Alleles in metabolic and oxygen-sensing genes are associated with antagonistic pleiotropic effects on life history traits and population fitness in an ecological model insect.

Genes with opposing effects on fitness at different life stages are the mechanistic basis for evolutionary theories of aging and life history. Examples come from studies of mutations in model organisms, but there is little knowledge of genetic bases of life history tradeoffs in natural populations. Here, we test the hypothesis that alleles affecting oxygen sensing in Glanville fritillary butterflies have opposing effects on larval versus adult fitness-related traits. Intermediate-frequency alleles in Succinate dehydrogenase d, and to a lesser extent Hypoxia inducible factor 1α, are associated in larvae with variation in metabolic rate and activation of the hypoxia inducible factor (HIF) pathway, which affects tracheal development and delivery of oxygen to adult flight muscles. A dominant Sdhd allele is likely to cause antagonistic pleiotropy for fitness through its opposing effects on larval metabolic and growth rate versus adult flight and dispersal, and may have additional effects arising from sensitivity to low-iron host plants. Prior results in Glanville fritillaries indicate that fitness of alleles in Sdhd and another antagonistically pleiotropic metabolic gene, Phosphoglucose isomerase, depend strongly on the size and distribution of host plant patches. Hence, these intermediate-frequency alleles are involved in ecoevolutionary dynamics involving life history tradeoffs.

DNA barcoding and species delimitation of butterflies (Lepidoptera) from Nigeria.

Accurate identification of species is a prerequisite for successful biodiversity management and further genetic studies. Species identification techniques often require both morphological diagnostics and molecular tools, such as DNA barcoding, for correct identification. In particular, the use of the subunit I of the mitochondrial cytochrome c oxidase (COI) gene for DNA barcoding has proven useful in species identification for insects. However, to date, no studies have been carried out on the DNA barcoding of Nigerian butterflies. We evaluated the utility of DNA barcoding applied for the first time to 735 butterfly specimens from southern Nigeria. In total, 699 DNA barcodes, resulting in a record of 116 species belonging to 57 genera, were generated. Our study sample comprised 807 DNA barcodes based on sequences generated from our current study and 108 others retrieved from BOLD. Different molecular analyses, including genetic distance-based evaluation (Neighbor-Joining, Maximum Likelihood and Bayesian trees) and species delimitation tests (TaxonDNA, Automated Barcode Gap Discovery, General Mixed Yule-Coalescent, and Bayesian Poisson Tree Processes) were performed to accurately identify and delineate species. The genetic distance-based analyses resulted in 163 well-separated clusters consisting of 147 described and 16 unidentified species. Our findings indicate that about 90.20% of the butterfly species were explicitly discriminated using DNA barcodes. Also, our field collections reported the first country records of ten butterfly species-Acraea serena, Amauris cf. dannfelti, Aterica galena extensa, Axione tjoane rubescens, Charaxes galleyanus, Papilio lormieri lormeri, Pentila alba, Precis actia, Precis tugela, and Tagiades flesus. Further, DNA barcodes revealed a high mitochondrial intraspecific divergence of more than 3% in Bicyclus vulgaris vulgaris and Colotis evagore. Furthermore, our result revealed an overall high haplotype (gene) diversity (0.9764), suggesting that DNA barcoding can provide information at a population level for Nigerian butterflies. The present study confirms the efficiency of DNA barcoding for identifying butterflies from Nigeria. To gain a better understanding of regional variation in DNA barcodes of this biogeographically complex area, future work should expand the DNA barcode reference library to include all butterfly species from Nigeria as well as surrounding countries. Also, further studies, involving relevant genetic and eco-morphological datasets, are required to understand processes governing mitochondrial intraspecific divergences reported in some species complexes.

Multiple roles forlaccase2 in butterfly wing pigmentation, scale development, and cuticle tanning.

