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Leaf choice in Dryas iulia (Nymphalidae: Heliconiinae): cyanide content and toughness Ashley Arthur Department of Genetics, University of Wisconsin Madison ABSTRACT Vines in the Passifloraceae synthesize cyanogenic glycosides that deter general herbivo res, but Heliconiinae butterfly larvae such as Dryas iulia have overcome this and utilize Passiflora leaves as larval food. Ovipositing adult females and larvae may access the suitability of leaves caused by various plant defenses such as cyani de content and leaf toughness D. iulia adult females show no preference in cyanide content (9.01 g 28.3, 5.77 g 12.6) or toughness (238.67g 78.4, 266.58g 123.1) for ovipostion, yet larvae prefer leaves with a significantly lower cyanide content (9.01 g 28 .3, 0.47 g 0.51) then the average available leaf but average toughness (238.67g 78.4, 227.23g 80.7). This indicates that larvae are assessing plants to maximize fitness and D. iulia ovipositon is determined by more factors then simply Passiflora leaf cyanide content and toughness. RESUMEN Lianas en la famili a Passifloraceae sintetizan glucosas de cianuro que disuaden herbvoros, pero larvas de la subfamilia Heliconiinae como Dryas iulia pueden comer las hojas de Passiflora Es posible que las hembras adultas y las larvas puedan evaluar la presencia de varias defensas en las hojas como cianuro y grosor. Las hembras de D.iulia no muestran preferencia en el contenido de cianuro (9.01 g 28.3, 5.77 g 12.6) o grosor (238.67g 78.4, 266.58g 123.1) para la oviposicion (t=1.02; p=0.307; df=67), aun as las larvas prefieren hojas con significativamente menor contenido de cianuro que las hojas promedio (9.01 g 28.3, 0.47 g 0.51) y grosor promedio (238.67g 78.4, 227.23g 80.7) Esto indica que las larvas estn evaluando las plantas para incrementar el xito reproductivo y la oviposicion de D. iulia esta determinada por ms factores que simplemente el contenido de cianuro y el gro sor de Passiflora. INTRODUCTION Vines of the family Passifloraceae synthesis cyanogenic glycosides to deter general herbivores (Seigler 1991, Benson et al.. 1975) and Heliconiinae is a diverse neotropical subfamily of that has species adapted to overcome s ome of the defenses of Passifloraceae. Passiflora shows specific adaptations to deter ovipositon and herbivory of these butterflies and larvae (Gilbert and Singer 1957, Freitas and Brown 2004). Heliconiinae adult females avoid direct Passiflora defenses, s uch as predacious ants attracted by extra floral nectarines (EFNs) that eat larvae and eggs on the leaves, by ovipositing not on leaves, but meristems, tendrils and the leaves of plants adjacent to their host Passiflora (Flemming et al 2005, Benson et al 1975). Females also have chemoreceptors on their forelegs to help determ.ine cyanide content of Passiflora (Benson et al.. 1975). Some plant species have altered leaf morphology to confuse females from ovipostion on the correct plant (Hae Roe and Nation 2006). Passiflora plants of various species also employ
trichomes, or small hooks, which trap larvae, prevent them from crawling, and cause starvation (Gilbert 1983). Additional physical cues to deter herbivory and ovipostion include stipules, glands, a nd egg mimics; the female butterflies are less likely to oviposit on plants that already have butterfly eggs or mimics (Futuyma 1983). The interaction has been described as a coevolutionary arms race (Spencer 1988; Ehrilich and Raven 1964) and the battle b etween species in this system is ongoing as butterflies adapt to their host and plants evolve defenses to escape herbivory and oviposition. Passiflora vines have a tradeoff between their herbivory defenses and growth and reproductive success (Coley et al. 1985). Allocating resources to secondary compounds may reduce overall plant fitness, (Seigler 1991) but herbivore defense is crucial to avoid herbivory especially in the tropics where the incident of herbivory is great (Price 1995). Plants have tradeoffs to maximize defense and use limited resources efficiently. The most effective defense a plant can employ against most herbivory is tough, nutrient poor tissue and toughness is usually a factor of age (Coley and Aide 1991). Passiflora show varying degrees o f the production of cyanogenic compounds; young leaves have much higher concentrations and this trend decreases with leaf age and toughness in most species (Benson et al. 1975, Hay Roe and Nation 2004). Although younger leaves typically have more