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El papel de cianuro en la oviposicin de Heliconius (Nymphalidae: Heliconiinae) sobre Passiflora (Passifloraceae)
The role of cyanide in oviposition by Heliconius (Nymphalidae: Heliconiinae) on Passiflora (Passifloraceae)
Vines of the Passifloraceae contain cyanide that deters most potential herbivores but not Heliconius spp.
(Nymphalidae: Heliconiinae). Despite their ability to cope with cyanide in Passifloraceae, Heliconius spp
may preferentially oviposit on leaves with lower cyanide concentrations, as young larval stages may still be
subject to toxicity. Leaf samples both with and without eggs were collected from Passiflora spp. from the
Monteverde Butterfly Garden and were assayed for cyanide concentration. There was no significant
relationship between cyanide concentration and oviposition (mean concentration with eggs = 6.04 + 9.41
(g/mg), mean concentration = 11.13 + 18.84 (g/mg); t = 1.15, p = 0.26, df = 42). Thus, ovipositing
Heliconius are either indiscriminate in their choice of leaves or are selecting leaves based upon other traits that are more important to determining egg survivorship or early larval development.
Los bejucos en Passifloraceae contienen cianuro que ahuyenta a la mayora de los herbvoros potenciales, pero no a Heliconius spp (Nymphalidae: Heliconiinae). A pesar de su habilidad para enfrentar al cianuro en Passifloraceae, Heliconius spp puede de manera preferencial poner huevos en las hojas con concentraciones mas bajas de cianuro; como las etapas larvales jvenes pueden ser todava susceptibles a la toxicidad. Se recolectaron muestras de hojas con y sin huevos de Passiflora spp del jardn de Mariposas en Monteverde y fueron analizadas para la concentracin de cianuro.
Text in English.
Tropical Ecology 2007
Ecologa Tropical 2007
t Monteverde Institute : Tropical Ecology
1 The role of cyanide in oviposition by Heliconius Nymphalidae: Heliconiinae on Passiflora Passifloraceae Zach Sheff Department of Biology, Indiana University ABSTRACT Vines of the Passifloraceae contain cyanide that deters most potential herbivore s but not Heliconius spp. Nymphalidae: Heliconiinae. Despite their ability to cope with cyanide in Passifloraceae, Heliconius spp . may preferentially oviposit on leaves with lower cyanide concentrations, as young larval stages may still be subject to t oxicity. Leaf samples both with and without eggs were collected from Passiflora spp. from the Monteverde Butterfly Garden and were assayed for cyanide concentration. There was no significant relationship between cyanide concentration and oviposition mea n concentration with eggs = 6.04 + 9.41 Âµg/mg, mean concentration = 11.13 + 18.84 Âµg/mg; t = 1.15, p = 0.26, df = 42. Thus, ovipositing Heliconius are either indiscriminate in their choice of leaves or are selecting leaves based upon other traits tha t are more important to determining egg survivorship or early larval development. RESUMEN Los bejucos en Passifloraceae contienen cianuro que ahuyenta a la mayorÃa de los herbÃvoros potenciales pero no a Heliconius spp. Nymphalidae: Heliconiinae. A pe sar de su habilidad para enfrentar cianuro en Passifloraceae, Heliconius spp. puede de manera preferencial ovipositar en hojas con conce ntraciones mÃ¡s bajas de cianuro; como jÃ³venes etapas larvales pueden ser todavÃa s usceptibles a la toxicidad. H oja s con y sin huevos fueron recolectadas de Passiflora spp. en el JardÃn de Mariposa s de Monteverde y fueron probada s para determinar la concentraciÃ³n de l cianuro. No hubo relaciÃ³n significativa entre la conce ntraciÃ³n de cianuro y ovoposiciÃ³ n la concentraciÃ³n m edia con huevos = 6,04 + 9,41 Âµg/mg, la concentraciÃ³n media sin huevos = 11,13 + 18,84 Âµg/mg. AsÃ, Heliconius n o discrimina en su elecciÃ³n de hojas o escoge las hojas basadas en otros rasgos que son mÃ¡s importantes a la supervivencia del huevo o el d esarrollo temprano larval . INTRODUCTION Heliconius butterflies Nymphalidae: Heliconiinae range from the southern United States through South America, reaching their highest density in the Amazon Basin DeVries 1987. Eggs are laid exclusively on vines in the family Passifloraceae, its larval host plant, commonly on leaves or meristems near the tip of a shoot Benson et al. 1975; Williams and Gilbert 1981. Almost all generalist herbivores are deterred from Passifloraceae because it contains noxious cy anogenic glycosides Benson et al. 1975. The specialization by Heliconius larvae on Passifloraceae, due to their ability to cope with the increased cyanide concentration, has resulted in a series of coevolutionary chemical, physical, and behavioral adapt ations in both Heliconius spp and Passifloracae Gilbert 1971; Benson et al . 1975; Smiley 1978; Williams and Gilbert 1981. The plant
