Imidacloprid-induced pathophysiological damage in the midgut of Locusta migratoria (Orthoptera: Acrididae) in the field.

Neonicotinoids are modern insecticides widely used in agriculture worldwide. Their impact on target (nervous system) and non-target (midgut) tissues has been well studied in beneficial insects including honeybees under controlled conditions. However, their detailed effects on pest insects on the field are missing to date. Here, we have studied the effects of the neonicotinoid imidacloprid on the midgut of the pest insect Locusta migratoria caught in the field. We found that in the midgut of imidacloprid-exposed locusts the activity of enzymes involved in reactive oxygen metabolism was perturbed. By contrast, the activity of P450 enzymes that have been shown to be activated in a detoxification response and that were also reported to produce reactive oxygen species was elevated. Probably as a consequence, markers of oxidative stress including protein carbonylation and lipid peroxidation accumulated in midgut samples of these locusts. Histological analyses revealed that their midgut epithelium is disorganized and that the brush border of the epithelial cells is markedly reduced. Indeed, microvilli are significantly shorter, misshapen and possibly non-functional in imidacloprid-treated locusts. We hypothesize that imidacloprid induces oxidative stress in the locust midgut, thereby changing the shape of midgut epithelial cells and probably in turn compromising their physiological function. Presumably, these effects reduce the survival rate of imidacloprid-treated locusts and the damage they cause in the field.

New Zealand Tree and Giant Weta (Orthoptera) Transcriptomics Reveal Divergent Selection Patterns in Metabolic Loci.

Exposure to low temperatures requires an organism to overcome physiological challenges. New Zealand weta belonging to the genera Hemideina and Deinacrida are found across a wide range of thermal environments and therefore subject to varying selective pressures. Here we assess the selection pressures across the weta phylogeny, with a particular emphasis on identifying genes under positive or diversifying selection. We used RNA-seq to generate transcriptomes for all 18 Deinacrida and Hemideina species. A total of 755 orthologous genes were identified using a bidirectional best-hit approach, with the resulting gene set encompassing a diverse range of functional classes. Analysis of ortholog ratios of synonymous to nonsynonymous amino acid changes found 83 genes that are under positive selection for at least one codon. A wide variety of Gene Ontology terms, enzymes, and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways are represented among these genes. In particular, enzymes involved in oxidative phosphorylation, melanin synthesis, and free-radical scavenging are represented, consistent with physiological and metabolic changes that are associated with adaptation to alpine environments. Structural alignment of the transcripts with the most codons under positive selection revealed that the majority of sites are surface residues, and therefore have the potential to influence the thermostability of the enzyme, with the exception of prophenoloxidase where two residues near the active site are under selection. These proteins provide interesting candidates for further analysis of protein evolution.

Calcite formation induced by Ensifer adhaerens, Microbacterium testaceum, Paeniglutamicibacter kerguelensis, Pseudomonas protegens and Rheinheimera texasensis.

A wide range of bacterial species are able to induce calcium carbonate precipitation. Using our own laboratory-preserved strains, we have newly discovered that Ensifer sp. MY11e, Microbacterium sp. TMd9a1, Paeniglutamicibacter sp. MSa1a, Pseudomonas sp. GTc3, and Rheinheimera sp. ATWe6 can induce the formation of calcite crystals on an agar medium. Type strains of their closely related species (Ensifer adhaerens, Microbacterium testaceum, Paeniglutamicibacter kerguelensis, Pseudomonas protegens, and Rheinheimera texasensis) could also induce calcite formation. Although the initial pH value of the agar medium was 6.1, the pH of the agar media containing calcite, induced by cultivation of the 10 bacterial strains, increased to 8.0-8.4. The ammonification (oxidative deamination) of amino acids may been responsible for this increase in pH. The crystals formed both on and around the bacterial colonies. Furthermore, when these strains (excepting two Microbacterium strains) were cultivated on a cellulose acetate membrane filter (0.20 µm pore size) resting on the surface of the agar medium (i.e., in the membrane filter culture method), the crystals formed on the agar medium separate from the bacterial cells. These results indicate that the bacterial cells did not necessarily become nucleation sites for these crystals. We also investigated whether the studied strains could be applied to the biocementation of sand, and found that only two Ensifer strains were able to form large sand lumps.

