suggesting defensive and nutritional benefits o f CN when Heliconiinae is able to sequester cyanogenic c ompounds. Many times there are trade-offs in the defenses of young leaves, which might suggest that cyanide indicates fewer defenses. This study exami nes the role of cyanide (CN) concentrations in Passiflora biflora on ovipostion by Heliconiinae. Two studies were performed on cyanide preference. First, an ana lysis of cyanide concentration in similar leaves wi th and without eggs was conducted. Second, leaves had their cyanide concentrations artificially increase d with CN/methanol extract and were then monitored fo r oviposition. When analyzing the cyanide concentrations of similar leaves with and without e ggs, a trend of preferential oviposition on leaves of higher cyanide concentration was observed. There a lso seemed to be a two-fold difference, on average, between leaves with and without eggs, 0.50g and 0. 25g CN respectively. The second study showed a preference for leaves with CN/methanol extract to l eaves with extract alone and leaves with no alterat ion with eggs totaling nine, one, and one, respectively This study suggests the importance of cyanide concentration for oviposition by heliconiines. Thi s behavior might be explained by the protection pro vided from non-heliconiine herbivores or by possible nutr itional and chemical benefits associated with the sequestration of CN. Additionally, if trade-offs a re present, cyanide might indicate fewer non-cyanid e defenses. RESUMEN Passiflora spp produce glicosidos de cianuro para prevenir la he rbivora. La subfamilia de mariposas Heliconiinae (Nymphalidae) ha traspasado esta defen sa con la habilidad de ingerir compuestos con cianu ro. Una carrera armamentista coevolucionaria de adaptac iones y contra adaptaciones seguidas, en la cual se cree que Passiflora spp evoluciona una serie de defensas adaptativas, como mimetismo de huevos, forma de la hoja, y nectarios extraflorales, para combati r especficamente a estas mariposas. Mientras algu nas veces las mariposas sobrepasan estas adaptaciones c onsiderando estas plantas para ovipositar. Adicionalmente, el rol del cianuro puede tener un e fecto en la oviposicin. Se ha sugerido que en lug ar de destruir la larva, el cianuro provee una proteccin impulsando la oviposicin. Existen tambien numero sos estudios que sugieren beneficios nutricionales y de fensives del CN cuando Heliconiinae es capaz de tom ar compuestos cianognicos. Algunas veces existen comp ensaciones en la defensa de las hojas jovenes, lo q ue
2puede indicar menos defensas. Este estudio examena el rol d concentraciones del cianurao (CN) en Passiflora biflora en la oviposicin por Heliconiinae. Dos estudios fueron realizados en la preferencia del cianuro. El primero, un anlisis de la concentraci n de cianuro en hojas similares con y sin huevos. Segundo, se incremento artificialmente la concentra cin de cianuro en hojas con extractos de CN/methan ol que fueron monitoreadas para ver oviposicin. Cuan do se analizo las concentraciones de cianuro en hoj as similares con y sin huevos, existe una tendencia a preferir ovipositar en hojas con altas concentracio nes de cianuro. Tambien parece haber una diferencia, en p romedio, entre hojas con y sin huevos en concentraciones de 0.50g y 0.25g CN respectvament e. El Segundo estudio muestra una preferencia por hojas con el extracto de CN/etanol sobre hojas con solo el extracto y hojas sin alteracin, con un tot al de nueve, uno y uno huevos respectivamente. Este estu dio sugiere que la importancia de las concentracion es de cianuro en la oviposicin por Heliconiinae. Est e comportamiento puede ser explicado por la protecc in provista para herbivoros diferentes a estas maripos as o por posibles beneficios qumicos y nutricional es asociados con la secuenciacin del CN. Adicionalme nete, si hay una compensacin presente, el cianuro puede indicar menores defensas no relacionadas con el cianuro. INTRODUCTION Many plants have evolved secondary compounds to com bat herbivory, including Passiflora spp. vines which produce noxious cyanogenic glycosides that are converted to HCN during consumption (Adsersen and Adsersen 1993; Benson et al. 1975; Levin 1976). This cyanogenic defense deters most herbivo rous insects except for nymphalid butterflies in the subfamily Heliconiinae. Heliconiines have a similar cyanogen ic defense against predators, and are able to synthesize the c hemicals de novo (Engler et al. 2000; Feuillet 2004). These butterflies are able to inge st CN containing compounds, which has been suggested as a result of their defense against auto-toxicty (Benson et al. 1975). Thus, the Heliconiinae unlike most herbivores, overcame the CN defense of Passiflora spp. It is widely believed that this, in turn, caused Passiflora spp to evolve defenses solely against Heliconiinae egg-mimics, hairs, le af shape , which heliconiines have sometimes overcome. The series of adaptations and counter-adaptations has been termed a coevolutionary arms race (Benson et al. 1975; DeVries 1987; Ehrlich and Raven 1964; Futuyma 1986; Turner 1981). Female heliconiine butterflies consider many of th e adaptations of Passiflora spp. when