2 has evolved more specific defenses against heliconiine oviposition to reduce herbivory: glands on leaves that mimic eggs, extrafloral nectaries to attract predacious ants, and irregular leaf shape to disguise the plant; to which the butterflies have responded in turn Gilbert 1971; Benson et al. 1975. As a result, there exists a coevolution between these two organisms. Mor e specifically I am investigating whether the female shows a preference for leaves with lower cyanide concentrations. The Â€toxic effectÂ states that compounds such as cyanogenic glycosides deter herbivory primarily through direct harm to organisms which in gest the compounds Adsersen and Adsersen 1993. However, under this model the time span between the consumption of the toxin and feeling its negative effects is too long for the herbivore to learn to avoid the plant, assuming it survives. Alternatively, A dsersen and Adsersen 1993 also propose that the Â€toxic effectÂ could function in concert with a Â€repellent effectÂ which deters animals from even initiating herbivory, thus reducing damage to both organisms and allowing for coevolution and, ultimately, c oexistence. The latter seems more appropriate for the instance of Heliconius and Passifloraceae because the Heliconius have chemoreceptors positioned on their forelegs and often inspect potential oviposition sites thoroughly before actually laying any egg s Benson et al. 1975; Williams and Gilbert 1981. Females may select a specific leaf for oviposition based on several different criteria Freitas et al. 1999. These include, looking for the presence of other eggs or egg mimic glands, testing the presen ce of cyanide, choosing leaves that are younger and softer, or choosing plants that are growing more vigorously Freitas et al. 1999. However, the choice to oviposit on a leaf must also account for circumstantial factors such as the presence of competiti on or parasites. If there is higher competition then Heliconius may choose leaves with more cyanide to avoid other herbivores; if there are parasites present then Heliconius may choose leaves with more cyanide to avoid parasites that are unable to withsta nd large amounts of cyanide. On the other hand, larvae may be particularly vulnerable to the effects of cyanide, which would lead to oviposition on leaves with lower cyanide concentration. Furthermore, the amount of cyanide in a leaf is regulated by seve ral factors including leaf age Copp and Davenport 1978; Futuyma 1983; Woodman and Fernandez 1991. Clearly, the relationship between Heliconius and Passifloraceae is a complex one deserving a closer look. I expect that, due to the vulnerability of Heli conius larvae, the butterflies will oviposit more frequently on leaves that are younger and have a lower cyanide concentration. The younger leaves should be easier for the larvae to eat because they have not yet toughened. Additionally, lower cyanide con tent is not as strenuous on the newly hatched larvae. The purpose of this study is to determine what, if any, connection exists between the cyanide concentration of leaves and oviposition. METHODS Study Site I performed this experiment at the Montever de Butterfly Garden in Monteverde, Costa Rica. Data collection was carried out from October 25 th to November 14 th during the fall of 2007. Leaf samples were collected from several vines in Heliconius garden and one vine in the Large Garden. The species o f Passiflraceae used was Passiflora biflora . The
3 species of Heliconius present in the gardens were H. sapho , H. melpomene , H. sara , and H. doris . Leaf Collection and Cyanide Determination I selected an equal number of leaves with eggs and without. Leav es were gathered from two individuals of Passiflora biflora in the Heliconius garden and one individual in the Large Garden . Eggs were all from the genus Heliconius, but were not identified to species. All leaves were of similar age; none had toughened b eyond the initial stage of leaf development and they were still unfolding or had just unfolded. Leaves that exhibited herbivory were excluded. Leaves were selected from new growth shoots of the plant within 15 cm of the tip. I weighed each leaf sample, composed of the entire sample leaf, and then placed it in separate vials, which were approximately five mL in size, to determine the concentration of cyanide. I crushed the leaf samples inside the vials and then added three drops of toluene. After this a strip of Watman No. 1 filter paper, one cm by five cm, was soaked in sodium picrate solution by dipping the paper into a small amount, approximately three mL, poured into a Petri dish. Once the filter paper had been dipped in the solution, it was gently passed against the edge of the Petri dish to remove any excess sodium picrate. The saturated paper was then suspended five mm above the leaf sample in each vial and secured with the lid. Then each sample was left in a dry box at approximately 33 Ã°Â° C for t wo hours. The