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Aposematic coloration offers an opportunity to explore the molecular mechanisms underlying canalization. In this study, the role of epigenetic regulation underlying robustness was explored in the aposematic coloration of the milkweed bug, Oncopeltus fasciatus. Polycomb (Pc) and Enhancer of zeste (E(z)), which encode components of the Polycomb repressive complex 1 (PRC1) and PRC2, respectively, and jing, which encodes a component of the PRC2.2 subcomplex, were knocked down in the fourth instar of O. fasciatus. Knockdown of these genes led to alterations in scutellar morphology and melanization. In particular, when Pc was knocked down, the adults developed a highly melanized abdomen, head and forewings at all temperatures examined. In contrast, the E(z) and jing knockdown led to increased plasticity of the dorsal forewing melanization across different temperatures. Moreover, jing knockdown adults exhibited increased plasticity in the dorsal melanization of the head and the thorax. These observations demonstrate that histone modifiers may play a key role during the process of canalization to confer robustness in the aposematic coloration. [ABSTRACT FROM AUTHOR]

Hemiptera insects exhibit a close relationship to plants and demonstrate a diverse range of dietary preferences, encompassing phytophagy as the predominant feeding habit while a minority engages in carnivorous or haematophagous behaviour. To counteract the challenges posed by phytophagous insects, plants have developed an array of toxic compounds, causing significant evolutionary selection pressure on these insects. In this study, we employed a comparative genomics approach to analyse the expansion and contraction of gene families specific to phytophagous insect lineages, along with their adaptive evolutionary traits, utilising representative species from the Hemiptera order. Our investigation revealed substantial expansions of gene families within the phytophagous lineages, especially in the Pentatomomorpha branch represented by Oncopeltus fasciatus and Riptortus pedestris. Notably, these expansions of gene families encoding enzymes are potentially involved in hemipteran-plant interactions. Moreover, the adaptive evolutionary analysis of these lineages revealed a higher prevalence of adaptively evolved genes in the Pentatomomorpha branch. The observed branch-specific gene expansions and adaptive evolution likely contribute significantly to the diversification of species within Hemiptera. These results help enhance our understanding of the genomic characteristics of the evolution of different feeding habits in hemipteran insects. [ABSTRACT FROM AUTHOR]

Aposematic animals rely on diverse secondary metabolites for defence. Various hypotheses, such as competition, life history and multifunctionality, have been posited to explain defence variability and diversity. We investigate the compound selectivity hypothesis using large milkweed bugs, Oncopeltus fasciatus, to determine if distinct cardenolides vary in toxicity to different predators. We quantify cardenolides in the bug’s defensive secretions and body tissues and test the individual compounds against predator target sites, the Na+/K+-ATPases, that are predicted to differ in sensitivity. Frugoside, gofruside, glucopyranosyl frugoside and glucopyranosyl gofruside were the dominant cardenolides in the body tissues of the insects, whereas the two monoglycosidic cardenolides—frugoside and gofruside—were the most abundant in the defensive fluid. These monoglycosidic cardenolides were highly toxic (IC50

Milkweed–herbivore systems are characterized by cardenolide chemical defenses and specialized herbivore adaptations such as physiological target site insensitivity. Cardenolide defenses in milkweeds can vary in terms of the total concentration, differences in the polarity of individual cardenolides, and the substitution of the steroidal structures that can contribute to the molecule's reactivity. The variability in cardenolide defenses could represent the plant's response to natural selection and adaptation of resistant herbivores and is a characteristic of phenotype-matching between defensive and offensive traits resulting from coevolution. Here, we test the phenotypic match of the cardenolide composition of seeds of Asclepias curassavica and those sequestered by nymphs and adults of the specialized seed herbivore Oncopeltus fasciatus, combined with tests of the inhibitory capacity of a subset of seed cardenolides against the Na+/K+-ATPase of O. fasciatus and a non-adapted insect (Drosophila melanogaster). We compare this with the inhibitory capacity against the highly sensitive porcine Na+/K+-ATPase. Among the five most abundant cardenolides present in milkweed seeds, glucopyranosyl frugoside, glucopyranosyl gofruside, and glucopyranosyl calotropin were significantly more abundant in the seeds than in the adults and nymphs; the bugs contained higher concentrations of the deglucosylated compounds. The most abundant compound, glucopyranosyl frugoside, was also the most inhibitory for O. fasciatus, but O. fasciatus was significantly more tolerant to all compounds compared to D. melanogaster and the highly sensitive porcine enzyme. Our results add to the evidence that O. fasciatus sequesters specific individual cardenolides from its Asclepias host plants that are not directly linked to the concentration and inhibitory potency.

