Larvae Of Swallowtail Research Articles

Does group feeding by toxic prey confer a defensive benefit? Aristolochic acid content, group size and survival of first‐instar pipevine swallowtail (Battus philenor L.) larvae

1. Aggregative feeding of larvae is widespread in the Lepidoptera, and many hypotheses have been proposed to explain the adaptive significance of this trait. Group feeding occurs disproportionately more in species with aposematic, chemically defended larvae compared with species with cryptic, non‐chemically defended larvae, consistent with the hypothesis that group feeding provides an enhanced aposematic signal to natural enemies. Most species characterised as having chemically defended larvae are cryptic during the first instar, when they are most highly aggregated and most vulnerable to predation.2. The benefits of group feeding in terms of decreased predation were explored for first‐instar larvae of the pipevine swallowtail, Battus philenor, a species that sequesters aristolochic acids from its Aristolochia host plant and exhibits aposematism in later instars and as adults. We found that groups of larvae with experimentally enhanced aristolochic acid content had significantly lower survivorship due to predation both in the field and in laboratory experiments compared with groups of larvae without enhanced chemical defence.3. A laboratory experiment found that aristolochic acid does not deter the generalist predator Hippodamia convergens.4. No evidence was found that was consistent with the hypothesis that group feeding and increased sequestered chemical defence interact to decrease rates of larval mortality in non‐aposematic, first‐instar larvae. Future research on chemical defence, aposematism, and aggregative feeding should continue to appreciate that particular chemical defences and feeding behaviours are not universally effective against all natural enemies.

Molecular Cloning and Characterization of Attacin from the Swallowtail Butterfly, Papilio xuthus

Attacin is an insect antibacterial protein that plays an important role in immune response to injury and infection. In this report, we have isolated and characterized of cDNA encoding for the attacin from the immunized larvae of swallowtail butterfly, <TEX>$Papilio$</TEX> <TEX>$xuthus$</TEX>. A full length cDNA of <TEX>$P.$</TEX> <TEX>$xuthus$</TEX> attacin was obtained by employing annealing control primer (ACP)-based differential display PCR and 5' RACE. The complete <TEX>$P.$</TEX> <TEX>$xuthus$</TEX> attacin cDNA was comprised of 949 bp encoding a 250 amino acid precursor. It contains a putative 18 amino acid signal peptide sequence, a 42 amino acid propeptide sequence, and a 190 amino acid mature protein with a theoretical molecular mass of 19904.01 and a pI of 9.13. The putative mature protein of <TEX>$P.$</TEX> <TEX>$xuthus$</TEX> attacin showed 48-52% and 24-30% identity in amino acid sequences with that of lepidopteran and dipteran insects, respectively. Semiquantitive RT-PCR results revealed that the transcript of <TEX>$P.$</TEX> <TEX>$xuthus$</TEX> attacin gene was up-regulated at significant levels after injection with bacterial lipopolysaccharide (LPS). We sub-cloned cDNA fragment encoding mature <TEX>$P.$</TEX> <TEX>$xuthus$</TEX> attacin into the expression vector, highly expressed in <TEX>$E.$</TEX> <TEX>$coli$</TEX> BL21 cells, and its antibacterial activity was analyzed. Recombinant <TEX>$P.$</TEX> <TEX>$xuthus$</TEX> attacin evidenced considerably antibacterial activity against Gram-negative bacteria, <TEX>$E.$</TEX> <TEX>$coli$</TEX> ML 35 and <TEX>$Klebsiella$</TEX> <TEX>$pneumonia$</TEX>.