filter paper begins yellow and in the presence of cyanide turns purple. It was removed and soaked in five mL of distilled water for 30 seconds. The samples were placed in a Sequoia Turner spectrophotometer, which had been zeroed using a cu vette containing five mL of distilled water that had a strip of filter paper saturated with sodium picrate Â‚ but not exposed to cyanide Â‚ soaked in it for 30 seconds, and the transmittance was recorded at a wavelength of 540 nm. Once all of the samples ha d been analyzed the concentration of cyanide was determined using a standard transmittance curve obtained from a previous student Morris 2005; however, I also created my own standard curve see Results. The concentration is given in micrograms of cyani de per mg of leaf. Standard Curve The standard curve for cyanide concentration was created using a serial dilution technique. I made an initial concentration by adding 2000 mg of potassium cyanide to two mL of distilled water. This created a concentrat ion of 1000 mg/mL. A serial dilution was performed by using a micropipette to put 0.2 mL of the original solution into 1.8 mL of distilled water to reduce each successive concentration by a factor of ten. In this manner concentration of 1000 mg/mL, 100 mg/mL, ten mg/mL, one mg/mL, 0.1 mg/mL, and 0.01 mg/mL were created. Once each concentration had been made approximately 100 ÂµL of each was placed in the same vials as were used for the leaf samples along with three drops of toluene. Then one cm wide by six cm long strips of Watman No. 1 filter paper were soaked in sodium picrate and secured five mm above the cyanide solution following the same procedure from above. The samples were then placed in a dry box at approximately 33 Ã°Â° C for two hours as before. After removing the samples from the dry box, the strips of filter paper were each soaked in five mL of distilled water for 30
4 seconds. The spectrophotometer was zeroed, using the same technique as above, and set to a wavelength of 540 nm. The transmitt ance for each five mL sample was recorded. Once the transmittances were recorded they were entered into an Excel spreadsheet with the corresponding concentrations and a curve was constructed plotting transmittance against concentration. RESULTS No tre nd between leaf cyanide concentration and oviposition was found in this experiment. The mean cyanide concentration for leaves that had eggs present was 6.04 + 9.41 Âµg/mg, and the mean concentration for leaves that did not have eggs was 11.13 + 18.84 Âµg /mg. While the mean cyanide concentration for leaves on which Heliconius did oviposit was lower than for those which did not contain eggs, the standard deviations were too large to extrapolate a reliable trend t test, t = 1.15, p = 0.26, df = 42; Fig. 1 . 0 5 10 15 20 25 30 35 no yes Eggs Present Concentration of Cyanide Âµg/mg FIGURE 1. Mean cyanide concentration Âµg/mg for Passiflora leaves with and leaves without Heliconius eggs. Standard deviation bars are shown. Mean value for leaves without eggs is 11.13 + 18.84 Âµg/mg, and mean value f or leaves with eggs is 6.04 + 9.41 Âµg/mg. The total number of samples collected was 44, consisting of 23 samples containing Heliconius eggs and 21 samples that did not. However, when an analysis of covariance was performed there was statistical sign ificance for the entire model ANCOVA, model: F = 2.95, p = 0.04, df = 43. This was due to a significant trend for the covariate, leaf weight, and for the combined effects of cyanide concentration and leaf weight ANCOVA, leaf weight: t = 2.09, p = 0.04, n = 44; combined: t = 1.98, p = 0.05, n = 44. The relationship derived from the combined effect of cyanide concentration and weight was that leaves with a higher cyanide concentration and larger size were less likely to contain eggs Regression, R 2 = 0 .53, p = 0.0002, n = 21; Fig. 2, while no reliable trend was present for leaves that did contain
5 eggs Regression, R 2 < 0.0001, p = 0.9047, n = 23; Fig. 3. Even though there was generally little variation in cyanide content, leaves that were heavier, an d by inference older, showed a higher concentration of cyanide than lighter, younger leaves Regression, R 2 = 0.16, p = 0.0068, n = 44; Fig. 3. Additional Results The standard curve that I constructed was not used to analyze any of the data because I di d not have high confidence in its accuracy. This is due to inadequate equipment in the lab to accurately create the small measurements needed in the serial dilution. 0 10 20 30 40 50 60 70 80 0 0.05 0.1 0.15 0.2 0.25 Weight g Concentration Âµg/mg With Eggs Without Eggs FIGURE 2. Weight g is plotted against cyanide concentratio n Âµg/mg and trendlines are compared from linear regressions for leaves with eggs y = 1.2296x + 6.1007, R 2 < 0.0001, p = 0.9047, n = 23 and leaves without eggs y = 331.24x Âƒ 9.8628, R 2 = 0.53, p = 0.0002, n = 21.