Structural, tribological, and mechanical properties of the hind leg joint of a jumping insect: Using katydids to inform bioinspired lubrication systems.

This study investigates the structural properties of the hind leg femur-tibia joint in adult katydids (Orthoptera: Tettigoniidae), including its tribological and mechanical properties. It is of particular interest because the orthopteran (e.g., grasshoppers, crickets, and katydids) hind leg is highly specialized for jumping. We show that the katydid hind leg femur-tibia joint had unique surfaces and textures, with a friction coefficient (µ) at its coupling surface of 0.053±0.001. Importantly, the sheared surfaces at this joint showed no sign of wear or damage, even though it had undergone thousands of external shearing cycles. We attribute its resiliency to a synergistic interaction between the hierarchical surface texture/pattern on the femoral surfaces, a nanograded internal nanostructure of articulating joints, and the presence of lubricating lipids on the surface at the joint interface. The micro/nanopatterned surface of the katydid hind leg femur-tibia joint enables a reduction in the total contact area, and this significantly reduces the adhesive forces between the coupling surfaces. In our katydids, the femur and tibia joint surfaces had a maximum effective elastic modulus (Eeff) value of 2.6GPa and 3.9GPa, respectively. Presumably, the decreased adhesion through the reduction of van der Waals forces prevented adhesive wear, while the contact between the softer textured surface and harder smooth surface avoided abrasive wear. The results from our bioinspired study offer valuable insights that can inform the development of innovative coatings and lubrication systems that are both energy efficient and durable. STATEMENT OF SIGNIFICANCE: Relative to body length, insects can outjump most animals. They also accelerate their bodies at a much faster rate. Orthopterans (e.g., grasshoppers, crickets, and katydids) have hind legs that are specialized for jumping. Over an individual's lifetime, the hind leg joint endures repeated cycles of flexing and extending, including jumping, and its efficiency and durability easily surpass that of most mechanical devices. Although the efficient functioning of insect joints has long been recognized, the mechanism by which insect joints experience friction/adhesion/wear, and operate efficiently/reliably is still largely unknown. Our study on the structural, tribological, and mechanical properties of the orthopteran hind leg joints reveals the potential of katydid bioinspired research leading to more effective coatings and lubrication systems.

Rampant nuclear insertion of mtDNA across diverse lineages within Orthoptera (Insecta).

Nuclear mitochondrial pseudogenes (numts) are non-functional fragments of mtDNA inserted into the nuclear genome. Numts are prevalent across eukaryotes and a positive correlation is known to exist between the number of numts and the genome size. Most numt surveys have relied on model organisms with fully sequenced nuclear genomes, but such analyses have limited utilities for making a generalization about the patterns of numt accumulation for any given clade. Among insects, the order Orthoptera is known to have the largest nuclear genome and it is also reported to include several species with a large number of numts. In this study, we use Orthoptera as a case study to document the diversity and abundance of numts by generating numts of three mitochondrial loci across 28 orthopteran families, representing the phylogenetic diversity of the order. We discover that numts are rampant in all lineages, but there is no discernable and consistent pattern of numt accumulation among different lineages. Likewise, we do not find any evidence that a certain mitochondrial gene is more prone to nuclear insertion than others. We also find that numt insertion must have occurred continuously and frequently throughout the diversification of Orthoptera. Although most numts are the result of recent nuclear insertion, we find evidence of very ancient numt insertion shared by highly divergent families dating back to the Jurassic period. Finally, we discuss several factors contributing to the extreme prevalence of numts in Orthoptera and highlight the importance of exploring the utility of numts in evolutionary studies.

Acquisition of Cry1Ac protein by non-target arthropods in Bt soybean fields.