Lepidopteran wing scales play important roles in a number of functions including color patterning and thermoregulation. Despite the importance of wing scales, however, we still have a limited understanding of the genetic mechanisms that underlie scale patterning, development, and coloration. Here, we explore the function of the phenoloxidase-encoding gene laccase2 in wing and scale development in the nymphalid butterfly Vanessa cardui. Somatic deletion mosaics of laccase2 generated by CRISPR/Cas9 genome editing presented several distinct mutant phenotypes. Consistent with the work in other nonlepidopteran insect groups, we observed reductions in melanin pigmentation and defects in cuticle formation. We were also surprised, however, to see distinct effects on scale development including complete loss of wing scales. This study highlights laccase2 as a gene that plays multiple roles in wing and scale development and provides new insight into the evolution of lepidopteran wing coloration.

Comparison of Genetic Structure between Endangered and Common Butterflies, Ypthima multistriata and Y. argus (Lepidoptera; Nymphalidae), Inhabiting Japan.

Genetic structures of two closely related butterflies, Ypthima multistriata and Y. argus, inhabiting Japan were compared based on the mitochondrial cytochrome c oxidase subunit I (COI) sequences. The former species is classified as a vulnerable species and exhibits a characteristic pattern of voltinism: univoltine and bivoltine populations are distributed in a scattered manner. The latter species is common and has a normal geographical pattern of voltinism: the number of annual generations is correlated with latitude. Our genetic analyses of these two species yielded contrasting results: a spatial analysis of molecular variance (SAMOVA) and FST between each pair of populations revealed a locally fragmented genetic structure for Y. multistriata, compared to three distinct geographic groups of Y. argus within which range-wide gene flow occurs. Although Y. argus is a common species, only the southernmost populations in Japan had higher genetic diversity, while the other populations had the same or lower levels of genetic diversity, compared to Y. multistriata. These results indicate that: 1) the degree of fragmentation of Y. multistriata populations was higher; however, markedly lower genetic diversity was not found, and 2) although Y. argus is a common species, its populations may not be genetically robust. In addition, AMOVA revealed a relationship between voltinism and genetic variation in Y. multistriata. This result suggests a phylogenetic constraint of voltinism in this butterfly.

The dynamics of cyanide defences in the life cycle of an aposematic butterfly: Biosynthesis versus sequestration.

Heliconius butterflies are highly specialized in Passiflora plants, laying eggs and feeding as larvae only on them. Interestingly, both Heliconius butterflies and Passiflora plants contain cyanogenic glucosides (CNglcs). While feeding on specific Passiflora species, Heliconius melpomene larvae are able to sequester simple cyclopentenyl CNglcs, the most common CNglcs in this plant genus. Yet, aromatic, aliphatic, and modified CNglcs have been reported in Passiflora species and they were never tested for sequestration by heliconiine larvae. As other cyanogenic lepidopterans, H. melpomene also biosynthesize the aliphatic CNglcs linamarin and lotaustralin, and their toxicity does not rely exclusively on sequestration. Although the genes encoding the enzymes in the CNglc biosynthesis have not yet been biochemically characterized in butterflies, the cytochromes P450 CYP405A4, CYP405A5, CYP405A6 and CYP332A1 have been hypothesized to be involved in this pathway in H. melpomene. In this study, we determine how the CNglc composition and expression of the putative P450s involved in the biosynthesis of these compounds vary at different developmental stages of Heliconius butterflies. We also establish which kind of CNglcs H. melpomene larvae can sequester from Passiflora. By analysing the chemical composition of the haemolymph from larvae fed with different Passiflora diets, we show that H. melpomene is able to sequestered prunasin, an aromatic CNglcs, from P. platyloba. They are also able to sequester amygdalin, gynocardin, [C13/C14]linamarin and [C13/C14]lotaustralin painted on the plant leaves. The CNglc tetraphyllin B-sulphate from P. caerulea is not detected in the larval haemolymph, suggesting that such modified CNglcs cannot be sequestered by Heliconius. Although pupae and virgin adults contain dihydrogynocardin resulting from larval sequestration, this compound was metabolized during adulthood, and not used as nuptial gift or transferred to the offspring. Thus, we speculate that dihydrogynocardin is catabolized to recycle nitrogen and glucose, and/or to produce fitness signals during courtship. Mature adults have a higher concentration of CNglcs than any other developmental stages due to increased de novo biosynthesis of linamarin and lotaustralin. Accordingly, all CYP405As are expressed in adults, whereas larvae mostly express CYP405A4. Our results shed light on the importance of CNglcs for Heliconius biology and their coevolution with Passiflora.