cyanide, some Passiflora species invest less energy and have smaller concentrations of cyanide while others invest more energy in secondary compounds and have much higher concentrations of cyanide. High cyanide plants have higher rates of larvae and pupae (Hay Roe and Nation 2004) and cyanogenic glycoside digestion reduces growth rate, even in Heliconiines (Gilbert 1983). Heliconiinae larvae do not avoid cyanogenic compounds, however, they eat assiflora leaves and have gut enzymes to break down volatile HCN as well as sequester valuable nitrogen (Engler et al. 2000). In the larval stage Heliconiinae accumulate cyanide as a defense mechanism to avoid predators by being unpalatable (Brower 1964). They seek a balance to between obtaining sufficient cyanogenic glycoside s to be toxic to their predators and avoiding self poisoning or reduced growth rate (Gilbert 1983). Dryas iulia (Heliconiinae) has several host species of Passiflora including Passiflora biflora P. biflora vines have altered leaf morphology, with several leaf morphs on the same vine. The morphs of P. biflora deceive adult females and can prevent them from laying eggs. P. biflora also has EFNs on new growth to attract ants (Durkee 1983). P. biflora lack trichomes and egg mimics that are other common Pass ifloraceae adaptations. D. iulia females lay eggs singly on leaf tendrils (Benson et al. 1975). Larvae are often cannibalistic and eat eggs and other larvae (Gilbert and Singer 1975). Once hatched, larvae eat leaves until they pupate and will travel for an extended time to locate an appropriate leaf (Benson et al. 1975). Heliconius spp, like D. iulia seems to make careful assessments of oviposition sites (Benson et al. 1978). They use chemoreceptors, leaf shape, presence of other eggs and presence of EF Ns to determine suitability (Benson et al. 1975). Larvae may also determine the suitability of leaves in terms of cyanogenic glycoside content and other nutritional factors such as cellulose (toughness) and nitrogen (Gilbert 1983). I hypothesize that leav es of P. biflora will show a trade
off between defense mechanisms, and both D. iulia adult females and that larvae will assess this trade off to optimize success of oviposition and foraging. MATERIALS AND METHODS Study Site My experiments were conducted i n the Monteverde Butterfly Garden in Monteverde, Costa Rica. This is located at approximately 1400m. My study was conducted in Garden 2, which is a covered garden meant to represent the forest canopy. Leaf Choice In order to study the relationship betw een larval leaf preference and maternal oviposition preference, I first needed to survey Passiflora leaves for characteristics of defenses. I examined both cyanide content and toughness. I randomly collected 60 leaves of a large vine in the Monteverde bu tterfly garden. I tested cyanide content and toughness in each of these leaves using the Sodium Picrate Test and leaf penetrometer, respectively (see below). I used these vine leaves as a baseline to compare the other groups. To determine larval leaf pref erence I located D. iulia larva foraging on P. biflora leaves. For each larva I found, I took a leaf sample and moved the larva to a different garden to avoid re sampling the preference of that larva. These leaf samples were analyzed for cyanide content and leaf toughness. In the same garden where larvae were sampled, I found D. iulia eggs on leaf tendrils. There were approximately 15 butterflies of this species in the garden, but the number of females was unknown. I examined the vines for eggs and sam pled leaves adjacent to tendrils where females had deposited an egg and marked the leaf to avoid re sampling. I tested these leaf samples for cyanide content and toughness. The final component to my experiment was to examine if larvae moved to leaves ba sed on leaf toughness or cyanide content. I put each of ten larvae on a random leaf tendril of its own small (10 15 leaves), potted P. biflora vine in the plant nursery and recorded the placement. After 48 hours returned and I sampled the leaf they were fo und on for cyanide content and toughness and sampled the cyanide and toughness for the leaf adjacent to the tendril they began on. The Sodium Picrate Test Protocol In order to determine the concentration of cyanide in the leaf material I used the Sodium P icrate Test (Seigler 1991). To make Picric acid test solution I dissolved 5 g of sodium bicarbonate and .5g of picric acid in 100mL of water and kept the solution in a brown glass bottle due to light sensitivity. To prepare picrate test strips I soaked 1c m x 6cm strips of filter paper in picrate solution and let excess liquid evaporate. I added 0.055 g of leaf material to the bottom of a small