Soybean tissue and arthropods were collected in Bt soybean fields in China at different times during the growing season to investigate the exposure of arthropods to the plant-produced Cry1Ac toxin and the transmission of the toxin within the food web. Samples from 52 arthropod species/taxa belonging to 42 families in 10 orders were analysed for their Cry1Ac content using enzyme-linked immunosorbent assay (ELISA). Among the 22 species/taxa for which three samples were analysed, toxin concentration was highest in the grasshopper Atractomorpha sinensis and represented about 50% of the concentration in soybean leaves. Other species/taxa did not contain detectable toxin or contained a concentration that was between 1 and 10% of that detected in leaves. These Cry1Ac-positive arthropods included a number of mesophyll-feeding Hemiptera, a cicadellid, a curculionid beetle and, among the predators, a thomisid spider and an unidentified predatory bug belonging to the Anthocoridae. Within an arthropod species/taxon, the Cry1Ac content sometimes varied between life stages (nymphs/larvae vs. adults) and sampling dates (before, during, and after flowering). Our study is the first to provide information on Cry1Ac-expression levels in soybean plants and Cry1Ac concentrations in non-target arthropods in Chinese soybean fields. The data will be useful for assessing the risk of non-target arthropod exposure to Cry1Ac in soybean.

De novo transcriptome hybrid assembly and validation in the European earwig (Dermaptera, Forficula auricularia).

BACKGROUND: The European earwig (Forficula auricularia) is an established system for studies of sexual selection, social interactions and the evolution of parental care. Despite its scientific interest, little knowledge exists about the species at the genomic level, limiting the scope of molecular studies and expression analyses of genes of interest. To overcome these limitations, we sequenced and validated the transcriptome of the European earwig. METHODOLOGY AND PRINCIPAL FINDINGS: To obtain a comprehensive transcriptome, we sequenced mRNA from various tissues and developmental stages of female and male earwigs using Roche 454 pyrosequencing and Illumina HiSeq. The reads were de novo assembled independently and screened for possible microbial contamination and repeated elements. The remaining contigs were combined into a hybrid assembly and clustered to reduce redundancy. A comparison with the eukaryotic core gene dataset indicates that we sequenced a substantial part of the earwig transcriptome with a low level of fragmentation. In addition, a comparative analysis revealed that more than 8,800 contigs of the hybrid assembly show significant similarity to insect-specific proteins and those were assigned for Gene Ontology terms. Finally, we established a quantitative PCR test for expression stability using commonly used housekeeping genes and applied the method to five homologs of known sex-biased genes of the honeybee. The qPCR pilot study confirmed sex specific expression and also revealed significant expression differences between the brain and antenna tissue samples. CONCLUSIONS: By employing two different sequencing approaches and including samples obtained from different tissues, developmental stages, and sexes, we were able to assemble a comprehensive transcriptome of F. auricularia. The transcriptome presented here offers new opportunities to study the molecular bases and evolution of parental care and sociality in arthropods.

The correlation of genome size and DNA methylation rate in metazoans.

Total DNA methylation rates are well known to vary widely between different metazoans. The phylogenetic distribution of this variation, however, has not been investigated systematically. We combine here publicly available data on methylcytosine content with the analysis of nucleotide compositions of genomes and transcriptomes of 78 metazoan species to trace the evolution of abundance and distribution of DNA methylation. The depletion of CpG and the associated enrichment of TpG and CpA dinucleotides are used to infer the intensity and localization of germline CpG methylation and to estimate its evolutionary dynamics. We observe a positive correlation of the relative methylation of CpG motifs with genome size. We tested this trend successfully by measuring total DNA methylation with LC/MS in orthopteran insects with very different genome sizes: house crickets, migratory locusts and meadow grasshoppers. We hypothesize that the observed correlation between methylation rate and genome size is due to a dependence of both variables from long-term effective population size and is driven by the accumulation of repetitive sequences that are typically methylated during periods of small population sizes. This process may result in generally methylated, large genomes such as those of jawed vertebrates. In this case, the emergence of a novel demethylation pathway and of novel reader proteins for methylcytosine may have enabled the usage of cytosine methylation for promoter-based gene regulation. On the other hand, persistently large populations may lead to a compression of the genome and to the loss of the DNA methylation machinery, as observed, e.g., in nematodes.

Genesis of spermatodesms in Tylopsis liliifolia (Orthoptera: Phaneropterinae) and their transit in the male genital tract.