Characterization of Female Reproductive Proteases in a Butterfly from Functional and Evolutionary Perspectives.

Molecules that mediate reproductive interactions are some of the most rapidly evolving traits. Researchers have often suggested that this is due to coevolution at key physiological interfaces. However, very few of these interfaces are well understood at the functional level. One such interface is the digestion of the spermatophore in Lepidoptera. Female Lepidoptera have a specialized reproductive organ called the bursa copulatrix that receives and processes the male spermatophore, a complex proteinaceous ejaculate. In the cabbage white butterfly, Pieris rapae, the bursa secretes a mixture of proteases hypothesized to digest the spermatophore. However, these proteases remain biochemically uncharacterized. Using a zymogram approach, we identified six proteases in bursal extracts at sufficiently high concentrations to characterize their in vitro activity. We assessed the modes of action of these bursal enzymes by quantifying their activity following exposure to diagnostic protease inhibitors. A serine protease-specific inhibitor failed to reduce bursal protease digestion of casein. However, a cysteine protease-specific inhibitor did decrease the activity of some proteases. To explore the possible molecular mechanisms responsible for these responses, we created protease homology models. The models mirrored the results of our in vitro experiments, indicating that protease homology models may offer insight into underlying functional mechanisms. Whether the observed bursal protease resistance to known inhibitors is important in the context of spermatophore digestion remains to be tested. However, our results suggest the exciting possibility that bursal protease specificity may have evolved in response to interactions with various proteins and inhibitors present within the female tract during the reproductive process.

Gene duplication and subsequent functional diversification of sucrose hydrolase in Papilio xuthus.

Sucrose is the main product of photosynthesis in plants, providing a rich carbon and energy source for the physiological growth and development of insects. In a previous study, we identified a novel sucrose hydrolase (SUH) in the larval midgut of moths. Intriguingly, there are two copies of Suh, namely Suh1 and Suh2, in several species of butterflies. However, the biochemical characteristics of SUHs in butterflies remain unclear. In this study, we found that this duplication and subsequent diversification produced two Suh genes in Papilio xuthus. These two PxSuh genes were significantly divergent in terms of their expression pattern and enzyme properties. PxSuh messenger RNA expression was highest during the larval stage, reduced in the prepupal and pupal stages and, for PxSuh1, slightly increased again in the adult. The observed levels of PxSuh2 were overall below those of PxSuh1 amongst the development stages examined. Compared with PxSUH2, which has maintained the original gene function of maltose hydrolysis, PxSUH1 exhibits substrate specificity for sucrose with an optimum enzyme activity occurring at an alkaline pH. The data show that PxSuh1 is evolutionarily adapted for effective functioning in an alkaline digestive system. Furthermore, we find that functional diversification of Suh facilitates P. xuthus to digestive carbohydrate of host plants. Thus, our findings offer new insights into the ecological and evolutionary adaptation of digestive enzymes in butterflies.

Cross-continental phylogeography of two Holarctic Nymphalid butterflies, Boloria eunomia and Boloria selene.

