The Enhanced Expression of Cruzipain-Like Molecules in the Phytoflagellate Phytomonas serpens Recovered From the Invertebrate and Plant Hosts

Phytomonas serpens is a protozoan parasite that alternates its life cycle between two hosts: an invertebrate vector and the tomato fruit. This phytoflagellate is able to synthesize proteins displaying similarity to the cysteine peptidase named cruzipain, an important virulence factor from Trypanosoma cruzi, the etiologic agent of Chagas disease. Herein, the growth of P. serpens in complex medium (BHI) supplemented with natural tomato extract (NTE) resulted in the increased expression of cysteine peptidases, as verified by the hydrolysis of the fluorogenic substrate Z-Phe-Arg-AMC and by gelatin-SDS-PAGE. Phytoflagellates showed no changes in morphology, morphometry and viability, but the proliferation was slightly reduced when cultivated in the presence of NTE. The enhanced proteolytic activity was accompanied by a significant increase in the expression of cruzipain-like molecules, as verified by flow cytometry using anti-cruzipain antibodies. In parallel, parasites incubated under chemically defined conditions (PBS supplemented with glucose) and added of different concentration of NTE revealed an augmentation in the production of cruzipain-like molecules in a typically dose-dependent way. Similarly, P. serpens recovered from the infection of mature tomatoes showed an increase in the expression of molecules homologous to cruzipain; however, cells showed a smaller size compared to parasites grown in BHI medium. Furthermore, phytoflagellates incubated with dissected salivary glands from Oncopeltus fasciatus or recovered from the hemolymph of infected insects also showed a strong enhance in the expression of cruzipain-like molecules that is more relevant in the hemolymph. Collectively, our results showed that cysteine peptidases displaying similarities to cruzipain are more expressed during the life cycle of the phytoflagellate P. serpens both in the invertebrate and plant hosts.

Out from under the wing: reconceptualizing the insect wing gene regulatory network as a versatile, general module for body-wall lobes in arthropods

Body plan evolution often occurs through the differentiation of serially homologous body parts, particularly in the evolution of arthropod body plans. Recently, homeotic transformations resulting from experimental manipulation of gene expression, along with comparative data on the expression and function of genes in the wing regulatory network, have provided a new perspective on an old question in insect evolution: how did the insect wing evolve? We investigated the metamorphic roles of a suite of 10 wing- and body-wall-related genes in a hemimetabolous insect, Oncopeltus fasciatus . Our results indicate that genes involved in wing development in O. fasciatus play similar roles in the development of adult body-wall flattened cuticular evaginations. We found extensive functional similarity between the development of wings and other bilayered evaginations of the body wall. Overall, our results support the existence of a versatile development module for building bilayered cuticular epithelial structures that pre-dates the evolutionary origin of wings. We explore the consequences of reconceptualizing the canonical wing-patterning network as a bilayered body-wall patterning network, including consequences for long-standing debates about wing homology, the origin of wings and the origin of novel bilayered body-wall structures. We conclude by presenting three testable predictions that result from this reconceptualization.

Structural Analysis Revealed the Interaction of Cardenolides from Calotropis procera with Na+/K+ ATPases from Herbivores

Background : The herbivores Danaus plexippus (Lepidoptera), Oncopeltus fasciatus and Aphis nerii (Hemiptera) are specialist insects that feed on Calotropis procera (Apocynaceae) (Sodom Apple). At least 35 chemically distinct cardenolides have been reported in C. procera. Objective We aimed to evaluate the interaction between cardenolides and Na+/K+ ATPases from herbivores. Methods : The Na+/K+ ATPases from these insects were modeled and docking studies were performed with cardenolides from C. procera. Results : The replacement of serine in sensitive Na+/K+ ATPase with histidine, phenylalanine and tyrosine in the structures examined suggests spatial impairment caused by interaction, probably making the herbivorous insects resistant against the cardenolides of C. procera. In addition, the ability of the insects to avoid cardenolide toxicity was not correlated with cardenolide polarity. Therefore, the plant fights predation through molecular diversity and the insects, regardless of their taxonomy, face this molecular diversity through amino acid replacements at key positions of the enzyme targeted by the cardenolides. Conclusions : The results show the arsenal of chemically distinct cardenolides synthesized by C. procera.