6 0 10 20 30 40 50 60 70 80 0 0.05 0.1 0.15 0.2 0.25 Leaf Weight g Concentration Âµg/mg FIGURE 3. A linear regression of all Passiflora leaf samples comparing leaf weight g to cyanide concentration Âµg/mg. y = 127.21x + 1.1567, R 2 = 0.1615, p = 0.0068, n = 44 DISCUSSION My initial expectation that Heliconius butterflies would preferentiall y oviposit on leaves with lower cyanide concentration and on younger leaves is partially supported by the results from this experiment. There is no significant relationship between cyanide levels of leaves that contain eggs and those that do not. While t he mean cyanide concentration for leaves lacking eggs is higher than the mean for leaves with eggs 11.13 + 18.84 Âµg/mg compared to 6.04 + 9.41 Âµg/mg, the standard deviation is much too large to extrapolate a reliable trend Fig. 1. However, there was a trend for leaves that did not contain eggs to generally have higher cyanide concentration and higher weight Fig. 2. The reason that this trend did not correspond to a trend of leaves containing eggs having less cyanide and lower weight could be that th e correlation came primarily from one parameter, but not both. This means that the weight, or age, of the leaf could be more important to the butterfly than the cyanide concentration. Alternatively, this could mean that Heliconius larvae can cope with to ugher leaves or higher cyanide content, but suffer when presented with both deterrents simultaneously. On the other hand, there is evidence that other adaptations by both Passiflora and Heliconius could be more important than the chemical defense afford ed by cyanogenic glycosides Gilbert 1971; Benson et al. 1975; Smiley 1978; Williams and Gilbert 1981. Gilbert 1971, citing the effectiveness of hook shaped trichomes at trapping and killing Heliconius larvae, suggested that physical adaptations by Pas sifloraceae represent a stronger deterrent for herbivory than its secondary compounds. Past experiments have shown that Heliconius have keen vision facilitating a strong image recognition system for locating potential leaves for oviposition. Not only has this precipitated further evolution in Heliconius , possessing the most advanced nervous system of any genus in Lepidoptera,
7 but it has also encouraged further adaptation in Passifloraceae, variable leaf shapes Benson et al. 1975. There is also evidence that leaf age contributes largely to choice of oviposition site Benson et al. 1975; Copp and Davenport 1978; Freitas et al . 1999. Taking into account the possibility that cyanide defense is not as effective as other defenses utilized by Passiflora and the fact that studies have shown that Heliconius oviposition is influenced by leaf age, it seems plausible that the cyanide content alone is not a determining factor for oviposition choice. Leaf age could have more implications to Heliconius larvae than j ust the toughness of the leaf. The physical defenses described by Gilbert 1971 may not be fully effective until the leaf has had sufficient time to develop. This may provide an even stronger advantage to ovipositing on young leaves. In Passifloraceae, younger leaves contain a greater amount of cyanide than older ones, possibly because they are of higher value to the plant than older ones and are particularly prone to herbivory because they have not yet toughened Futuyma 1983; Woodman and Fernandez 1991 . However, the leaves in my study exhibit the opposite trend, actually becoming more toxic as they grow older Fig. 3. This could be due to the fact that all of the leaves included in my study were of a young age, some weighing as little as 0.005 g, and the smallest had not been given enough time to become maximally toxic. In these extremely young leaves that generally show low cyanide content, the most critical criteria could be toughness. Ultimately, the role of cyanide is complex as are the interacti ons between Passifloraceae and Heliconius . A butterflyÂ„s