Pleistocene glaciations had significant effects on the distribution and evolution of species inhabiting the Holarctic region. Phylogeographic studies concerning the entire region are still rare. Here, we compared global phylogeographic patterns of one boreo-montane and one boreo-temperate butterflies with largely overlapping distribution ranges across the Northern Hemisphere, but with different levels of range fragmentation and food specialization. We reconstructed the global phylogeographic history of the boreo-montane specialist Boloria eunomia (n = 223) and of the boreo-temperate generalist Boloria selene (n = 106) based on mitochondrial and nuclear DNA markers, and with species distribution modelling (SDM). According to the genetic structures obtained, both species show a Siberian origin and considerable split among populations from Nearctic and Palaearctic regions. According to SDMs and molecular data, both butterflies could inhabit vast areas during the moderate glacials. In the case of B. selene, high haplotype diversity and low geographic structure suggest long-lasting interconnected gene flow among populations. A stronger geographic structuring between populations was identified in the specialist B. eunomia, presumably due to the less widespread, heterogeneously distributed food resources, associated with cooler and more humid climatic conditions. Populations of both species show opposite patterns across major parts of North America and in the case of B. eunomia also across Asia. Our data underline the relevance to cover entire distribution ranges to reconstruct the correct phylogeographic history of species.

Viral and metazoan poxins are cGAMP-specific nucleases that restrict cGAS-STING signalling.

Cytosolic DNA triggers innate immune responses through the activation of cyclic GMP-AMP synthase (cGAS) and production of the cyclic dinucleotide second messenger 2',3'-cyclic GMP-AMP (cGAMP)1-4. 2',3'-cGAMP is a potent inducer of immune signalling; however, no intracellular nucleases are known to cleave 2',3'-cGAMP and prevent the activation of the receptor stimulator of interferon genes (STING)5-7. Here we develop a biochemical screen to analyse 24 mammalian viruses, and identify poxvirus immune nucleases (poxins) as a family of 2',3'-cGAMP-degrading enzymes. Poxins cleave 2',3'-cGAMP to restrict STING-dependent signalling and deletion of the poxin gene (B2R) attenuates vaccinia virus replication in vivo. Crystal structures of vaccinia virus poxin in pre- and post-reactive states define the mechanism of selective 2',3'-cGAMP degradation through metal-independent cleavage of the 3'-5' bond, converting 2',3'-cGAMP into linear Gp[2'-5']Ap[3']. Poxins are conserved in mammalian poxviruses. In addition, we identify functional poxin homologues in the genomes of moths and butterflies and the baculoviruses that infect these insects. Baculovirus and insect host poxin homologues retain selective 2',3'-cGAMP degradation activity, suggesting an ancient role for poxins in cGAS-STING regulation. Our results define poxins as a family of 2',3'-cGAMP-specific nucleases and demonstrate a mechanism for how viruses evade innate immunity.

Toxicity of Milkweed Leaves and Latex: Chromatographic Quantification Versus Biological Activity of Cardenolides in 16 Asclepias Species.

Cardenolides are classically studied steroidal defenses in chemical ecology and plant-herbivore coevolution. Although milkweed plants (Asclepias spp.) produce up to 200 structurally different cardenolides, all compounds seemingly share the same well-characterized mode of action, inhibition of the ubiquitous Na+/K+ ATPase in animal cells. Over their evolutionary radiation, milkweeds show a quantitative decline of cardenolide production and diversity. This reduction is contrary to coevolutionary predictions and could represent a cost-saving strategy, i.e. production of fewer but more toxic cardenolides. Here we test this hypothesis by tandem cardenolide quantification using HPLC (UV absorption of the unsaturated lactone) and a pharmacological assay (in vitro inhibition of a sensitive Na+/K+ ATPase) in a comparative study of 16 species of Asclepias. We contrast cardenolide concentrations in leaf tissue to the subset of cardenolides present in exuding latex. Results from the two quantification methods were strongly correlated, but the enzymatic assay revealed that milkweed cardenolide mixtures often cause stronger inhibition than equal amounts of a non-milkweed reference cardenolide, ouabain. Cardenolide concentrations in latex and leaves were positively correlated across species, yet latex caused 27% stronger enzyme inhibition than equimolar amounts of leaf cardenolides. Using a novel multiple regression approach, we found three highly potent cardenolides (identified as calactin, calotropin, and voruscharin) to be primarily responsible for the increased pharmacological activity of milkweed cardenolide mixtures. However, contrary to an expected trade-off between concentration and toxicity, later-diverging milkweeds had the lowest amounts of these potent cardenolides, perhaps indicating an evolutionary response to milkweed's diverse community of specialist cardenolide-sequestering insect herbivores.