Sexual dimorphism is common in animals. The most complete model of sex determination comes from Drosophila melanogaster, where the relative dosage of autosomes and X chromosomes leads indirectly to sex-specific transcripts of doublesex (dsx). Female Dsx interacts with a mediator complex protein encoded by intersex (ix) to activate female development. In males, the transcription factor encoded by fruitless (fru) promotes male-specific behaviour. The genetics of sex determination have been examined in a small number of other insects, yet several questions remain about the plesiomorphic state. Is dsx required for female and male development? Is fru conserved in male behaviour or morphology? Are other components such as ix functionally conserved? To address these questions, we report expression and functional tests of dsx, ix and fru in the hemipteran Oncopeltus fasciatus, characterizing three sexual dimorphisms. dsx prevents ix phenotypes in all sexes and dimorphic traits in the milkweed bug. ix and fru are expressed across the body, in females and males. fru and ix also affect the genitalia of both sexes, but have effects limited to different dimorphic structures in different sexes. These results reveal roles for ix and fru distinct from other insects, and demonstrate distinct development mechanisms in different sexually dimorphic structures. [ABSTRACT FROM AUTHOR]

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, quantitative polymerase chain reaction shows 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. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

Aposematic animals rely on diverse secondary metabolites for defence. Various hypotheses, such as competition, life history and multifunctionality, have been posited to explain defence variability and diversity. We investigate the compound selectivity hypothesis using large milkweed bugs, Oncopeltus fasciatus , to determine if distinct cardenolides vary in toxicity to different predators. We quantify cardenolides in the bug's defensive secretions and body tissues and test the individual compounds against predator target sites, the Na + /K + -ATPases, that are predicted to differ in sensitivity. Frugoside, gofruside, glucopyranosyl frugoside and glucopyranosyl gofruside were the dominant cardenolides in the body tissues of the insects, whereas the two monoglycosidic cardenolides-frugoside and gofruside-were the most abundant in the defensive fluid. These monoglycosidic cardenolides were highly toxic (IC 50  < 1 μM) to an invertebrate and a sensitive vertebrate enzyme, in comparison to the glucosylated compounds. Gofruside was the weakest inhibitor for a putatively resistant vertebrate predator. Glucopyranosyl calotropin, found in only 60% of bugs, was also an effective inhibitor of sensitive vertebrate enzymes. Our results suggest that the compounds sequestered by O. fasciatus probably provide consistency in protection against a range of predators and underscore the need to consider predator communities in prey defence evolution., Competing Interests: We declare we have no competing interests., (© 2024 The Authors.)

DNA methylation is an important chromatin modification that can stably alter gene expression in cells and maintain genome integrity in plants and vertebrates. The function of DNA methylation outside of these well-studied systems, however, is unclear. Insects, in particular, represent an understudied group. Variation in the level of DNA methylation and gains and losses in the maintenance methyltransferase, DNMT1, across the insect tree of life suggests that there is much we don't understand about DMNT1 function and evolution. One constant across the studies examining patterns of Dnmt1 expression in insects is that expression is consistently high in reproductive tissues compared to somatic tissue. The explanation for this has been that DNMT1 is required in tissues that have high levels of cell division. Our previous study found that downregulation of Dnmt1 expression in the milkweed bug Oncopeltus fasciatus results in the expected reduction of DNA methylation, no global changes in gene expression reflecting changes in DNA methylation, and the loss of the ability to produce viable oocytes. Here, we show that females treated with ds-Dnmt1 RNA during larval development have a more extreme phenotype; they lack oocytes entirely but develop a normal somatic ovary. Our results indicate a specific role for DNMT1 in the formation of gametes and are consistent with data from other systems, including Tribolium castaneum, a species does not have DNA methylation. We propose that DNMT1 has multiple functional roles in addition to methylating DNA, which explains its complex patterns of evolution.