The spermatodesms of Tylopsis liliifolia form in the most proximal follicular cysts and are composed of a large number of sperm held together by a cap located in the anterior region of the acrosome. The cap is formed by short thin fibrils, loosely arranged at random, probably derived from secretory activity of cells of the cyst wall. Compared to other Tettigoniidae, a peculiar feature is acrosomal wings that twist gradually around the anterior region of the nucleus; at the end of the twisting process, the region of the sperm acrosome, observed in cross section, shows a typical spiral form. Spermatodesms do not undergo any substantial changes in the spermiduct. The epithelial cells of the wall have secretory activity and many show marked spermiophagic activity, which is conducted by epithelial cell protrusions that envelop the gametes, taking them into the cytoplasm. When removed from seminal vesicles and observed in vivo, spermatodesms show accentuated corkscrew movement, and when observed by SEM, slight torsion. Thus organized, spermatodesms are transferred to the spermatophore during mating, where they are transformed before reaching the seminal receptacle.

Role of the fat body in the cave crickets Troglophilus cavicola and Troglophilus neglectus (Rhaphidophoridae, Saltatoria) during overwintering.

The cave crickets Troglophilus cavicola and Troglophilus neglectus are the most widely distributed European species of the family Rhaphidophoridae. Their life cycles span two years. They overwinter twice in caves in 4-6 months lasting diapause, T. cavicola in warmer microhabitats. In caves, older T. cavicola undergo sexual maturation, while T. neglectus do not. We hypothesized that the use of energy-supplying compounds and reserve proteins in the fat body is more extensive in T. cavicola than in T. neglectus. We analyzed the contents and morphology of lipid droplets, glycogen rosettes and protein granula at the beginning, the middle and the end of overwintering applying optic, TEM and biochemical methods. In all individuals, the fat body is composed of about 40 oval ribbons consisted of gradually changing adipocytes and urocytes. T. cavicola use glycogen continuously, and stop using lipids in the middle of overwintering, while this is inverse in T. neglectus. Till the middle of overwintering, all individuals exploit proteins, afterwards they are unevenly exploited. We found that the fat body is differently engaged in metabolism of both cave crickets during overwintering, supporting a more glycogen-dependent metabolism in T. cavicola, and a more lipid-dependent one in T. neglectus.

A novel member of the adipokinetic peptide family in a "living fossil", the ice crawler Galloisiana yuasai, is the first identified neuropeptide from the order Grylloblattodea.

This is the first report on the structural identity of a neuropeptide of the insect order Grylloblattodea. A peptide was isolated and sequenced from the retrocerebral corpora cardiaca-corpora allata complex of the ice crawler, Galloisiana yuasai. The sequence of the peptide was deduced from the multiple MS(N) electrospray mass data as that of an octapeptide: pGlu-Val-Asn-Phe-Ser-Pro-Thr-Trp amide. The retention time on reversed-phase HPLC and the CID MS(2) mass spectra of a synthetic peptide with the same primary structure were exactly the same as of the natural peptide. The sequence represents a novel peptide of the adipokinetic hormone family which contains presently 50 members. The primary structure differs in only one position to a few previously discovered AKHs. A scenario is outlined that makes it likely that the most recently discovered insect order, the Mantophasmatodea, and the Grylloblattodea are closely related.

Structure of male accessory glands of Bolivarius siculus (fischer) (Orthoptera, Tettigoniidae) and protein analysis of their secretions.

In Tettigoniidae (Orthoptera), male reproductive accessory glands are involved in the construction of a two-part spermatophore; one part, the spermatophylax, is devoid of sperm and considered a nuptial gift. The morphology, ultrastructure, and secretion protein content of the male reproductive accessory glands from Bolivarius siculus were investigated. Two main groups of gland tubules open into the ejaculatory duct: the "first-order" glands, a number of large anterior tubules, and the "second-order" glands, smaller and more numerous tubules positioned posteriorly. Along with a further subdivision of the gland tubules, we here describe for the first time an additional gland group, the intermediate tubules, which open between first and second-order glands. The mesoderm-derived epithelium of all glands is a single layer of microvillated cells, which can be either flattened or cylindric in the proximal or distal region of the same gland. Epithelial cells, very rich in RER and Golgi systems, produce secretions of both electron-dense granules and globules or electron-transparent material, discharged into the gland lumen by apocrine or merocrine mechanisms, respectively. With one exception, a unique electrophoresis protein profile was displayed by each of the gland types, paralleling their unique morphologies. To assess the contribution of different types of accessory glands to the construction of the spermatophore, the protein patterns of the gland secretions were compared with those of the extracts from the two parts of the spermatophore. All samples showed bands distributed in a wide range of molecular weight, including proteins of very low molecular mass. However, one major high molecular weight protein band (>180 kDa) is seen exclusively in extracts from the first-order glands, and corresponds to an important protein component of the spermatophylax.