Identification of putative cytochrome P450 monooxygenase genes from the small white butterfly, Pieris rapae (Lepidoptera: Pieridae), and their response to insecticides.

The small white butterfly, Pieris rapae (Lepidoptera: Pieridae), is an important pest on Brassicaceae plants, causing heavy crop loss each year. Cytochrome P450 monooxygenase (CYP) is a superfamily of enzymes involved in the detoxification of various xenobiotic compounds, including insecticides. However, little is known about the role of CYP genes in P. rapae. In this study, we identified 63 CYP genes in P. rapae, and analyzed their phylogenetic relationships, exon-intron structures and genomic locations. Moreover, our insecticide-response transcription profiling showed that LD5 doses of lambda-cyhalothrin, chlorantraniliprole, and abamectin significantly increased expression of five (CYP4M59, CYP6AE119, CYP6AE120, CYP6AE121, and CYP6BD18), three (CYP4AU1, CYP6AE120, and CYP6AW1), and five (CYP4L40, CYP4AU1, CYP6AE119, CYP6AW1, and CYP6BD19) CYP genes, respectively; and LD20 doses of the three pesticides significantly upregulated six (CYP4M59, CYP6AE119, CYP6AE120, CYP6AE121, CYP4AU1, and CYP6BD18), six (CYP4G168, CYP4L40, CYP4AU1, CYP6AE120, CYP6AW1, and CYP6BD19), and five (CYP4L40, CYP4AU1, CYP6AB108, CYP6AE119, and CYP6BD19) genes, respectively. When we used LD50 doses of the three insecticides, we reported significantly elevated expression levels of five (CYP4M59, CYP6AE119, CYP6AE120, CYP6BD17, and CYP6BD18), eight (CYP4G168, CYP4L40, CYP4AU1, CYP6AE120, CYP6AE121, CYP6AW1, CYP6BD18, and CYP6BD19), and six (CYP4L40, CYP4S34, CYP6AB108, CYP6AE119, CYP6AE120, and CYP6BD19) genes, respectively. Our expression analysis also revealed that five (CYP4G168, CYP4G169, CYP4S34, CYP6AW1, and CYP6CT3) and three (CYP4L40, CYP6AN33, and CYP6BD17) CYP genes were mainly expressed in the midgut and fat body, respectively, and one CYP gene (CYP6AE119) in the Malpighian tubules. This is the first large-scale report into the characterization of CYP genes in P. rapae.

Gut peptidases from a specialist herbivore of latex plants are capable of milk protein hydrolysis: Inputs for hypoallergenic milk formulas.

Transitory allergies to cow milk proteins in infants or adults have become a public health problem. Although extensively or partially hydrolyzed cow milk protein formulas are available, these products are costly. Therefore, studies into innovative enzymes to digest cow milk proteins are needed. Danaus plexippus gut peptidases were purified and examined with regard to cow milk protein hydrolysis. The peptidases hydrolyzed caseins and whey proteins. However, after heat treatment, there was a significant improvement in ß-lactoglobulin hydrolysis. The hydrolyzed cow milk proteins were not recognized by anti-casein antibodies and only reacted slightly with antibodies against whey proteins. This performance was better than that of partially hydrolyzed formulas and similar to that of an extensively hydrolyzed formula. These results suggest that D. plexippus gut peptidases are suitable and innovative enzymes to produce hypoallergenic cow milk protein formulas.