Despite its history as a developmental and evolutionary model organism, gene expression analysis in the large milkweed bug, Oncopeltus fasciatus , has rarely been explored using quantitative real-time PCR. The strength of this method depends greatly on the endogenous controls used for normalization, which are lacking for the milkweed bug system. Here, to fill in this gap in our knowledge, we validated the stability of a set of ten candidate reference genes identified from the O. fasciatus transcriptome, and did so upon exposure to a dietary toxin, a cardiac glycoside, and across four different exposure periods. To increase robustness against gDNA contaminants, genome resources were used to design intron-bridging primers. A comprehensive stability validation by the Bestkeeper, Normfinder, geNorm and comparative ΔCt methods identified ef1a and tubulin as the most stable genes across treatments and time points, whereas 18S rRNA was the most unstable. However, accounting for the temporal scale indicated that time point confined normalizers might enable higher quantification accuracy for treatment comparison. Overall this study demonstrates: (i) a robust RT-qPCR primer design approach is possible for non-model organisms where genome annotation is often incomplete, and (ii) the importance of detailed reference gene stability exploration in multifactorial experimental designs. • 10 potential references genes were identified in O. fasciatus. • Ef1a and tubulin are suitable normalizers upon temporal cardiac glycoside challenge. • Robust RT-qPCR primer design is also feasible in non-model organisms. • Multifactorial designs allow for detailed reference gene stability exploration. [ABSTRACT FROM AUTHOR]

Abstract Background Organisms develop and evolve in a modular fashion, but how individual modules interact with the environment remains poorly understood. Phenotypically plastic traits are often under selection, and studies are needed to address how traits respond to the environment in a modular fashion. In this study, tissue-specific plasticity of melanic spots was examined in the large milkweed bug, Oncopeltus fasciatus. Results Although the size of the abdominal melanic bands varied according to rearing temperatures, wing melanic bands were more robust. To explore the regulation of abdominal pigmentation plasticity, candidate genes involved in abdominal melanic spot patterning and biosynthesis of melanin were analyzed. While the knockdown of dopa decarboxylase (Ddc) led to lighter pigmentation in both the wings and the abdomen, the shape of the melanic elements remained unaffected. Although the knockdown of Abdominal-B (Abd-B) partially phenocopied the low-temperature phenotype, the abdominal bands were still sensitive to temperature shifts. These observations suggest that regulators downstream of Abd-B but upstream of DDC are responsible for the temperature response of the abdomen. Ablation of wings led to the regeneration of a smaller wing with reduced melanic bands that were shifted proximally. In addition, the knockdown of the Wnt signaling nuclear effector genes, armadillo 1 and armadillo 2, altered both the melanic bands and the wing shape. Thus, the pleiotropic effects of Wnt signaling may constrain the amount of plasticity in wing melanic bands. Conclusions We propose that when traits are regulated by distinct pre-patterning mechanisms, they can respond to the environment in a modular fashion, whereas when the environment impacts developmental regulators that are shared between different modules, phenotypic plasticity can manifest as a developmentally integrated system.

Oncopeltus fasciatus males fed the ancestral diet of milkweed seeds prioritize reproduction over lifespan as evidenced by higher rates of fertility and shorter lifespans than males from the same population fed the adapted diet of sunflower seeds. We examined the proximate mechanisms by which milkweed‐fed males maintained late‐life fertility. We tested the hypothesis that older milkweed‐fed males maintained fertility by producing more, higher quality sperm. Our results, that older males have more sperm, but their sperm do not have higher viability, are in general agreement with other recent studies on how nutrition affects male fertility in insects. We further examined the mechanisms by which sperm are produced by examining the progression of spermatogonial cells through the cell cycle during the transit amplification divisions. We demonstrated that diet affects the likelihood of a spermatocyst being in the S‐phase or M‐phase of the cell cycle. Given work in model systems, these results have implications for subtle effects on sperm quality either through replication stress or epigenetic markers. Thus, viability may not be the best marker for sperm quality and more work is called for on the mechanisms by which the germline and the production of sperm mediate the cost of reproduction. A fundamental tenant of life‐history theory is that there is a trade‐off between reproduction and somatic maintenance, but organisms can respond to variable environments by shifting the balance between these two critical activities. We have applied a developmental approach to attempt to examine the proximal mechanisms by which males vary life‐history allocation under variable diets. Our results show that in milkweed bugs, males invest in reproduction by investing in sperm numbers. However, examining spermatogenesis leads to further questions about investment in sperm quality. [ABSTRACT FROM AUTHOR]