Relationship between incomplete synapsis and chiasma localization.

One of the subjects within the meiotic field that has been actively investigated in the recent years is the temporal and functional relationships between meiotic recombination, cohesin loading and synaptonemal complex (SC) assembly. Although the study of meiotic mutants has shed some light, many questions remain to be answered. Here, we have studied this topic in the orthopteran Paratettix meridionalis, a species with telocentric chromosomes, which shows two unusual cytological features: pairing and synapsis of homologues during prophase I are restricted to the non-centromeric distal regions and extremely distal chiasma localization in metaphase I bivalents. In order to determine whether there is a relationship between both phenomena, we have used: (1) a spreading technique for following the ultrastructure of SC assembly and (2) immunofluorescence for SMC3 and SMC1alpha cohesin subunits, which mark the development of the axial element (a SC component); the histone gamma-H2AX, which mostly labels the sites of double-strand breaks; and the recombinase RAD51. Spermatocytes showed conspicuous polarization of both the maturation of cohesin axes and the initiation of meiotic recombination events. Consequently, it is proposed that maturation of cohesin axes, which begins in very distal regions, could drive the latter loading of recombinases to such regions. This restricted distribution of recombination events along homologues would finally be responsible for the incomplete pairing and synapsis observed in all autosomes of the complement and hence for chiasma localization.

Peptides of the adipokinetic hormone/red pigment-concentrating hormone family with special emphasis on Caelifera: primary sequences and functional considerations contrasting grasshoppers and locusts.

The presented work is a hybrid of an overview and an original research paper. First, we review briefly the structure, biosynthesis, release, mode of action and function of those peptides that constitute the adipokinetic/red pigment-concentrating family. Second, we collate the data on primary sequences available for caeliferan orthoptera, i.e. grasshoppers and locusts, and add a number of new data from previously unpublished work. The data are interpreted in conjunction with morphological and molecular biology data with respect to phylogenetic relationships of these various taxa. Finally, we discuss the differences between the adipokinetic response of grasshoppers and locusts to corpus cardiacum extract or synthetic adipokinetic hormone with regard to flight ability, phase polymorphism, age, presence of adipokinetic hormones, lipophorin system and other parameters. It appears that the higher hyperlipaemic response is always correlated with pronounced flight ability.

Female bushcrickets fuel their metabolism with male nuptial gifts.

In many arthropods, such as bushcrickets, males donate protein-rich nuptial gifts-so-called spermatophores-to females, which females ingest while the sperm enter the female's reproductive tract. Previously, it was shown that females route spermatophore nutrients over the course of hours and days to egg production or body synthesis. We investigated whether female bushcrickets fuel their metabolism with spermatophores immediately after consumption. We fed two male groups diets that were either enriched or depleted in 13C, and then tracked the isotopic changes in exhaled breath in female bushcrickets after spermatophore consumption. Within 3 hours, the stable carbon isotope ratio (delta13C) of female breath converged on the ratio of the male donor of the nuptial gift. This supports the idea that females quickly routed nutrients to metabolism, receiving immediate benefits from spermatophore feeding.

The presence and distribution of crustacean cardioactive peptide in the central and peripheral nervous system of the stick insect, Baculum extradentatum.

Crustacean cardioactive peptide (CCAP)-like immunoreactivity was localized and quantified in the central and peripheral nervous system of the Vietnamese stick insect, Baculum extradentatum, using immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). The brain, frontal ganglion, suboesophageal ganglion and ventral nerve cord displayed neurons and processes with CCAP-like immunoreactivity. The brain, in comparison to the other parts of the central nervous system, contained the greatest amount of CCAP (167 +/- 18 fmol), and showed CCAP-like staining in neurons, neuropil regions and the central complex. There were also CCAP-like varicosities and processes associated with the corpus cardiacum. The alimentary canal of B. extradentatum contained CCAP with the largest amount localized in the midgut (1110 +/- 274 fmol CCAP equivalents). The midgut contained numerous endocrine-like cells which stained positively for CCAP, whereas the foregut and hindgut revealed an extensive network of CCAP-like immunoreactive axons and varicosities. Based on physiological assays, the hindgut of the stick insect was found to be sensitive to CCAP, showing dose-dependent increases in contractions with threshold at 10(-10) M CCAP and maximal response at 5 x 10(-7) M CCAP. There were negligible quantities of CCAP in the oviducts and no CCAP-like immunoreactivity was associated with the oviducts. CCAP had no effect on spontaneous contractions of the oviducts. The presence of CCAP in the central nervous system, the stomatogastric nervous system, the corpus cardiacum and the alimentary canal, suggest broad ranging roles for CCAP in B. extradentatum.