glass vial and used a stirring rod to crush the material with 4 drops of toluene as a solvent. Then I suspended 3 cm of a picrate strip in the vial with a rubber stopper and put the vial at 37C for 60 minutes. After incubation, I removed the strips and rinsed them in a test tube with 5mL of water for 30 seconds. The picrate paper turns from yellow to red in the p resence of cyanide. I put the solution in a cuvet and read in an MRC UV 200 RS Ultraviolet and Visible spectrophotometer at 550nm and recorded transmittance. Transmittance was converted to absorbance using the formula absorbance = log(transmittance/100). The blank was prepared using the same procedure without a leaf sample. To interpret the absorbance measured with the leaf concentration of cyanide I used a published standard curve of absorption (Burkholder 2008). This was prepared by creating serial dilu t micropipette. The micrograms of cyanide per gram of leaf were determined from the slope of the line of the curve y=0.0284ln(x)+0.1665. The line cannot intersect wi th zero due to the of cyanide. Measuring toughness In order to measure the leaf toughness of my samples I used a leaf penetrometer. I put each leaf sample cent ered in between two metal plates with a 2.0mm hole in the center. I used a plastic plate with a 1.1mm diameter rod and set it on top of the sample, avoiding taking measurements at a vein. I very slowly added water to a graduated cylinder which was atop t he plate until the leaf was punctured. Then I weighed the water and graduated cylinder used to puncture the leaf and recorded the mass in grams as the measure of toughness (Image 1). RESULTS The mean cyanide concentration per gram of leaf material in a sur vey of P biflora leaves in the groups were statistically different (A NOVA: F=3.54; p=0.0447; df=2). Cyanide concentration of vines leaves 9.01 g 28.3 were higher then larval leaves 0.47 g 0.51 (t=2.49; p=00.015; df=71). This shows larva e are choosing leaves with lower cyanide concentrations The difference in average leaf toughness between leaves in the garden (238.67g 78.4) and leaves with larvae foraging (227.23g 80.7) was not significant (t=0.498; p=0.62 df=71). This indicates larvae are not choosing leaves based on toughness (Fig 1).
Figure 1. Average cyani de concentration and toughness of a sample of P. biflora leaves and leaves D. iulia larvae were found foraging on. Larvae in the garden were found on leaves of less cyanide concentrations and equal toughness then the average leaf available. Cyanide concen trations of vines leaves (9.01 g 28.3) were higher then leaves with eggs 5.77 g 12.6 but the trend was not significant (t= 1.02, p=0.31; df=67). The difference in average leaf toughness between leaves in the garden (238.67g 78.4) and leaves with eggs on adjacent tendrils (266.58g 123.1) was not significant (t=1.02; p=0.31 df=67). This indicates that ovipostion is not determined by cyanide concentration or toughness (Fig 2).
Figure 2. Average cyanide concentration and toughness of a sample of P. bi flora leaves and leaves with D. iulia eggs on adjacent tendrils. Ovipositon sites were not a factor cyanide concentration or toughness. The average cyanide concentration in a leaf adjacent to an egg was higher then the average leaf being foraged on by a la rva, but the trend was not significant (F=1.8; p=0.078, df=28), nor was toughness (t=1.42; p=0.167, df=28). In the larval preference experiment seven out of ten of the larvae moved to a different The average start leaf cyanide concentration (0.19 g 0.17) was not different than the leaf concentration they were found foraging on after the trial (0.3 g 0.57; t =1.204; p=0.259), nor was the toughness (225.05 g 115.5, 207.73 g 90.67; t=1.298; p=0.227), (Fig 3).
Figure 3. Larval preference of cya nide content and toughness. The first measurment is the characterists of the leaves larvae were placed on, the second is leaf choice after 48 hours. Larvae did not prefer a significantly different leaf from what they started in respect to cyanide content or leaf toughness. maximum toughness in grams necessary to puncture the leaf with a penetrom eter was 402.50g and the minimum was 55.00g. A linear regression of CN and toughness indicates no trend between toughness and cyanide content (y= 0.0907x+30.64, R 2 =0.063; p=0.65), (Fig 4).