A novel trade-off of insect diapause affecting a sequestered chemical defense

Diapause allows insects to temporally avoid conditions that are unfavorable for development and reproduction. However, diapause may incur a cost in the form of reduced metabolic energy reserves, reduced potential fecundity, and missed reproductive opportunities. This study investigated a hitherto ignored consequence of diapause: trade-offs involving sequestered chemical defense. We examined the aristolochic acid defenses of diapausing and non-diapausing pipevine swallowtail butterflies, Battus philenor. Pipevine swallowtail larvae acquire these chemical defenses from their host plants. Butterflies that emerge following pupal diapause have significantly less fat, a female fitness correlate, compared to those that do not diapause. However, butterflies emerging from diapaused pupae are more chemically defended compared to those that have not undergone diapause. Furthermore, non-diapausing butterflies are confronted with older, lower quality host plants on which to oviposit. Thus, a trade-off exists where butterflies may have greater energy reserves at the cost of less chemical defense and sub-optimal food resources for their larvae, or have substantially less energetic reserves with the benefit of greater chemical defense and plentiful larval food resources.

Inhibition of CYP6B1-Mediated Detoxification of Xanthotoxin by Plant Allelochemicals in the Black Swallowtail (Papilio polyxenes)

The structural and biosynthetic diversity of allelochemicals in plants is thought to arise from selection for additive toxicity as a consequence of toxin mixture or for enhanced toxicity as a result of synergism. In order to understand how insects cope with this type of plant defense, we tested the effects of some allelochemicals in host plants of the black swallowtail Papilio polyxenes on the xanthotoxin-metabolic activity of CYP6B1, the principal enzyme responsible for the detoxification of furanocoumarins in this caterpillar. Additionally, the effects of some synthetic compounds not normally encountered by P. polyxenes on CYP6B1 were tested. These studies demonstrate that the integrity of furanocoumarin structure is important for competitive binding to the active site of CYP6B1, even though the carbonyl group on the pyranone ring apparently does not affect its inhibitory capacity, as in the case of furanochromones. Angular furanocoumarins are generally less phototoxic to many organisms than linear furanocoumarins due to their reduced capacity for cross-linking DNA strands, yet they are more toxic than linear furanocoumarins to black swallowtail larvae. This enhanced toxicity in vivo may be due to the ability of angular furanocoumarins to bind to the active site of CYP6B1 without being rapidly metabolized. This binding reduces the availability of CYP6B1 to metabolize other linear furanocoumarins. The structure-activity relationships for methylenedioxyphenyl compounds, flavonoids, imidazole, and imidazole derivatives are also discussed in light of their capacity to inhibit the xanthotoxin-metabolic activity of CYP6B1.

Between‐clutch interactions affect a benefit of group feeding for pipevine swallowtail larvae

Abstract. 1. In California, early instar larvae of the pipevine swallowtail (Battus philenor) develop at an accelerated rate when feeding in large groups compared with small groups due to a plant‐mediated response to feeding group size. Larvae benefit from accelerated growth because the time larvae remain in early stages, where mortality is highest, is reduced. Occasionally, multiple clutches are laid on the same plant stem. Clutch size modification by females ovipositing on plant with previously laid clutches and the effect of kinship and group size on larval behaviour was examined. The direct and indirect interactions between clutches were investigated to determine if group size and time between clutch establishment affects the performance of early instar larvae.2. Larger groups consume the young foliage more quickly and develop at an accelerated rate compared with smaller groups. Older foliage available to later clutches is an inferior food resource compared with younger foliage.3. There was no evidence that females adjust clutch size in response to the presence of conspecific clutches.4. Second groups of larvae readily joined previously established feeding groups. There were no observed behavioural differences between sibling and mixed‐family groups.5. The effect of a second group on the growth of the initial group was dependent on the size of both groups and the time interval between the arrival of the two groups.6. Accelerated growth associated with larger feeding aggregations was absent when these groups were introduced to plants with previously established groups.7. It is beneficial for ovipositing females to avoid plants with previously laid clutches because direct and indirect interactions with established clutches compromises larval performance.