Heavy metal accumulation, heat shock protein expression and cytogenetic changes in Tetrix tenuicornis (L.) (Tetrigidae, Orthoptera) from polluted areas.

The orthopteran insect Tetrix tenuicornis, collected from polluted and unpolluted areas, was used to study heavy metal accumulation and its impact on stress protein levels and on changes in the number and morphology of chromosomes in mitotic and meiotic cells. During two consecutive years, insects were collected from polluted areas of zinc-lead mine spoils near Boleslaw (Poland) and from unpolluted areas near Busko and Staszów (Poland). T. tenuicornis from the polluted area showed 1.5, 4.03, 4.32 and 41.73 times higher concentrations of copper (Cu), zinc (Zn), lead (Pb) and cadmium (Cd), respectively, than insects of the same species collected from unpolluted areas. Insects exposed to heavy metals showed only small changes, and rather a decrease in the concentration of constitutive and inducible heat shock proteins Hsp70, the level of which increases under stress conditions. A cytogenetic study of T. tenuicornis revealed intra-population anomalies in chromosome number and morphology in mitotic and meiotic cells and the presence of an additional B chromosome in germinal cells. In 50% of females collected from polluted areas, mosaic oogonial mitotic chromosome sets and diploid, hypo- or hypertetraploid, tetraploid, and octoploid chromosome numbers were detected. In turn, 14.6% of males showed a heterozygous deficiency of chromatin in L2 and M3 bivalents in addition to the presence of B chromosomes.

A conserved domain of alkaline phosphatase expression in the Malpighian tubules of dipteran insects.

Malpighian (renal) tubules are key components of the insect osmoregulatory system and show correspondingly great diversity in both number and length. Recently, the organisation of the Drosophila melanogaster tubule has been elucidated by enhancer trapping, and an array for functional properties has been shown to align with the functional domains. In Drosophila, there is a lower tubule domain, which coincides with expression of alkaline phosphatase and delineates the absorptive region of the tubule. Here, these observations are extended to three dipteran vectors of disease (Aedes aegypti, Anopheles stephensii and Glossina morsitans) and a non-dipteran out-group, Schistocerca gregaria (Orthoptera). Despite a huge range in cell number and size, alkaline phosphatase was found on the apical surface of the lower 10% of each of the dipteran tubules but nowhere within the orthopteran tubule. An alkaline phosphatase lower tubule domain is thus conserved among Diptera. Cell counts are also provided for each species. As in Drosophila, stellate cells are not found in the lower tubule domain of Anopheles or Aedes tubules, confirming the unique genetic identity of this domain. As previously reported, we failed to find stellate cells in Schistocerca but, remarkably, also failed to find them in Glossina, the dipteran most closely related to Drosophila. The orthodoxy that stellate cells are unique to, and general among, Diptera may thus require revision.

The two facies of pyrrolizidine alkaloids: the role of the tertiary amine and its N-oxide in chemical defense of insects with acquired plant alkaloids.