Enzyme polymorphism, oxygen and injury: a lipidomic analysis of flight-induced oxidative damage in a succinate dehydrogenase d (Sdhd)-polymorphic insect.

When active tissues receive insufficient oxygen to meet metabolic demand, succinate accumulates and has two fundamental effects: it causes ischemia-reperfusion injury while also activating the hypoxia-inducible factor pathway (HIF). The Glanville fritillary butterfly (Melitaea cinxia) possesses a balanced polymorphism in Sdhd, shown previously to affect HIF pathway activation and tracheal morphology and used here to experimentally test the hypothesis that variation in succinate dehydrogenase affects oxidative injury. We stimulated butterflies to fly continuously in a respirometer (3 min duration), which typically caused episodes of exhaustion and recovery, suggesting a potential for cellular injury from hypoxia and reoxygenation in flight muscles. Indeed, flight muscle from butterflies flown on consecutive days had lipidome profiles similar to those of rested paraquat-injected butterflies, but distinct from those of rested untreated butterflies. Many butterflies showed a decline in flight metabolic rate (FMR) on day 2, and there was a strong inverse relationship between the ratio of day 2 to day 1 FMR and the abundance of sodiated adducts of phosphatidylcholines and co-enzyme Q (CoQ). This result is consistent with elevation of sodiated lipids caused by disrupted intracellular ion homeostasis in mammalian tissues after hypoxia-reperfusion. Butterflies carrying the Sdhd M allele had a higher abundance of lipid markers of cellular damage, but the association was reversed in field-collected butterflies, where focal individuals typically flew for seconds at a time rather than continuously. These results indicate that Glanville fritillary flight muscles can be injured by episodes of high exertion, but injury severity appears to be determined by an interaction between SDH genotype and behavior (prolonged versus intermittent flight).

Evolutionary and structural analyses uncover a role for solvent interactions in the diversification of cocoonases in butterflies.

Multi-omic approaches promise to supply the power to detect genes underlying disease and fitness-related phenotypes. Optimal use of the resulting profusion of data requires detailed investigation of individual candidate genes, a challenging proposition. Here, we combine transcriptomic and genomic data with molecular modelling of candidate enzymes to characterize the evolutionary history and function of the serine protease cocoonase. Heliconius butterflies possess the unique ability to feed on pollen; recent work has identified cocoonase as a candidate gene in pollen digestion. Cocoonase was first described in moths, where it aids in eclosure from the cocoon and is present as a single copy gene. In heliconiine butterflies it is duplicated and highly expressed in the mouthparts of adults. At least six copies of cocoonase are present in Heliconius melpomene and copy number varies across H. melpomene sub-populations. Most cocoonase genes are under purifying selection, however branch-site analyses suggest cocoonase 3 genes may have evolved under episodic diversifying selection. Molecular modelling of cocoonase proteins and examination of their predicted structures revealed that the active site region of each type has a similar structure to trypsin, with the same predicted substrate specificity across types. Variation among heliconiine cocoonases instead lies in the outward-facing residues involved in solvent interaction. Thus, the neofunctionalization of cocoonase duplicates appears to have resulted from the need for these serine proteases to operate in diverse biochemical environments. We suggest that cocoonase may have played a buffering role in feeding during the diversification of Heliconius across the neotropics by enabling these butterflies to digest protein from a range of biochemical milieux.

Comparative transcriptome analysis provides insights of anti-insect molecular mechanism of Cassia obtusifolia trypsin inhibitor against Pieris rapae.