Immune function of the serosa in hemimetabolous insect eggs

Insects comprise more than a million species and many authors have attempted to explain this success by evolutionary innovations. A much overlooked evolutionary novelty of insects is the serosa, an extraembryonic epithelium around the yolk and embryo. We have shown previously that this epithelium provides innate immune protection to eggs of the beetle Tribolium castaneum. It remained elusive, however, whether this immune competence evolved in the Tribolium lineage or is ancestral to all insects. Here, we expand our studies to two hemimetabolous insects, the bug Oncopeltus fasciatus and the swarming grasshopper Locusta migratoria. For Oncopeltus, RNA sequencing reveals an extensive response upon infection, including the massive upregulation of antimicrobial peptides (AMPs). We demonstrate antimicrobial activity of these peptides using in vitro bacterial growth assays, and describe two novel AMP families called Serosins and Ovicins. For both insects, qPCRs show immune competence of the eggs when the serosa is present, and in situ hybridizations demonstrate that immune gene expression is localized in the serosa. This first evidence from hemimetabolous insect eggs suggests that immune competence is an ancestral property of the serosa. The evolutionary origin of the serosa with its immune function might have facilitated the spectacular radiation of the insects.

Development of the Pre-gnathal Segments in the Milkweed Bug Oncopeltus fasciatus Suggests They Are Not Serial Homologs of Trunk Segments

The three anterior-most segments in arthropods contain the ganglia that make up the arthropod brain. These segments, the pre-gnathal segments (PGS), are known to exhibit many developmental differences to other segments, believed to reflect their divergent morphology. We have analyzed the expression and function of the genes involved in the conserved segment-polarity network, including genes from the Wnt and Hedgehog pathways, in the PGS, compared with the trunk segments, in the hemimetabolous insect Oncopeltus fasciatus. Gene function was tested by manipulating expression through RNA interference against components of the two pathways. We show that there are fundamental differences in the expression patterns of the segment polarity genes, in the timing of their expression and in the interactions among them in the process of pre-gnathal segment generation, relative to all other segments. We argue that given these differences, the PGS should not be considered serially homologous to trunk segments. This realization raises important questions about the differing evolutionary ancestry of different regions of the arthropod head.

Atividade inseticida da neolignana Burchelina sobre Oncopeltus fasciatus

Insetos praga são modelos de estudo na busca de métodos alternativos de controle que não agridam o homem e o meio ambiente, devido aos efeitos nocivos às plantações de grãos causando grandes impactos econômicos. O estudo buscou avaliar o potencial inseticida e ovicida da neolignana Burchelina sobre Oncopeltus fasciatus. Os bioensaios foram conduzidos no Laboratório de Insetos Vetores, da Universidade de Vassouras sobre ninfas de 5º estádio, em triplicatas por grupo e com três repetições. Nas ninfas, a substância foi utilizada por tratamento tópico nas concentrações de 1, 10, 50, 100 e 200 µg/mL. Após o tratamento, as ninfas foram mantidas em temperatura ambiente, dieta habitual (água e semente de girassol) e observadas durante 20 dias. Os bioensaios sobre a massa de ovos foi realizada por aplicação tópica na concentração de 82 µg/mL (DL50). Foram utilizados 20 mg de ovos/20 µL da substância, em triplicata. O tratamento tópico com Burchelina resultou na mortalidade de ninfas de 5º estádio em 73% (200µg) e de 100% de toxicidade (82µg) sobre os ovos de O. fasciatus. Estes resultados demonstraram o potencial inseticida da Burchelina e sua importância no controle do Hemiptera Lygaidae.

Distinct developmental mechanisms influence sexual dimorphisms in the milkweed bug Oncopeltus fasciatus

Sexual dimorphism is a common feature of animals. Sex determination mechanisms vary widely among species and evolve rapidly. Until recently studies have found consistent mechanisms across the body of each individual determine female or male dimorphic body structures. In sexually dimorphic cells throughout the body of Drosophila, the relative dosage of autosomes and X chromosomes leads indirectly to alternatively spliced transcripts from the gene doublesex. The female Dsx isoform interacts with the mediator complex protein encoded by intersex to activate female development in flies. In males the transcription factor encoded by fruitless promotes male-specific behavior. In the milkweed bug Oncopeltus fasciatus, we find a requirement for different combinations of these genes during development of distinct dimorphic structures, within the same sex, suggesting a previously unappreciated level of diversity in sex determination. While intersex and fruitless are structurally conserved, doublesex has a history of duplication and divergence among Paraneoptera. Three doublesex paralogs in O. fasciatus produce multiple transcripts with sex- and tissue-specific expression. intersex and fruitless are expressed across the body, in females and males. RNA interference reveals only one doublesex paralog functions in somatic sex determination. Knockdown of doublesex and fruitless produces intersex phenotypic conditions in two sexually dimorphic structures: genitalia and abdominal sternites. In contrast, intersex is required for dimorphic development of female and male genitalia, but not for sternite dimorphism. These results reveal sex determination roles for intersex and fruitless distinct from their orthologs in other insects. Our results illuminate a novel form of developmental diversity in insect sex determination.