Background: The Na,K-ATPase is a vital animal cell-membrane protein that maintains the cell's resting potential, among other functions. Cardenolides, a group of potent plant toxins, bind to and inhibit this pump. The gene encoding the α-subunit of the pump has undergone duplication events in some insect species known to feed on plants containing cardenolides. Here we test the function of these duplicated gene copies in the cardenolide-adapted milkweed bug, Oncopeltus fasciatus, which has three known copies of the gene: α1A, α1B and α1C. Results: Using RT-qPCR analyses we demonstrate that the α1C is highly expressed in neural tissue, where the pump is generally thought to be most important for neuron excitability. With the use of in vivo RNAi in adult bugs we found that α1C knockdowns suffered high mortality, where as α1A and α1B did not, supporting that α1C is most important for effective ion pumping. Next we show a role for α1A and α1B in the handling of cardenolides: expression results find that both copies are primarily expressed in the Malpighian tubules, the primary insect organ responsible for excretion and when we injected either α1A or α1B knockdowns with cardenolides this proved fatal (whereas not in controls). Conclusions: These results show that the Na,K-ATPα gene-copies have taken on diverse functions. Having multiple copies of this gene appears to have allowed the newly arisen duplicates to specialize on resistance to cardenolides, whereas the ancestral copy of the pump remains comparatively sensitive, but acts as a more efficient ion carrier. Interestingly both the α1A and α1B were required for cardenolide handling, suggesting that these two copies have separate and vital functions. Gene duplications of the Na,K-ATPase thus represent an excellent example of subfunctionalization in response to a new environmental challenge. [ABSTRACT FROM AUTHOR]

Iron is an essential nutrient needed for multiple biological processes, but it is also an effective pro-oxidant in its reduced form. Environmental sources of iron toxic species include reduced soils from rice plantations, polluted natural areas from metal industry waste, or iron oxides used in soil bioremediation. Few studies have been conducted to assess the toxicity of iron species in insects. The present work aims to assess the oxidative stress effects of ferrous sulfate administered in drinking water after acute exposure (96 h) to adults of the insect model Oncopeltus fasciatus (Dallas). Mortality was higher in exposed groups and significantly associated with iron treatment (OR [95% CI]; 11.8 [6.1-22.7]). Higher levels of body iron content were found in insects exposed to ferrous sulfate, with an increase of 5-6 times with respect to controls. Catalase activity and lipid peroxidation (TBARS content), but not glutathione S-transferase activity, were significantly higher in exposed insects and significantly correlated with body iron content (Pearson coefficient of 0.68 and 0.74, respectively) and between them (0.78). The present work demonstrates that, despite the disruption in water and food intake caused by iron administration, this metal is accumulated by insect causing lipid peroxidation and eliciting an antioxidant response mediated by catalase. [ABSTRACT FROM AUTHOR]

Morphogenesis involves the dynamic reorganization of cell and tissue shapes to create the three-dimensional body. Intriguingly, different species have evolved different morphogenetic processes to achieve the same general outcomes during embryonic development. How are meaningful comparisons between species made, and where do the differences lie? In this Perspective, we argue that examining the evolution of embryonic morphogenesis requires the simultaneous consideration of different levels of biological organization: (1) genes, (2) cells, (3) tissues, and (4) the entire egg. To illustrate the importance of integrating these levels, we use the extraembryonic epithelia of insects – a lineage-specific innovation and evolutionary hotspot – as an exemplary case study. We discuss how recent functional data, primarily from RNAi experiments targeting the Hox3/ Zen and U-shaped group transcription factors, provide insights into developmental processes at all four levels. Comparisons of these data from several species both challenge and inform our understanding of homology, in assessing how the process of epithelial morphogenesis has itself evolved.