Larvae of Creatonotos transiens (Lepidoptera, Arctiidae) and Zonocerus variegatus (Orthoptera, Pyrgomorphidae) ingest 14C-labeled senecionine and its N-oxide with the same efficiency but sequester the two tracers exclusively as N-oxide. Larvae of the non-sequestering Spodoptera littoralis eliminate efficiently the ingested alkaloids. During feeding on the two alkaloidal forms transient levels of senecionine (but not of the N-oxide) are built up in the haemolymph of S. littoralis larvae. Based on these results, senecionine [18O]N-oxide was fed to C. transiens larvae and Z. variegatus adults. The senecionine N-oxide recovered from the haemolymph of the two insects shows an almost complete loss of 18O label, indicating reduction of the orally fed N-oxide in the guts, uptake of the tertiary alkaloid and its re-N-oxidation in the haemolymph. The enzyme responsible for N-oxidation is a soluble mixed function monooxygenase. It was isolated from the haemolymph of the sequestering arctiid Tyria jacobaeae and purified to electrophoretic homogeneity. The enzyme is a flavoprotein with a native Mr of 200000 and a subunit Mr of 51000. It shows a pH optimum at 7.0, has its maximal activity at a temperature of 40-45 degrees C and an isoelectric point at pH 4.9. The reaction is strictly NADPH-dependent (Km 1.3 microM). From 20 pyrrolizidine alkaloids so far tested as substrates, the enyzme N-oxidizes only alkaloids with structural elements which are essential for hepatotoxic and genotoxic pyrrolizidine alkaloids (i.e. 1,2-double bond, esterification of the allylic hydroxyl group, presence of a second free or esterified hydroxyl group at carbon 7). A great variety of related alkaloids and xenobiotics were tested as substrate, none was accepted. The Km values of senecionine, monocrotaline and heliotrine, representing the three main types of pyrrolizidine alkaloids, are 1.3 microM, 12.5 microM and 290 microM, respectively. The novel enzyme was named senecionine N-oxygenase (SNO). The enzyme was partially purified from two other arctiids. The three SNOs show the same general substrate specificity but differ in their affinities towards the main structural types of pyrrolizidine alkaloids. The enzymes from the two generalists (Creatonotos transiens and Arctia caja) display a broader substrate affinity than the enzyme from the specialist (Tyria jacobaeae). The two molecular forms of pyrrolizidine alkaloids, the lipophilic protoxic tertiary amine and its hydrophilic nontoxic N-oxide are discussed in respect to their bioactivation and detoxification in mammals and their role as defensive chemicals in specialized insects. Pyrrolizidine-alkaloid-sequestering insects store the alkaloids as nontoxic N-oxides which are reduced in the guts of any potential insectivore. The lipophilic tertiary alkaloid is absorbed passively and then bioactivated by cytochrome P-450 oxidase.

Native vitellins are modified during ovarian development in the stick insect Carausius morosus (Br.).

Vitellins from ovarian follicles and newly laid eggs of the stick insect Carausius morosus were examined by ion exchange chromatography on a HPLC Mono Q column. Under these conditions, vitellins from newly laid eggs resolved as two distinct peaks, referred to as VtA and VtB, that eluted at 8.5 and 12.0 min, respectively. On native gels, both VtA and VtB separated into two different variant forms (VtA' and VtA", VtB' and VtB"). By two-dimensional gel electrophoresis, VtA' and VtA" were shown to contain polypeptides A1, A2 and A3. On the other hand, VtB' and VtB" appeared to comprise polypeptides B1 and B2 and B1, A1, A2, B2 and A3*, respectively. A similar Vt polypeptide composition was also observed by size-exclusion chromatography of vitellins from newly laid eggs. Vitellins from early vitellogenic ovarian follicles resolved into a single chromatographic peak at 7.5 min that coeluted with a major peak from the hemolymph of egg-laying females. Ovarian follicles progressively more advanced in development exhibited a more complex chromatographic profile, consisting of three separate peaks. By two-dimensional gel immunoelectrophoresis, vitellins from ovarian follicles appeared to consist of two closely related, immunologically cross-reacting antigens that gradually shifted apart as ovarian development proceeded to completion. By size-exclusion chromatography, each Vt from ovarian follicles was shown to consist of a unique set of polypeptides different from those listed above. Single ovarian follicles were fractionated into yolk granules and yolk fluid ooplasm and tested by immunoblotting against Mab 12. Under these conditions, VtA variant forms in yolk granules and yolk fluid ooplasm reacted differently. Sections from ovarian follicles in different developmental stages were exposed to Mab 12 and stained with a peroxidase-conjugated, goat anti-mouse antibody. Regardless of the developmental stage attained, staining for peroxidase was restricted to free yolk granules, suggesting that native vitellins in stick insects are structurally modified upon fusion into the yolk fluid ooplasm.