ovipositing. For example, they avoid leaves w ith eggs or egg-mimics, as heliconiine larvae are cannibalistic (Gilbert and S inger 1975). Additionally, they prefer young growth that is easier for small caterpillars to consume (Coley and Aide 1991). While it has been shown that egg mimics are able to deter females from oviposition, Heliconiinae has developed tremendous eyesight to d istinguish the egg-mimics and have been known to probe them to test for authenticity ( Benson et al. 1975; DeVries 1987; Williams and Gilbert 1981). Additionaly, Heliconius sara will often oviposit in the presence of eggs or egg-mimics (Benson et al. 1975). Passiflora spp. has also changed its leaf shape in order to escape oviposition. Lea f shape is often irregular to avoid recognition and sometimes similar to leaves of comm on species to avoid detection (Benson et al. 1975). The female overcomes the changing leaf sha pe of Passiflora spp because she has chemoreceptors on her forelegs (Ben son et al. 1975), and the sensing of secondary compounds, like cyanogenic glycosides, is frequently used to recognize host plants when visual cues are unavailable (Ehrlich an d Raven 1964). Here, I investigated whether or not a female butter fly prefers to oviposit on leaves with higher cyanide concentration than those withou t. Leaves with more cyanide are less likely to be attacked by non-heliconiine herbivores may offer a higher nutritional reward
3 as CN contains nitrogen, which is generally limitin g in plant material, and provide precursors for the larval and/or adults own defense s. In addition, there might be tradeoffs, in that more cyanide may mean fewer other def enses affecting oviposition. This is especially true in young leaves, which have not yet developed persistent mechanical defenses that compete against secondary compounds d uring production (Koricheva et al. 2004). Higher cyanide concentration may deter potential co mpetition and predation, offering protection to the larvae. It has been dem onstrated that certain butterfly species, including heliconiine, oviposit preferentially for protection, even if it means forfeiting efficiency and nutrition (Karban and Agrawal 2002). For example, butterfly species Pieris napi (F. Pieridae) oviposits off its larval food plant t o avoid parasitoid wasps (Ohsaki and Sato 1994). Additionally, the heliconi ine species Dryas iulia has been observed ovipositing on adjacent plants, dead leave s, or other obects away from host plants to avoid predation by ants attracted by EFNs (Benson et al. 1975). Occasionally, higher cyanide concentrations have been shown to be detrimental to some species of heliconiine. For example, larval mortality of Heliconius erato increases significantly when fed high CN leaves (Hay-Roe and Nation 2007). So the question is why endure increased mortality for higher CN concentration. C yanide provides protection against both competition against non-heliconiine herbivores and mortality from large generalist herbivores and omnivorous mammals. Many non-helico niine herbivores cannot ingest CN and are unable to compete for larval food source s (Adserson and Adserson 1993; Benson et al 1975; Levin 1976). Whereas many large herbivores and omnivorous mammals are able to metabolize small quantities of cyanogenic compounds, their bitter taste often deters ingestion (Gleadow and Woodrow 2 002). Moreover, the absence of these animals might reduce direct harm on eggs or l arvae, which could occur from the consumption of leaves. A number of species in the Heliconiinae have develo ped the ability to sequester cyanogenic compounds for nutrition by exchanging th e nitrogen with a thiol group, thereby releasing nitrogen for its own use while de activating the noxious compound (Engler et al 2000; Nishida 2002). In addition, several studie s suggest sequestered CN can be used directly for chemical defense and that higher a sequestered concentration in the larval stage directly relates to the concentrat ion in the adult stage (Engler et al. 2000; Gleadow and Woodrow 2002; Karban and Agrawal 2002; Nishida 2002). Moreover, if reduced de novo CN synthesis translates to increased energy, then sequestering CN might allow energy to be redirected to larval growth and development. Therefore, I expect that female heliconiine butter flies will oviposit with greater frequency on leaves with higher cyanide concentrati ons to give their larvae access to more nitrogen for use in both growth and their own chemical protection. Additionally, if decreased larval growth and development from CN-rel ated toxicity occurs, I still expect to find a higher frequency of oviposition due to th e protective benefits that CN provides. However, if the detrimental effects lower frequency it would still indicate that CN concentration plays a significant role in ovipositi on. There is also the case as with Heliconius erato favorinus that neither suffers or benefits by CN (Hay-Roe an d Nation 2007). If there is indeed a trade-off in P. biflora defenses, then higher CN concentrations might indicate to the butterfly that there are fewe r defenses. Thus, a heliconiine like H. erato favorinus could potentially prefer a higher CN leaf.