How caterpillars avoid overheating: behavioral and phenotypic plasticity of pipevine swallowtail larvae

We tested the hypothesis that larvae of the pipevine swallowtail butterfly, Battus philenor, employ behavioral and phenotypic plasticity as thermoregulatory strategies. These larvae are phenotypically varied across their range with predominantly black larvae (southeastern USA and California) and red larvae (western Texas, Arizona) occurring in different regions. Two years of field observations in south Texas indicate that the proportion of red larvae increases with increasing daily temperatures as the growing season progresses. Larvae were also observed to shift their microhabitats by climbing on non-host vegetation and avoided excessive heat in their feeding microhabitat. Larvae of ten half-sib families from populations in south Texas and California, reared under different temperature regimes in common garden experiments, exhibited plasticity in larval phenotype, with larvae from both populations producing the red phenotype at temperatures greater than 30 degrees C and maintaining the black phenotype at cooler temperatures. However, larvae from Texas were more tolerant of higher temperatures, showing no decrease in growth rate in the highest temperature (maximum seasonal temperature) treatment, compared to the California population. In a field experiment, black larvae were found to have higher body temperatures when exposed to sunlight compared to red larvae. These results suggest that microhabitat shifts and the color polyphenism observed in pipevine swallowtail larvae may be the adaptive strategies that enable larvae to avoid critical thermal maximum temperatures.

Conserved regulatory elements in the promoters of two allelochemical-inducible cytochrome P450 genes differentially regulate transcription

CYP6B4, a cytochrome P450 gene from the tiger swallowtail Papilio glaucus, is transcriptionally induced in the midgut by dietary furanocoumarins, plant allelochemicals that can crosslink DNA in their UV-activated form. The CYP6B4 promoter contains an overlapping EcRE/ARE/XRE-xan element similar to that used for basal and xanthotoxin-inducible expression of the CYP6B1 promoter from the black swallowtail Papilio polyxenes. Transfection of the CYP6B4 promoter:CAT reporter construct into Sf9 cells demonstrates that the basal and xanthotoxin-inducible expression levels observed reflect the relative expression levels of this gene in the midguts of tiger swallowtail larvae. Transfections of mutant CYP6B4 promoter constructs into Sf9 cells indicate that the EcRE/ARE/XRE-xan element is necessary for CYP6B4 induction by xanthotoxin but not for its minimal basal expression. In addition to these elements, the CYP6B4 and CYP6B1 promoters also contain putative XRE-AhR elements identical to the aryl hydrocarbon response elements present in mammalian phase I detoxification genes. Transfections of CYP6B4 and CYP6B1 promoters containing EcRE/ARE/XRE-xan and XRE-AhR elements indicate that both are induced significantly by benzo(α)pyrene, an aryl hydrocarbon widespread in the environment, as well as by xanthotoxin, an allelochemical encountered in their hostplants.

Aggregative feeding of pipevine swallowtail larvae enhances hostplant suitability.

Many hypotheses have been proposed to explain the adaptive significance of aggregative feeding in the Lepidoptera. One hypothesis that has received little attention is how induced plant responses may be influenced by aggregative feeding, as compared to feeding by solitary larvae. This study investigated the role of aggregative feeding of the pipevine swallowtail, Battus philenor, in California with special emphasis on the induced responses to herbivory of its hostplant. Here, I show that first-instar larvae develop faster when feeding in a large aggregation compared to solitary or small groups of larvae. Furthermore, I show that this effect is mediated by a larval-density-dependent response in the plant and is independent of prior larval experience and direct interaction among larvae. These results indicate that large groups of larvae can effectively enhance hostplant suitability. A separate experiment showed that larvae feeding on previously damaged leaves had a reduced growth rate. Thus, following initial damage a plant first goes through a period of increased suitability, followed by induced resistance against subsequent herbivory. Aggregative feeding in this system may be an adaptive strategy for larvae to manipulate hostplant suitability, adding a new dimension to the role of aggregative feeding for the Lepidoptera.