Herbivorous insects and their adaptations against plant toxins provide striking opportunities to investigate the genetic basis of traits involved in coevolutionary interactions. Target site insensitivity to cardenolides has evolved convergently across six orders of insects, involving identical substitutions in the Na,K-ATPase gene and repeated convergent gene duplications. The large milkweed bug, Oncopeltus fasciatus, has three copies of the Na,K-ATPase α-subunit gene that bear differing numbers of amino acid substitutions in the binding pocket for cardenolides. To analyze the effect of these substitutions on cardenolide resistance and to infer possible trade-offs in gene function, we expressed the cardenolide-sensitive Na,K-ATPase of Drosophila melanogaster in vitro and introduced four distinct combinations of substitutions observed in the three gene copies of O. fasciatus. With an increasing number of substitutions, the sensitivity of the Na,K-ATPase to a standard cardenolide decreased in a stepwise manner. At the same time, the enzyme's overall activity decreased significantly with increasing cardenolide resistance and only the least substituted mimic of the Na,K-ATPase α1C copy maintained activity similar to the wild-type enzyme. Our results suggest that the Na,K-ATPase copies in O. fasciatus have diverged in function, enabling specific adaptations to dietary cardenolides while maintaining the functionality of this critical ion carrier. [ABSTRACT FROM AUTHOR]

Background: Organisms develop and evolve in a modular fashion, but how individual modules interact with the environment remains poorly understood. Phenotypically plastic traits are often under selection, and studies are needed to address how traits respond to the environment in a modular fashion. In this study, tissue-specific plasticity of melanic spots was examined in the large milkweed bug, Oncopeltus fasciatus. Results: Although the size of the abdominal melanic bands varied according to rearing temperatures, wing melanic bands were more robust. To explore the regulation of abdominal pigmentation plasticity, candidate genes involved in abdominal melanic spot patterning and biosynthesis of melanin were analyzed. While the knockdown of dopa decarboxylase (Ddc) led to lighter pigmentation in both the wings and the abdomen, the shape of the melanic elements remained unaffected. Although the knockdown of Abdominal-B (Abd-B) partially phenocopied the low-temperature phenotype, the abdominal bands were still sensitive to temperature shifts. These observations suggest that regulators downstream of Abd-B but upstream of DDC are responsible for the temperature response of the abdomen. Ablation of wings led to the regeneration of a smaller wing with reduced melanic bands that were shifted proximally. In addition, the knockdown of the Wnt signaling nuclear effector genes, armadillo 1 and armadillo 2, altered both the melanic bands and the wing shape. Thus, the pleiotropic effects of Wnt signaling may constrain the amount of plasticity in wing melanic bands. Conclusions: We propose that when traits are regulated by distinct pre-patterning mechanisms, they can respond to the environment in a modular fashion, whereas when the environment impacts developmental regulators that are shared between different modules, phenotypic plasticity can manifest as a developmentally integrated system. [ABSTRACT FROM AUTHOR]

Diversity in insect pigmentation, encompassing a wide range of colors and spatial patterns, is among the most noticeable features distinguishing species, individuals, and body regions within individuals. In holometabolous species, a significant portion of such diversity can be attributed to the melanin synthesis genes, but this has not been formally assessed in more basal insect lineages. Here we provide a comprehensive analysis of how a set of melanin genes (ebony, black, aaNAT, yellow, and tan) contributes to the pigmentation pattern in a hemipteran, Oncopeltus fasciatus. For all five genes, RNA interference depletion caused alteration of black patterning in a region-specific fashion. Furthermore, the presence of distinct nonblack regions in forewings and hindwings coincides with the expression of ebony and aaNAT in these appendages. These findings suggest that the region-specific phenotypes arise from regional employment of various combinations of the melanin genes. Based on this insight, we suggest that melanin genes are used in two distinct ways: a "painting" mode, using predominantly melanin-promoting factors in areas that generally lack black coloration, and, alternatively, an "erasing" mode, using mainly melanin-suppressing factors in regions where black is the dominant pigment. Different combinations of these strategies may account for the vast diversity of melanin patterns observed in insects. [ABSTRACT FROM AUTHOR]