4 MATERIALS AND METHODS Study Site Cyanide preference was analyzed using two studies. The first study compared concentrations of cyanide on similar leaves with an d without eggs, and the second study increased the concentration of CN on leaves and rec orded the frequency of oviposition. Both studies were performed at the butterfly-rearin g garden owned by Amabeliz Argueda in Cementario de Santa Elena de Monteverde, Costa R ica, at 1300 meters. The garden measured 18 x 24 x 8 m. Nearly a quarter of the ar ea composed of P. biflora to rear heliconiine larvae. Cyanide Concentration P. biflora leaves were collected throughout the garden. Eggs found on leaves were from one of several possible heliconiine species, based on reports of their host plant specificity (Benson et al. 1975; DeVries 1987; Smiley 1978). Possible specie s included: Heliconius sara fulgidas Heliconius charithonia charithonia Heliconius clysonymus montanus Dryas iulia monerata and Dione moneta poeyii When a leaf with an egg was located, the leaf was taken along with the nearest leaf of s imilar age and size. When possible, this leaf was taken from an adjacent vine, and when not possible, half of the time this leaf was the next youngest and half of the time this leaf wa s the next oldest. Age was determined by the distance from the meristem. If herbivore da mage was found on either leaf the pair was excluded to keep paired weights and sizes appro ximately equal. The pairs were analyzed for cyanide concentration. Whole leaf samples were first weighed and then crus hed to determine cyanide concentration. Cyanide was detected using the Sodi um Picrate Test (Seigler 1991) using three drops of toluene as the solvent. Each sample was allowed to react for ten minutes at room temperature (20-22C). The resulting picrate filter paper was soaked in three mL of distilled water for 30 seconds and the water analyz ed in a spectrophotometer (MRC, UV200-RS) at a wavelength of 540 nm. Recorded percen t transmittance was converted to absorbance, and the results were plotted against th e standard curve in order to obtain concentration. The standard curved was created usi ng serial dilutions of potassium cyanide and analyzed using the same conditions abov e, see sub-section: Standard Curve. Statistical analysis performed employed the paired t-test for significance. Additionally, mean cyanide concentrations of leaves with eggs vs. without were analyzed. Artificial Increase of Leaf Cyanide Concentration Leaf cyanide concentrations were artificially incre ased. A cyanide leaf extract was made with 10.5 g of ground leaf material from Passiflora collected in the garden and 20 mL of methanol. Young leaves, described in this study as within the first five open leaves of the meristem, were used to ensure CN was present, as yo ungest leaves have the highest cyanide concentrations (Hay-Roe and Nation 2007; We bber and Woodrow 2008). A
5 blank extract was made with only methanol. Leaves of P. biflora were tagged with a small green mark, slightly darker than natural leaf color, in one of three locations, bottom-left edge, bottom-right edge, or petiole, in dicating cyanide/methanol extract, extract alone, or control, respectively. Edge mark s were green and short to make them unlike eggs or egg-mimics, thus not interfering wit h oviposition. Petiole marks were short and along the underside to avoid detection. Young leaves were painted with cyanide/methanol extract and extract alone using a small paintbrush. The entire upperside of the leaf was painted with extract, and shortly after application no noticeable difference was observed between painted and unpaint ed leaves. Painted and unpainted leaves were randomly distributed within small patch es consisting of 50 to 100 leaves as well as on single vines throughout the garden. Lea ves that had eggs were excluded. Two separate paintings were conducted, one at the begin ning of the experiment and one eight days later. The leaves were monitored for oviposit ion everyday with eggs being removed when found. Leaves were recorded as either with or without eggs. Standard Curve The standard curve for cyanide concentration was cr eated using serial dilutions of potassium cyanide starting at 100 g/mL and ending at 0.1 g/mL. Dilutions were made with a micropipette. Technique was the same as lea f cyanide determination, but 100 m L of the potassium cyanide concentrations were tested in lieu of whole leaf. After obtaining readings from the spectrophotometer a cur ve of absorbance vs. concentration (g cyanide) was constructed. A blank utilizing al l methods without the addition of cyanide or plant material was used to zero the inst rument before beginning daily analysis. RESULTS Cyanide Concentration A total of 38 pairs of similar leaves with and with out eggs were analyzed. The observed trend is that Heliconiinae butterflies oviposited more frequently on leaves wi th higher cyanide concentrations (paired t-test, t = -2.43, d f = 37, p = 0.02). The average cyanide concentration for leaves of similar type with and w ithout eggs were 0.50 g/g leaf material and 0.25 g/g leaf material respectively ( figure 1).