ANOTHER PERSPECTIVE ON THE SLOW-GROWTH/HIGH-MORTALITY HYPOTHESIS: CHILLING EFFECTS ON SWALLOWTAIL LARVAE

The slower-growth/higher-mortality hypothesis proposes that reduced herbivore growth rates benefit plants because slower growing herbivores remain vulnerable to predator attack for an extended time period, resulting in a lower reduction in plant fitness attributed to herbivory. We propose cooling events as an alternative mechanism leading to higher mortality for slower growing larvae. We observed that, in the absence of predators, slower growing pipevine swallowtail (Battus philenor) larvae had higher mortality compared to faster growing larvae during the unseasonably cool spring temperatures of 1998. However, this was not true for the warmer spring of 1999. Laboratory experiments showed that the probability of surviving a chill coma increased with larval mass. We propose that smaller larvae are susceptible to chilling events because they have less energy reserved for metabolism during a chill coma, and that “warm” chill events near the activity threshold may be more lethal than “cold” chill events. Weather station data since 1951 and field data collected from 1976 to 2000 suggest that exposure to chill events below the chill coma threshold is not uncommon for these larvae. We propose that faster growth may be important for larvae because slower growing larvae remain at a chill-susceptible size for an extended period of time. Corresponding Author: S. J. Simpson.

Effects of exposure to event 176 Bacillus thuringiensis corn pollen on monarch and black swallowtail caterpillars under field conditions.

The widespread planting of corn genetically modified to produce Bacillus thuringiensis endotoxin has led to speculation that pollen from these fields might adversely affect nearby nontarget lepidopterans. A previous study of Bt corn engineered with Monsanto event 810 failed to detect an effect of pollen exposure on the black swallowtail, Papilio polyxenes, in either the field or the laboratory. Here, we report results of a field study investigating the impact of exposure to pollen from a Bt corn hybrid containing Novartis event 176 on two species of Lepidoptera, black swallowtails and monarch butterflies, Danaus plexippus. Nearly half of the 600 monarch larvae died within the first 24 h; this and subsequent mortality was not associated with proximity to Bt corn and may have been due in part to predation. Survivorship of black swallowtails was much higher than that of the monarchs and was also independent of proximity to the transgenic corn. However, despite five rainfall events that removed much of the pollen from the leaves of their host plants during the experiment, we observed a significant reduction in growth rates of black swallowtail larvae that was likely caused by pollen exposure. These results suggest that Bt corn incorporating event 176 can have adverse sublethal effects on black swallowtails in the field and underscore the importance of event selection in reducing environmental impacts of transgenic plants.

Competition between the gypsy moth, Lymantria dispar, and the northern tiger swallowtail, Papilio canadensis: interactions mediated by host plant chemistry, pathogens, and parasitoids

The gypsy moth, Lymantria dispar, and the northern tiger swallowtail, Papilio canadensis, overlap geographically as well as in their host ranges. Adult female swallowtails are incapable of distinguishing between damaged and undamaged leaves, and the opportunities for competition between these two species are numerous. We designed field and laboratory experiments to look for evidence of indirect competition between P. canadensis and L. dispar larvae. Swallowtail caterpillars were reared in the laboratory on leaves from gypsy-moth-defoliated and undefoliated trees to explore host-plant effects. We tested for pathogen-mediated interactions by rearing swallowtail larvae on both sterilized and unsterilized leaves from defoliated and undefoliated sources. In addition, we measured the effects of known gypsy moth pathogens, as well as gypsy moth body fluids, on the growth and survival of swallowtail larvae. Field experiments were designed to detect the presence of parasitoid-mediated competition, as well: we recorded parasitism of swallowtail caterpillars placed in the field either where there were no gypsy moth larvae present, or where we had artificially created dense gypsy moth populations. We found evidence that swallowtails were negatively affected by gypsy moths in several ways: defoliation by gypsy moths depressed swallowtail growth rate and survival, whether leaves were sterilized or not; sterilization significantly reduced the effect of defoliation, and gypsy moth body fluids proved lethal; and swallowtail caterpillars suffered significantly increased rates of parasitism when they were placed in the field near gypsy moth infestations.

Chemical defense in the zebra swallowtail butterfly, Eurytides marcellus, involving annonaceous acetogenins.

Few herbivores feed on the foliage of the North American paw paw tree, Asimina triloba; notable exceptions are the larvae of the zebra swallowtail butterfly, Eurytides marcellus. Toxic annonaceous acetogenins, produced by A. triloba, are responsible for the relative unpalatability of the leaves. Acetogenins found in A. triloba extracts are potent pesticidal and antineoplastic agents and have emetic activity in vertebrates. In this study, partitioned aqueous MeOH fractions of the bioactive CH2Cl2 extracts, of freeze-dried and pulverized larvae, and of mature butterflies revealed acetogenin content through the use of HPLC coupled to tandem MS (LC-MS/MS). This sensitive technique provides an uncomplicated method for the detection of trace compounds and, in this instance, has confirmed tissue presence of acetogenins that serve a probable role as chemical defense agents against bird predation in zebra swallowtail larvae and adults.