Extracts and secondary metabolites from plants can act as feeding deterrents, toxic agents or developmental disruptors in insects. In this study, the experimental milkweed bug Oncopeltus fasciatus (Dallas), (Hemiptera) was used to evaluate the activities of crude ethanol extract and fractions, obtained from flowers or stems of Eremanthus erythropappus (DC.) MacLeisch (Asteraceae), on its mortality and development. Butanol fraction from ethanol extract showed toxicity against nymphs of O. fasciatus as well as growth inhibition activity. The results imply that E. erythropappus butanol fraction has active substances on this insect growth development physiology. Atividade Antimuda de Eremanthus erythropappus (DC.) MacLeisch Resumo. Os extratos e metabólitos secundários de plantas podem agir como agentes tóxicos e inibidores do da alimentação e do desenvolvimento em insetos. Neste estudo foi utilizado Oncopeltus fasciatus (Dallas) (Hemiptera) como modelo experimental a fim de avaliar as atividades do extrato etanólico bruto e frações obtidos das flores ou caules de Eremanthus erythropappus (DC.) MacLeisch (Asteraceae), na mortalidade e sobre o desenvolvimento do inseto. A fração butanólica obtida do extrato etanólico mostrou toxidade sobre as ninfas de O. fasciatus, bem como atividade de inibição do crescimento. Os resultados sugerem que a fração butanólica de E. erythropappus possui substâncias ativas sobre a fisiologia, crescimento e desenvolvimento de insetos.

In protein-coding genes, synonymous codon usage and amino acid composition correlate to expression in some eukaryotes, and may result from translational selection. Here, we studied large-scale RNA-seq data from three divergent arthropod models, including cricket (Gryllus bimaculatus), milkweed bug (Oncopeltus fasciatus), and the amphipod crustacean Parhyale hawaiensis, and tested for optimization of codon and amino acid usage relative to expression level. We report strong signals of AT3 optimal codons (those favored in highly expressed genes) in G. bimaculatus and O. fasciatus, whereas weaker signs of GC3 optimal codons were found in P. hawaiensis, suggesting selection on codon usage in all three organisms. Further, in G. bimaculatus and O. fasciatus, high expression was associated with lowered frequency of amino acids with large size/complexity (S/C) scores in favor of those with intermediate S/C values; thus, selection may favor smaller amino acids while retaining those of moderate size for protein stability or conformation. In P. hawaiensis, highly transcribed genes had elevated frequency of amino acids with large and small S/C scores, suggesting a complex dynamic in this crustacean. In all species, the highly transcribed genes appeared to favor short proteins, high optimal codon usage, specific amino acids, and were preferentially involved in cell-cycling and protein synthesis. Together, based on examination of 1,680,067, 1,667,783, and 1,326,896 codon sites in G. bimaculatus, O. fasciatus, and P. hawaiensis, respectively, we conclude that translational selection shapes codon and amino acid usage in these three Pancrustacean arthropods. [ABSTRACT FROM AUTHOR]

Morphogenesis involves the dynamic reorganization of cell and tissue shapes to create the three-dimensional body. Intriguingly, different species have evolved different morphogenetic processes to achieve the same general outcomes during embryonic development. How are meaningful comparisons between species made, and where do the differences lie? In this Perspective, we argue that examining the evolution of embryonic morphogenesis requires the simultaneous consideration of different levels of biological organization: (1) genes, (2) cells, (3) tissues, and (4) the entire egg, or other gestational context. To illustrate the importance of integrating these levels, we use the extraembryonic epithelia of insects--a lineage-specific innovation and evolutionary hotspot--as an exemplary case study. We discuss how recent functional data, primarily from RNAi experiments targeting the Hox3/Zen and U-shaped group transcription factors, provide insights into developmental processes at all four levels. Comparisons of these data from several species both challenge and inform our understanding of homology, in assessing how the process of epithelial morphogenesis has itself evolved. [ABSTRACT FROM AUTHOR]