The essential role of Dnmt1 in gametogenesis in the large milkweed bug Oncopeltus fasciatus

Given the importance of DNA methylation in protection of the genome against transposable elements and transcriptional regulation in other taxonomic groups, the diversity in both levels and patterns of DNA methylation in the insects raises questions about its function and evolution. We show that the maintenance DNA methyltransferase, DNMT1, affects meiosis and is essential to fertility in milkweed bugs, Oncopeltus fasciatus, while DNA methylation is not required in somatic cells. Our results support the hypothesis that Dnmt1 is required for the transition of germ cells to gametes in O. fasciatus and that this function is conserved in male and female gametogenesis. They further suggest that DNMT1 has a function independent of DNA methylation in germ cells. Our results raise the question of how a gene so critical in fitness across multiple insect species can have diverged widely across the insect tree of life.

Dietary cardenolides enhance growth and change the direction of the fecundity-longevity trade-off in milkweed bugs (Heteroptera: Lygaeinae)

1. Sequestration, i.e., the accumulation of plant toxins into body tissues for defence, is primarily observed in specialised insects. Sequestration was frequently predicted to incur a physiological cost mediated by increased exposure to plant toxins and may require resistance traits different from those of non-sequestering insects. Alternatively, sequestering species could experience a cost in the absence of toxins due to selection on physiological homeostasis under permanent exposure of sequestered toxins in body tissues. 2. Milkweed bugs (Heteroptera: Lygaeinae) sequester high amounts of plant-derived cardenolides. Although being potent inhibitors of the ubiquitous animal enzyme Na+/K+-ATPase, milkweed bugs can tolerate cardenolides by means of resistant Na+/K+-ATPases. Both adaptations, resistance and sequestration, are ancestral traits shared by most species of the Lygaeinae. 3. Using four milkweed bug species and the related European firebug (Pyrrhocoris apterus) showing different combinations of the traits ′cardenolide resistance′ and ′cardenolide sequestration′, we set out to test how the two traits affect larval growth upon exposure to dietary cardenolides in an artificial diet system. While cardenolides impaired the growth of P. apterus nymphs neither possessing a resistant Na+/K+-ATPase nor sequestering cardenolides, growth was not affected in the non-sequestering milkweed bug Arocatus longiceps, which possesses a resistant Na+/K+-ATPase. Remarkably, cardenolides increased growth in the sequestering dietary specialists Caenocoris nerii and Oncopeltus fasciatus but not in the sequestering dietary generalist Spilostethus pandurus, which all possess a resistant Na+/K+-ATPase. 4. We then assessed the effect of dietary cardenolides on additional life history parameters, including developmental speed, the longevity of adults, and reproductive success in O. fasciatus. Remarkably, nymphs under cardenolide exposure developed substantially faster and lived longer as adults. However, fecundity of adults was reduced when maintained on cardenolide-containing diet for their entire life-time but not when adults were transferred to non-toxic sunflower seeds. 5. We speculate that the resistant Na+/K+-ATPase of milkweed bugs is selected for working optimally in a ′toxic environment′, i.e. when sequestered cardenolides are stored in the body tissues. Contrary to the assumption that toxins sequestered for defence produce a physiological burden, our data suggest that they can even increase fitness in specialised insects.

The trade-off between investment in weapons and fertility is mediated through spermatogenesis in the leaf-footed cactus bug Narnia femorata

Males have the ability to compete for fertilizations through both pre-copulatory and post-copulatory intrasexual competition. Pre-copulatory competition has selected for large weapons and other adaptations to maximize access to females and mating opportunities while post-copulatory competition has resulted in ejaculate adaptations to maximize fertilization success. Negative associations between these strategies support the hypothesis that there is a trade-off between success at pre- and post-copulatory mating success. Recently, this trade-off has been demonstrated with experimental manipulation. Male leaf-footed cactus bugs, Narnia femorata, that lose a weapon by autotomy during development invest instead in large testes. While evolutionary outcomes of the trade-offs between pre- and post-copulatory strategies have been identified, less work has been done to identify proximate mechanisms by which the trade-off might occur, perhaps because the systems in which the trade-offs have been investigated are not ones that have the molecular tools required for exploring mechanism. Here we applied knowledge from a related model species for which we have developmental knowledge and molecular tools, the milkweed bug Oncopeltus fasciatus, to investigate the proximate mechanism by which autotomized N. femorata males developed larger testes. Autotomized males had evidence of a higher rate of transit amplification divisions in the spermatogonia, which would result in greater sperm numbers. Identification of mechanisms underlying a trade-off can help our understanding of the direction and constraints on evolutionary trajectories and thus the evolutionary potential under multiple forms of selection.