6 Figure 1. Average cyanide concentration for P. biflora leaves with and without heliconiine eggs. Standard error bars are shown. 38 pairs of similar leaves were tested for a total of 76 analyses. Leaves with eggs were found by inspecting vines in a butterfly-rearing garden; leaves without eggs were of similar age and size on the nearest vine or adjacent to leaves with eggs, alternating o ne leaf older or younger. n r n r nrn
7 Cyanide Manipulation 237 leaves were monitored for oviposition. Of 101 leaves painted with cyanide/methanol extract, nine eggs were found. One egg was found o n the 102 methanol alone leaves, and one egg was found on the 34 control leaves (Figure 2). There was a non-random distribution with more eggs found on the leaves wit h cyanide/methanol extract than expected, indicating preference (g-test, g = 7.47, df = 2, p = 0.017). Figure 2. Frequency of heliconiine butterfly eggs on P. biflora leaves with cyanide/methanol extract, methanol extract alone, a nd control leaves with no alterations. r r
8 Standard Curve The following standard curve (figure 3) was constru cted to analyze percent transmittance data. There was a very high confidence in the accu racy of resulting function (R2 = 0.9805) used to obtain cyanide concentrations from the recorded absorbencies. Figure 3. Cyanide concentration standard curve for testing Passiflora leaves (y = 0.0325Ln(x) + 0.1722). The standard was a solution of potassium cyanide. DISCUSSION This study shows that heliconiine butterflies prefe rentially oviposit on P. biflora leaves with higher cyanide concentrations. Unaltered P. biflora leaves with eggs had higher CN concentrations, on average, than similar and adjace nt leaves. Likewise, leaves with artificially increased CN were chosen more often by ovipositing heliconiines. These trends indicate that heliconiine butterflies choose to lay eggs on more highly concentrated leaves. Heliconiine butterflies seem to prefer leaves with more cyanide for oviposition. There are many benefits that might be associated wi th higher CN concentrations. Even if CN-related toxicity occurs in larvae, the added pro tection provided might still promote oviposition. Because cyanogenic glycosides contain nitrogen, which is often limiting in
9 plant material, there are possible nutritional bene fits connected with more CN. Additionally, CN could provide the precursors to th e heliconiines own defense. Because of potential trade-offs, more cyanide might indicat e fewer defenses, in which case Heliconiinae might oviposit more frequently even if there are no direct benefits. One of the first benefits associated with higher C N is the added protection from herbivores that could possibly harm the larvae by f ood depletion or direct damage. Cyanide acts as a deterrent against most forms of n on-heliconiine herbivory, including herbivory from omnivorous mammals (Adsersen and Ads ersen 1993; Benson et al. 1975; Levin 1976). Many of these non-heliconiine herbivo res are unable to ingest CN so they cannot compete for larval resources. Additionally, they are able to sense, or smell, cyanide, which indicates unpalatable or dangerous l eaves (Ehrlich and Raven 1964). Large herbivores and omnivorous mammals, which are able to ingest CN in small quantities, are deterred by the bitter taste of cya nogenic compounds (Gleadow and Woodrow 2002). Large herbivores can harm larvae an d eggs by the removal of whole leaves during feeding. With higher cyanide concent rations, leaves might have a more prominent taste and smell thereby reducing non-he liconiine herbivory. However, some heliconiine larvae are vulnerable to CN-related tox ins. Studies on Heliconius erato showed that ingestion of high levels of cyanogenic glycosides increases larval mortality (Hay-Roe and Nation 2007). Nevertheless, this may not deter females from ovipositing on leaves with high cyanide. In effect, this may s ubstantiate the trend as numerous studies indicate that several species of butterfly, including those in Heliconiinae, forfeit nutrition and larval growth efficiency for protecti on (Benson et al. 1975; Karban and Agrawal 2002; Ohsaki and Sato 1994). Thus, it is p ossible that the butterflies in the garden benefit from the higher CN leaves and choose them for oviposition even if it causes them some harm. Alongside added protection, the garden heliconiine s could have chosen higher CN leaves for the purpose of nutrition or chemical def ense. Although heliconiines are known for their de novo synthesis of CN (Engler et al. 2000; Feuillet 2004), at least some species have more recently been shown to sequester CN from their Passiflora host plants (Gleadow and Woodrow 2002; Karban and Agrawal 2002; Nishida 2002). If this is the case for the heliconiines in the garden, selecting