Figure 4. Of a survey of leaves in the garden, toughness is not significantly correlated with [CN]. DISCUSSION The data presented here reveals that the cyanide content of P. biflora impacts larval foraging decisions. The data also suggest that cyanide concentration or tougness do not seem to be the only factors that D iulia assess when making oviposition decisions. Finally, I found no clear trend between toughness and cyanide concentration. This study shows that D. iulia typically forage on leaves in the lower range of cyanide concentration of all the leaves availabl e to them. Although some species of Heliconius, including D. iulia, can sequester CN to be unpalatable and metabolize it to release nitrogen in a useable form (Gleadow and Woodrow 2002) there are advantages foraging on leaves of lower CN concentration. H eliconiinae larvae have de novo synthesis of CN (in addition to their ability to sequester cyanide) and a lower concentration of cyanide is less of a stress to digest (Engler et al.. 2000). Foraging on leaves with a low CN concentration may be a way for t his species to prevent CN related toxicity due a limit on digestive capability cyanogenic glycosides Possible explanations for D. iulia leaf choice are: because P. biflora is not the favored host of D. iulia this species may be less efficient at metaboli zing P. biflora specific cyanogenic glycosides, or interspecific competition occurs on the vine such that niches are partitioned across leaves with different cyanide concentrations. If there are niches based on plant defenses, it is possible other
species are more specialized or more competitive to occupy and forage on the cyanide rich leaves of the vine. Adult females make ovipostion decisions that will maximize success of offspring (Benson et al. 1975). This study found no significant trend in oviposit on sites and cyanide content or toughness although the average leaf adjacent to an egg was lower in cyanide concentration then randomly sampled vine leaves. Cyanide content may play a role in ovipostion, especially in light of Heliconius having chemorecep tors on their legs; however, is not the only factor. Likely determinates of ovipostion are the presence of nearby eggs or larvae, intact nature of nearby leaves, vine density and tendril accessibility, EFN, trichomes, pubescence and other factors. Amount s of light or herbivory could alter the distribution of Heliconius eggs. Toughness is unlikely to affect ovipostion as this species lays eggs on tendrils, not leaves. Overall, ovipostion cannot be explained by only the level of cyanide or toughness. The di fference in cyanide content between leaves with eggs on nearby tendril and leaves with larvae on them was not statistically significant, nor was toughness although there was a trend to lay eggs on leaves with higher concentration. Leaves with eggs had CN c oncentrations more like average vine leaf concentrations than leaves with larvae. One possible explanation of the trend is that oviposition on leaves with CN concentration favored by larvae would have a greater chance to have a larva on them (or nearby) a nd therefore increase the likelihood of an egg being eaten by a cannibalistic larva. Another explanation is that ovipositing females need to lay eggs adjacent to leaves in an acceptable range of CN concentration but are not as selective as larvae and the a verage leaf is suitable. Previous studies have illustrated the trade off between cyanide content and toughness in Passiflora species (Burkholder 2008, Coley et al.. 1985). My study shows a wide range of toughness but no correlation between cyanide content and toughness (see Fig 4). This could indicate that while this particular bunch of vines or species of Passiflora may reduce cyanide production as the leaf toughens, the decrease of CN is small or at a variable rate between leaves. Another possibility fo r why my study shows no correlation between CN and toughness could be that these vines are in a garden with high herbivory. In the Butterfly Garden, multiple species and many individuals eat P. biflora and cause a lot of leaf tissue damage. High herbivory reduces the photosynthetic capability of the plants and with less resources the plant may be limited in its ability to properly synthesize adequate cyanide for defense of all its leaves. When assessing the effect of toughness on larval preferences, a typi cal prediction