Food utilization and antioxidant enzyme activities of black swallowtail in response to plant phototoxins

AbstractPhototoxic compounds are widely distributed among plant families; due to their ability to bind covalently to DNA or to react with oxygen and generate toxic oxyradicals, these compounds are toxic to a variety of herbivorous organisms. Black swallowtail (Papilio polyxenes) larvae feed exclusively on phototoxic host plants in the Apiaceae and Rutaceae. In this study, we examined the toxicity of four phototoxins—three furanocoumarins and one β‐carboline alkaloid—to P. polyxenes, as well as the inducibility of antioxidant enzyme defenses in response to these phototoxins. Neither the furanocoumarins nor the β‐carboline alkaloid demonstrated any toxic effect on digestive efficiencies of P. polyxenes in the presence of light; harmine, the alkaloid, did significantly reduce growth and consumption rates. None of the compounds had a significant effect on antioxidant enzyme levels. These findings contrast with those reported in earlier studies for Trichoplusia ni, a generalist noctuid sensitive to both furanocoumarins and β‐carboline alkaloids. Greater detoxicative metabolic capabilities, coupled with substantially higher constitutive levels of antioxidant enzyme activity, likely explain at least in part the absence of induced antioxidant enzyme responses in the specialist feeder on phototoxic plants. © 1993 Wiley‐Liss, Inc.

Do predators avoid groups of aposematic prey? An experimental test

This study investigated the widely held idea that predators are disinclined to attack groups of aposematic prey. There were four experimental situations. Great tits, Parus major, were presented with either grouped or solitary individuals of either larvae of the European pine sawfly, Neodiprion sertifer, or larvae of the European swallowtail, Papilio machaon. Sawfly larvae are naturally gregarious and the swallowtail larvae solitary. The sawfly larvae were attacked, killed and eaten more frequently than swallowtail larvae. The attack frequency was not independent of whether prey was grouped or solitary but groups of sawflies were attacked less than solitary individuals, whereas groups of swallowtails were attacked more than solitary individuals. The results imply that a high initial cost of group living may be reduced through the evolution of communal defence or display.

Immunochemical analysis of cytochrome P450 monooxygenase diversity in the black swallowtail caterpillar, Papilio polyxenes

Antisera to nineteen cytochrome P450 monooxygenases were assayed for cross-reactivity with microsomal and mitochondrial proteins of the black swallowtail caterpillar, Papilio polyxenes (Lepidoptera:Papilionidae), on Western blots. Antisera to P450s from a bacterium, an insect and three mammals showed selective cross-reactivity with seven bands in the P450 molecular weight range of 45–60 kDa. Of the five putative P450s identified in midgut microsomes, only two were induced in larvae reared on a diet supplemented with high levels of xanthotoxin, a host plant constituent metabolized by P450 in the black swallowtail. The results confirm that insect P450s share conserved epitopes with P450s from other taxa and provide further evidence of the selective induction of P450 isozymes by xanthotoxin in black swallowtail larvae.

Antioxidant enzyme activities in subcellular fractions of larvae of the black swallowtail butterfly, Papilio polyxenes