high CN leaves may mean better chemical protection for the developing larvae. Fur ther, if de novo synthesis is facultative, high CN leaves may save energy that can be diverted to growth. Engler et al. (2000) explored the effects of cyanide on Heliconius sara and discovered, for the first time, that it was able to metabolize the cyanogenic glycosides deactivating it while at the same time releasing nitrogen for nutritional use. While H. sara was not the only heliconiine present in the garden, the other species may also gain nutr ition from CN. Thus, higher CN leaves may offer a leaf of higher nutritional value. The use of plant-derived cyanogenic compounds by Heliconiicae creates an interesting paradox for host plants like P. biflora P. biflora has attempted to deter heliconiines from oviposition through egg-mimics an d extrafloral nectaries (EFNs), but research, however, has shown that heliconiines are not always deterred by these modifications (Benson et al. 1975; DeVries 1987; Feuillet 2004; Williams and Gil bert 1981). Furthermore, the young leaves of Passiflora vines are more susceptible to herbivory (Coley and Aide 1991) as well as more hig hly affected by trade-offs for defense. Young leaves have not yet developed persi stent mechanical defenses, such as
10 toughness, pubescence, or egg-mimics, all which com pete against secondary compounds for resources (Koricheva et al. 2004). Thus, young leaves with more cyanide might indicate fewer other defenses. This might explain the trend in the garden for higher CN preference, especially if the butterflies function like H. erato, as cyanide itself also benefits larval development. Nonetheless, there ar e butterflies that neither benefit nor suffer from CN in any studied concentration (Hay-Ro e and Nation 2007). These butterflies might also prefer higher cyanide leaves given the possible reduced defenses associated with trade-offs. However, some species of Passiflora have solved the problem of multiple defenses and limited energy pools. P. adenopoda has developed hooked trichomes, which provides an absolute defense against butterfl y larvae (Gilbert 1971). Currently only three species of Passiflora have developed this modification and it has been suggested that this is the next step for PassifloraHeliconiinae coevolution (Gilbert 1971). If hooked trichomes provide an absolute def ense against damage from larvae, a trade-off would only need to occur between cyanide and pubescence as long as cyanide alone was powerful enough to deter other potential herbivores. Currently, though, most Passiflora vines do not have hooked trichomes and are unable to reduce CN as a defense because it is needed for non-heliconiine herbivory. Additionally, because Passiflora may always use CN as its main generalist herbivory defe nse, it will continue to play an important role in Heliconiinae oviposition. Future Studies Having seen trends in preferential ovipostion on le aves with higher cyanide concentration (this study) and the deterrent effect of egg-mimics (DeVries 1987; Williams and Gilbert 1981), a suggested study would be the artificial in crease in cyanide concentrations on leaves with egg-mimics via extract or other method in order to compel Heliconiinae to oviposit on leaves with egg-mimics rather than thos e without. This might help to better understand which deterrent impacts oviposition more In addition, further studies could improve upon this study by utilizing known cyanide concentration manipulations in order to discover optimum cyanide concentrations for ovip osition. A more telling approach to this study would be to monitor every leaf available for oviposition rather than small portions. If cyanide does play a significant role in oviposition, this study would possibly have more pronounced trends, as butterflies would a lmost always be faced with a choice between painted and unpainted leaves. In regards t o the metabolic developments of H. sara studies similar to Engler et al. (2000) should be performed on many species of Heliconiinae to further understand their metabolism as well as learn how common it is among this subfamily of butterflies. ACKNOWLEDGEMENTS I would first like to thank Amabeliz Argueda for th e use of her garden. Without it, my research would have been much more difficult to conduct. I would like to thank the Estacion Biologica de Monteverde for providing me space to accomplish my goals. I would like to thank Alan Masters for his guidance throug h the project and his help with the spectrophotometer Thanks to Karen Masters for helping me determine my needed statistical analyses and to Pablo Allen f or walking me through them. Many thanks to Moncho Caldern for all the help translating my abstract.
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