is that they will choose younger, softer leaf tissue because cellulose is energy expensive to digest. Upon determining D. iulia preference for leaves with less cyanide I expected them to be adapted to tougher leaf tissue with higher cellulos e and lower nutritional value due to the previously determined tradeoff. While the foraging habits of D. iulia show their preference of lower amounts of cyanide, they do not eat tougher leaves. This is likely due to the options they have in their environm ent; the leaves with less cyanide are not necessarily tougher and therefore larvae chose leaves with low cyanide and average toughness. If D. iulia prefer lower concentrations of CN and they are not forced to eat very tough leaves they have a competitive a dvantage against
other species of a relative increase in growth rate as cyanide and cellulose are both developmental deterrents (Gilbert 1983). When I selected larvae and put them on random leaves to measure their food choice after 48 hours I saw no clear trend in preferences. Seventy percent of the sample (n=10) changed from their original start place. Larvae of this group did not always move to a leaf with less CN. This could be because larvae in the garden had more than 48 hours to forage and find an ideal leaf. Alternatively, it may be that there is a range of acceptable leaves and they stop searching once in that range. Another factor of this experiment is that the small plants were much more open and exposed compared to the vine in the garden and th ey had different abiotic factors. The small sample size makes assumptions difficult to draw with confidence but this may suggest that cyanide concentration is not the only factor of larvae foraging. Heliconiiae are dependant on Passiflora and cyanide conc entration seems to be an important contributor to leaf choice in D. iulia larvae Ovipostion sites are important for ensuring egg survival and maybe even early larval choices. More mature larvae as studied here showed leaf preference other then ovipositio n sites suggesting oviposition site is more likely for egg survival and larvae can assess their own food to maximize fitness. Ovipostion site suitability is not determined by cyanide concentration or leaf toughness, and more likely factors of other plant d efenses such as EFNs and egg mimics. In summary, Heliconiiae are adapted and dependent on their Passiflora host plant despite adaptations the plant shows to deter herbivory. Future Studies Future studies could determine the effects of higher cyanide on gr owth rate and survival in Dryas iulia to see if it is less adapted to metabolize cyanogenic glycosides. An experiment comparing this species of Heliconius to others with time it can survive in a cyanide kill jar will show if this particular species is les s efficient at metabolizing cyanide and would explain its choice of leaves with less cyanide. Additionally, a future experiment could study the factors that determine ovipostion if cyanide and toughness do not. This could include using other plant defens es such as egg mimics, EFNs to determine the occurrence and success of eggs in reference to these other plant defenses. ACKNOWLEDGEMENTS I would like to thank the Monteverde Butterfly Garden for allowing me to conduct my experiments there and use the plant s and butterflies. I would especially like to thank Julio, the gardener for his tremendous insight about my experiment and Marvin and Anna Luisa for all help and accommodations as well. I would also like to Yimen Araya and Moncho Caldren for help with s tatistics questions and my resumen Finally, I would like to thank Alan Masters for his help with my experiment at every step from designing the experiment, running chemical tests and running and understanding my statistical tests and my data significance.