Pieris rapae, a serious Lepidoptera pest of cultivated crucifers, utilizes midgut enzymes to digest food and detoxify secondary metabolites from host plants. A recombinant trypsin inhibitor (COTI) from nonhost plant, Cassia obtusifolia, significantly decreased activities of trypsin-like proteases in the larval midgut on Pieris rapae and could suppress the growth of larvae. In order to know how COTI took effect, transcriptional profiles of P. rapae midgut in response to COTI was studied. A total of 51,544 unigenes were generated and 45.86% of which had homologs in public databases. Most of the regulated genes associated with digestion, detoxification, homeostasis, and resistance were downregulated after ingestion of COTI. Meanwhile, several unigenes in the integrin signaling pathway might be involved in response to COTI. Furthermore, using comparative transcriptome analysis, we detected differently expressing genes and identified a new reference gene, UPF3, by qRT-polymerase chain reaction (PCR). Therefore, it was suggested that not only proteolysis inhibition, but also suppression of expression of genes involved in metabolism, development, signaling, and defense might account for the anti-insect resistance of COTI.

Inhibitory effects of an extract from non-host plants on physiological characteristics of two major cabbage pests.

The diamondback moth (Plutella xylostella) and small white cabbage butterfly (Pieris rapae) are the two main serious pests of cruciferous crops (Brassicaceae) that have developed resistance to chemical control methods. In order to avoid such resistance and also the adverse effects of chemical pesticides on the environment, alternative methods have usually been suggested, including the use of plant enzyme inhibitors. Here, the inhibitory effects of proteinaceous inhibitors extracted from wheat, canola, sesame, bean and triticale were evaluated against the digestive α-amylases, larval growth, development and nutritional indecs of the diamondback moth and small white cabbage butterfly. Our results indicated that triticale and wheat extracts inhibited α-amylolytic activity in an alkaline pH, which is in accordance with the moth and butterfly gut α-amylase optimum pH. Dose-dependent inhibition of two crucifer pests by triticale and wheat was observed using spectrophotometry and gel electrophoresis. Implementation of specificity studies showed that wheat and triticale-proteinaceous extract were inactive against Chinese and purple cabbage amylase. Triticale and wheat were resistant against insects' gut proteases. Results of the feeding bioassay indicated that triticale-proteinaceous extract could cause a significant reduction in survival and larval body mass. The results of the nutritional indecs also showed larvae of both species that fed on a Triticale proteinaceous inhibitor-treated diet had the lowest values for the efficiency of conversion of ingested food and relative growth rate. Our observations suggested that triticale shows promise for use in the management of crucifer pests.

Biochemical characterization a digestive trypsin in the midgut of large cabbage white butterfly, Pieris brassicae L. (Lepidoptera: Pieridae).

A comprehensive study on digestive trypsin was undertaken in the larval midgut of Pieris brassicae L. Results of enzymatic compartmentalization showed a significantly higher activity of crude trypsin in the anterior larval midgut rather than posterior-midgut. Using Diethylaminoethyl cellulose fast flow column chromatography a purified trypsin was obtained by specific activity of 21 U mg-1 protein, recovery of 22%, purification fold of 28-fold and molecular weight of 25 kDa. This purified enzyme showed the highest activity at pH 8 and the corresponding temperature of 40°C. However, the specific inhibitors used including 4-(2-Aminoethyl) benzenesulfonyl fluroride hydrochloride, N-p-Tosyl-L-lysine methyl ester hydrochloride and Soybean Trypsin Inhibitor significantly lowered the activity of the purified enzyme in vitro. Moreover, the activity of trypsin and likewise the nutritional indices were significantly altered in the larval midgut feeding upon the leaves treated by 1 mM concentration of each inhibitor in comparison with control. Determination of enzymatic characteristics of insect trypsins is crucial in paving the path for controlling pests by potential natural compounds via transgenic plants.

Structural basis of autoinhibition and activation of the DNA-targeting ADP-ribosyltransferase pierisin-1.