choice of oviposition site seems to be governed more by physical characteristics of the leaf rather than the chemical composition of that leaf. However, more research into this area needs to be don e to ascertain the specific roles of cyanide in this interaction. Future Studies Future studies in this area should preferably include Passiflora spp. bred for higher and lower cyanide content to ensure a wider variation in cyanide concentration from which to draw trends. This strategy might provide greater insight into the role the cyanide plays in interactions with Heliconius by magnifying the natural gradient of cyanide experienced by the butterflies . My study suffered from a low variation in cyan ide content that did not allow me to draw any concrete conclusions about the role of cyanide in oviposition, and I feel that a wider range of concentrations would allow for more definitive answers in this area. I would also recommend conducting a study th at examines more closely the connection between leaf age, cyanide concentration, and frequency of oviposition to determine if cyanide or leaf age is truly the more integral component. This study could be conducted by using two groups of Passiflora : one wh ich contains plants of low cyanide variation and one which contains plants of higher cyanide variation. Both groups would be offered to Heliconius , separately and in isolation, and this would allow for a separation of whether Heliconius prefer leaves with more or less cyanide, and leaves that are tougher. ACKNOWLEDGMENTS Many thanks to Alan Masters for helping me devise and execute an interesting and dynamic project. I also thank Karen Masters for her statistical genius and ability to turn
8 one figure in to thousands, as well as, the staff of the Monteverde Butterfly Garden, especially Zach and Eli for answering my overly specific and often superfluous questions. Finally, thanks to Taegan McMahon for proofreading my paper at midnight and Pablo Allen for h elping me with the statistics. LITERATURE CITED Adsersen, Anne, Henning Adsersen. 1993. Cyanogenic Plants in the Galapagos Islands: Ecological and Evolutionary Aspects. Oikos. 673: 511 520. Benson, Woodruff W., Keith S. Brown, Lawrence E. Gilbert. 1975. Coevolution of Plants and Herbivores: Passion Flower Butterflies. Evolution. 294: 659 680. Copp, Newton H., Demorest Davenport. 1978. Agraulis and Passiflora I. Control of Specificity. Biological Bulletin. 1551: 98 112. DeVries, Phillip J. 1987. The Butterflies of Costa Rica and Their Natural History. Princeton University Press, Princeton, New Jersey, USA. Ehrlich, Paul R., Peter H. Raven. 1964. Butterflies and Plants: A Study of Coevolution. Evolution. 184: 586 608. Fraenkel, Go ttfried S. 1959. The Raison dÂ„Â…tre of Secondary Plant Substances. Science. 1293361: 1466 1470. Freitas, Andre V.L., Inara R. Leal, Sirayama O. Ferreira. 1999. Selection of Oviposition Sites by a Lepidopteran Community of a Trophic Forest in Southea stern Brazil. Biotropica. 312: 372 375. Futuyma, Douglas J. 1983. Evolutionary Interactions Among Herbivorous Insects and Plants. In: Coevolution, Douglas J. Futuyma and Montgomery Slatkin, ed. Sinauer Associates, Inc., Sunderland, MA, pp. 207 231. Gilbert, Lawrence E. 1971. Butterfly Plant Coevolution: Has Passiflora adenopoda Won the Selection Race with Heliconiine Butterflies? Science. 1723983: 585 586. Morris, Melissa. 2005 Herbivore Induced Defenses in Passiflora biflora . CIEE, Spring 2005. Smiley, John. 1978. Plant Chemistry and the Evolution of Host Specificity: New Evidence from Heliconius and Passiflora . Science. 2014357: 745 747. Williams, Kathy S., Lawrence E. Gilbert. 1981. Insects as Selective Agents on Plant Vegetative Morphology: Egg Mimicry Reduces Egg Laying by Butterflies. Science. 2124493: 467 469. Woodman, Robert L., G. Wilson Fernandes. 1991. Differential Mechanical Defense: Herbivory, Evapotransporation, and Leaf Hairs. Oikos. 601: 11 19.