LITERATURE CITED BENSON, W.W. K.S BROWN, AND L.E GILBERT. 1975. Coevolution of Plants and Herbivores: Passion Flower Butterflies. Evolution 29: 659 680. BENSON, WOODRUFF W. 1978 Resource Partitioning in Passion Vine Butterflies. Evolution 32.3 493 518. BROWER, L. P., AND J. V. Z. BROWER. 1964. Birds, butterflies, and plant poisons: A study in ecological chemistry. Zoologica 49:137 159. BURKHOLDER, P. 2008. Preferential oviposition by Heliconiinae (Nymphalidae) butterflies on Passiflora biflora ( Passiflo ra ceae) leaves with higher cyanide concentrations. CIEE Spring 2008 TEC : 188 199. Print. COLEY, PHYLLIS D., JOHN P. BRYANT, AND STUART F. CHAPIN. 1985. Resource Availability and Plant Antiherbivore Defense. Science 230.4728: 895 99. DURKEE, Lenore T. 19 83. The Floral and Extra Floral Nectaries of Passiflora. II. The Extra Floral Nectary. American Journal of Botany. 69(9): 1420 1428. ENGLER, H. S., K. C. SPENCER, AND L. E. GILBERT. 2000. Insect metabolism: Preventing cyanide release from leaves. Nature 4 06. EHRILICH, P.R AND P.H RAVEN. 1964. Butterflies and Plants: A Study of Coevolution. Evolution. 18(4): 586 608. FLEMING, THEODORE H., DAVID SERRANO, JAFET NASSAR. 2005. Dynamics of a Subtropical Population of the Zebra Longwing Butterfly Heliconius chari thonia (Nymphalidae). The Florida Entomologist. 88(2): 169 179 FREITAS, A. V. L., AND K. S. J. BROWN. 2004. Phylogeny of the Nymphalidae (Lepidoptera: Papilionoidea). Systematic Biology 53:363 383 GILBERT, L.E. 1983. COEVOLUTION ED. DOUGLAS J. FUYUMA AND MANIGOMERY SLATKIN. Sunderland, Mass: Sinauer Associates. Print. GILBERT, L.E. AND M.C. SINGER. 1975. Butterfly Ecology. Annual Review of Ecology and Systematics. GLEADOW, R.M. AND I.E WOODROW. 2002. Constraints on Effectiveness of Cyanogenic Glycosides in Herbivore defense. Journal of Chemical Ecology. 28(7): 1301 1313. HAY ROE, MIRIAN M., AND JAMES NATION. 2006. Spectrum of Cyanide Toxicity and Allocation in Heliconius erato and Passiflora Host Plants." Journal of Chemical Ecology 33: 319 29. PRICE, PE TER W., IVONE R. DINIZ, HELENA C. MORAIS, AND EVELYN S. MARQUES. 1995. The Abundance of Insect Herbivore Species in the Tropics: The High Local Richness of Rare Species. Biotropica 27.4: 468 78. SEIGLER, DAVID S. 1991. Herbivores, their interactions with secondary plant metabolites 2nd ed. Vol. 1. San Diego: Academic. Print.
APPENDIX Figure 5. Standard curve of absorbance for testing cyanide content of leaves. Y=0.284ln(x)+0.1665. The standard curve was made from potassium cyanide. Image 1. Leaf Pen e trometer. This is the device used to measure leaf toughness. The top plant is placed on top of the leaf and the disk with small rod is balanced in the hole. A graduated cylinder rests on top of the plate and water is slowly added until the leaf is punctured. Water and cylinder are weighed and mass is recorded. Image 2. Dryas iulia larvae eating a P. biflora leaf. Image 3. Dryas iulia egg on a leaf tendril
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Eleccin de la hoja en Dryas iulia (Nymphalidae Heliconiinae) contenido de cianuro y dureza
Leaf choice in Dryas iulia (Nymphalidae Heliconiinae) cyanide content and toughness
Vines in the Passifloraceae synthesize cyanogenic glycosides that deter general herbivores, but Heliconiinae butterfly larvae such as Dryas iulia have overcome this and utilize Passiflora leaves as larval food. Ovipositing adult females and larvae may access the suitability of leaves caused by various plant defenses such as cyanide content and leaf toughness. D. iulia adult females show no preference in cyanide content (9.01g 28.3, 5.77g 12.6) or toughness (238.67g 78.4, 266.58g 123.1) for ovipostion, yet larvae prefer leaves with a significantly lower cyanide content (9.01g 28.3, 0.47g 0.51) then the average available leaf but average toughness
(238.67g 78.4, 227.23g 80.7). This indicates that larvae are assessing plants to maximize fitness and D. iulia ovipositon is determined by more factors then simply Passiflora leaf cyanide content and toughness.
Las lianas en la familia Passifloraceae sintetizan glucosas de cianuro que disuaden a los herbvoros, pero las larvas de la subfamilia Heliconiinae como Dryas iulia pueden comer las hojas de Passiflora. Es posible que las hembras adultas y las larvas puedan evaluar la presencia de varias defensas en las hojas como el cianuro y el grosor. Las hembras de D. iulia no muestran preferencia en el contenido de cianuro (9.01g 28.3, 5.77g 12.6) o grosor (238.67g 78.4, 266.58g 123.1) para la ovoposicin (t=1.02; p=0.307; df=67), aun as las larvas prefieren las hojas con significativamente menor contenido de cianuro que las hojas promedio (9.01g 28.3, 0.47g 0.51) y grosor promedio (238.67g 78.4, 227.23g 80.7). Esto indica que las larvas estn evaluando las plantas para incrementar el xito reproductivo y la ovoposicin de D. iulia est determinada por ms factores que simplemente el contenido de cianuro y el grosor de Passiflora.
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