ADP-ribosyltransferases transfer the ADP-ribose moiety of ßNAD+ to an acceptor molecule, usually a protein that modulates the function of the acceptor. Pierisin-1 is an ADP-ribosyltransferase from the cabbage butterfly Pieris rapae and is composed of N-terminal catalytic and C-terminal ricin B-like domains. Curiously, it ADP-ribosylates the DNA duplex, resulting in apoptosis of various cancer cells, which has raised interest in pierisin-1 as an anti-cancer agent. However, both the structure and the mechanism of DNA ADP-ribosylation are unclear. Here, we report the crystal structures of the N-terminal catalytic domain of pierisin-1, its complex with ßNAD+, and the catalytic domain with the linker connecting it to the ricin B-like domains. We found that the catalytic domain possesses a defined, positively charged region on the molecular surface but that its overall structure is otherwise similar to those of protein-targeting ADP-ribosyltransferases. Electrophoretic mobility shift assays and site-directed mutagenesis indicated that pierisin-1 binds double-stranded but not single-stranded DNA and that Lys122, Lys123, and Lys124, which are found in a loop, and Arg181 and Arg187, located in a basic cleft near the loop, are required for DNA binding. Furthermore, the structure of the catalytic domain with the linker revealed an autoinhibitory mechanism in which the linker occupies and blocks both the ßNAD+- and DNA-binding sites, suggesting that proteolytic cleavage to remove the linker is necessary for enzyme catalysis. Our study provides a structural basis for the DNA-acceptor specificity of pierisin-1 and reveals that a self-regulatory mechanism is required for its activity.

A possible genetic basis for vulnerability in Euphydryas maturna (Lepidoptera: Nymphalidae).

Nearly all of the known populations of Scarce Fritillary, Euphydryas maturna (Linnaeus, 1758), are declining in Western and Central Europe. In order to identify the possible reasons for its vulnerability we surveyed the population genetics of this butterfly species using multi-locus genotype data. Females of our target species lay lots of eggs in one or two batches only and pre-hibernation caterpillars live and feed gregariously in a nest. As a consequence, a random unfavourable event can eliminate most offspring of a particular female resulting in a strong genetic drift effect combined with inbreeding. Thus, our hypothesis regarding the genetic composition of Scarce Fritillary populations suggests that: (1) there will be random fluctuations in allele frequencies from generation to generation; (2) populations should exhibit small effective sizes and a relatively high level of heterozygote deficiency, and; (3) the majority of the individuals in a population will be composed of the offspring of just a few females. In order to test these hypotheses, fine-scale genetic structure was studied in two subpopulations of a Hungarian Scarce Fritillary population for 4 consecutive years (generations) using enzyme polymorphism data. The results supported all of our assumptions. We detected random fluctuation in the frequency of several alleles, small effective population size and the index of heterozygote deficiency (F IS) indicated a considerable level of inbreeding in most samples. Furthermore, average values of relatedness were also fairly high, and we were able to identify 17 putative sib families in total with the two subpopulations based on estimation of individual gametic phases. Thus, the present study suggests that intrinsic factors (e.g. specific life history) might increase the sensitivity of a species to various threatening factors (e.g. habitat loss or fragmentation) and result in the vulnerability of the given species.

Phylogenetic incongruence and the evolutionary origins of cardenolide-resistant forms of Na(+) ,K(+) -ATPase in Danaus butterflies.

Many distantly related insect species are specialized feeders of cardenolide-containing host plants such as milkweed (Asclepias spp.). Previous studies have revealed frequent, parallel substitution of a functionally important amino acid substitution (N122H) in the alpha subunit of Na(+) ,K(+) -ATPase in a number of these species. This substitution facilitates the ability of these insects to feed on their toxic hosts and sequester cardenolides for their own use in defense. Among milkweed butterflies of the genus Danaus, the previously established phylogeny for this group suggests that N122H arose independently and fixed in two distinct lineages. We reevaluate this conclusion by examining Danaus phylogenetic relationships using >400 orthologous gene sequences assembled from transcriptome data. Our results indicate that the three Danaus species known to harbor the N122H substitution are more closely related than previously thought, consistent with a single, common origin for N122H. However, we also find evidence of both incomplete lineage sorting and post-speciation genetic exchange among these butterfly species, raising the possibility of collateral evolution of cardenolide-insensitivity in this species group.