AbstractThe black swallowtail butterfly, Papilio polyxenes, larvae are specialized feeders of pro‐oxidant rich plants of Apiaceae and Rutaceae. An important defense against toxic forms of oxygen species generated by ingestion of the pro‐oxidants, are the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), GSH‐dependent glutathione peroxidases (selenium‐dependent glutathione peroxidase [GPOX] and peroxidase activity of selenium‐independent glutathione‐S‐transferase [GTpx]), and glutathione reductase (GR). The subcellular distribution of these enzymes in black swallowtail larvae was investigated and was found to resemble the patterns described for larvae of two other lepidopteran species: the southern armyworm, Spodoptera eridania, and the cabbage looper, Trichoplusia ni. The confinement of SOD in the cytosol and mitochondria was typically eukaryotic, but the relative proportion (1:1) was markedly different from the mammalian pattern (4:1; cytosol:mitochondria). The most obvious difference between the black swallowtail and other lepidoptera as a group, and mammalian species, is in very wide intracellular distributions of CAT, GTpx, and GR in insect species. Insects possess very low levels of a GPOX‐like activity which reduces both H2O2 and organic peroxides. Consequently, insects have elaborate activities with a wide subcellular distribution of both CAT which decomposes H2O2, and GTpx which decomposes organic peroxides. The reduction of peroxides is dependent on GSH, which in this process is oxidized to GSSG. GR which reduces GSSG to GSH is also of wide subcellular distribution, analogous to the distribution pattern of GTpx.

Interspecific differences among swallowtail larvae (Lepidoptera: Papilionidae) in susceptibility to aristolochic acids and berberine

Abstract. 1. The larvae of four swallowtail butterfly species, Papilio polyxenes Fabr. in the Papilionini, Parides bunichus (Hübner) and Battus polydamas (L.) in the Troidini, and Eurytides marcellus (Cramer) in the Graphiini, were grown on host leaves to which test compounds had been added.2. The test chemicals are biosynthetically related. Berberine, a 1‐benzyl‐tetrahydroisoquinoline‐related (1‐BTIQ) alkaloid, is found in many rutaceous hosts of the Papilionini; laudanosine, a simpler 1‐BTIQ alkaloid, is found in the Papaveraceae, plants on which no papilionids feed; aristolochic acids are found only in the Aristolochiaceae, hosts of the Troidini.3. Swallowtail larvae can tolerate the compounds characteristic of their host plants, but not chemicals found in the hosts of other papilionid groups. Diets with laudanosine did not affect the test species.4. Our results support the contention that plant secondary chemicals, such as berberine and aristolochic acids, are important feeding barriers, and are partially responsible for host specificity in swallowtails.

Constituents of osmeterial secretion of pre-final instar larvae of citrus swallowtail,Papilio demodocus (Esper) (Lepidoptera: Papilionidae)

The defensive osmeterial secretion of pre-final instar larvae of the citrus swallowtail,Papilio demodocus (Lepidoptera: Papilionidae) was found to contain methyl 3-hydroxybutanoate, 3-hydroxybutanoic acid, α-pinene, myrcene, limonene, β-phellandrene, (Z)-ocimene, (E)-ocimene, β-caryophyllene, (E)-β-farnesene, and germacrene-A, as well as a further number of unidentified sesquiterpenoid constituents. The presence of germacrene-A in the secretion was inferred from the formation of β-elemene under certain GC conditions. Larvae of the second, third, and fourth instars produce qualitatively similar secretions. Remarkable quantitative differences were found between the secretions of individual larvae. These variations could not be correlated with the diet on which the larvae were fed, their sex, instar, or color form. However, in a number of larvae the two prongs of the osmeterium were found to produce quantitatively different secretions.

The Evolution of Egg Clustering by Butterflies and Other Insects

On met en evidence le fait que la ponte en grappes chez de nombreux lepidopteres (et d'autres insectes) a evolue en reponse a la selection pour une fecondite accrue

Individual versus Kin Selection for Aposematic Coloration: A Reply to Harvey and Paxton

both brightly coloured and distasteful. In addition, there will have been slight differences in shape as well as considerable differences in strength of the integument between the two prey types. The authors argue that, because the great tits were able to distinguish between mealworms and swallowtail larvae, the latter qualify as being aposematic. The swallowtail larvae may, indeed, have been aposematic but the evidence presented does not demonstrate that. The basis for discrimination may not have been colour, it could have been shape. The reason for avoidance may not have been distaste, it could have been the tough integument. While we accept the importance of the experiments performed by Jdrvi et al. we emphasize the need for more detailed experiments that allow us to distinguish between some of the processes outlined above.