Maternal provisioning can have profound effects on offspring phenotypes, or maternal effects, especially early in life. One ubiquitous form of provisioning is in the makeup of egg. However, only a few studies examine the role of specific egg constituents in maternal effects, especially as they relate to maternal selection (a standardized selection gradient reflecting the covariance between maternal traits and offspring fitness). Here, we report on the evolutionary consequences of differences in maternal acquisition and allocation of amino acids to eggs. We manipulated acquisition by varying maternal diet (milkweed or sunflower) in the large milkweed bug, Oncopeltus fasciatus. Variation in allocation was detected by examining two source populations with different evolutionary histories and life-history response to sunflower as food. We measured amino acids composition in eggs in this 2 × 2 design and found significant effects of source population and maternal diet on egg and nymph mass and of source population, maternal diet, and their interaction on amino acid composition of eggs. We measured significant linear and quadratic maternal selection on offspring mass associated with variation in amino acid allocation. Visualizing the performance surface along the major axes of nonlinear selection and plotting the mean amino acid profile of eggs from each treatment onto the surface revealed a saddle-shaped fitness surface. While maternal selection appears to have influenced how females allocate amino acids, this maternal effect did not evolve equally in the two populations. Furthermore, none of the population means coincided with peak performance. Thus, we found that the composition of free amino acids in eggs was due to variation in both acquisition and allocation, which had significant fitness effects and created selection. However, although there can be an evolutionary response to novel food resources, females may be constrained from reaching phenotypic optima with regard to allocation of free amino acids. [ABSTRACT FROM AUTHOR]

The environmental influences of mothers on offspring traits, or maternal effects, often arise from dietary differences experienced by mothers. However, few studies have explored if and how maternal effects facilitate adaptation to new host plants. To address this, we compared the maternal and direct effects arising from dietary differences in two populations of the large milkweed bug, Oncopeltus fasciatus that live on and feed on the seeds from different hosts. We compared a laboratory population, which has been reared for over 400 generations on sunflower seeds and is now adapted to use these as a host, to the wild population, which is adapted to the ancestral diet of toxic milkweed seeds. We first tested for changes in maternal effects, and then examined offspring performance and survivorship. We found evidence for evolution of the maternal effect facilitating the use of a novel host. However, the strongest effects were population differences and direct dietary effects for all traits. Offspring performance was more strongly influenced by diet than maternal effects. Survivorship depended on population and offspring diet, and their interaction, but was unaffected by maternal diet or other interactions. In the artificially evolved population, diet breadth was increased rather than evolving specialization. Our results suggest changes in maternal effects are likely to be weak compared to direct effects of host plants and other adaptations in adaptation to a novel host. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 114, 202-211. [ABSTRACT FROM AUTHOR]

Migration evolves as a response to seasonally unfavourable environments but plasticity in reproductive physiology is another avenue by which insects can respond to resource-poor conditions. We investigated the relationship between individual variation in migratory propensity and the level of response to poor conditions modulated by the female reproductive physiology. We tested the hypothesis that, compared to migrants, residential behaviour is associated with a higher degree of phenotypic plasticity in oosorption, an adaptive physiological mechanism that allows females to recoup resources from undeveloped oocytes. Reallocation from reproduction to survival would allow females to skip migration and to cope with unfavourable environments. If this plasticity is evolved, we further predicted it would vary between as well as within populations. We examined variation associated with migratory behaviour in females from four populations of the milkweed bug, Oncopeltus fasciatus, using a behavioural assay to categorize females as either migrant or resident and observing the differences in oosorption between these groups. As expected, food availability, source population and wing length influenced the propensity for migratory flight, and food availability influenced levels of oosorption. We also found support for our key prediction that resident females are characterized by higher levels of ovarian oosorption than migrant females. Our study provides support for a physiological difference between migrant and resident females and suggests the presence of both physiological and behavioural tactics that interact with the potential for migration to provide adaptation to seasonally challenging environments. [ABSTRACT FROM AUTHOR]

BACKGROUND: Studies were carried out to evaluate the effects of Manilkara subsericea extracts and triterpenes on the development of two species of agricultural pest insects, Oncopelus fasciatus and Dysdercus peruvianus. RESULTS: All treatments of insects with M. subsericea extracts induced mortality, delayed development and inhibited moulting. Some extracts assayed (FH, FB and FD in D. peruvianus, and FH, FB and FEA in O. fasciatus) also produced body deformities in the few adults that emerged. Other extracts (FH, FEA, FB, FD and LET in both insects), however, induced either permanent (overaged) or extranumerary nymphs, both of which were unable to achieve the adult stage and reproductive status. The insects were also treated with triterpenes (α- and β-amyrin acetates) which showed high lethality at 30 days after treatment and delayed the intermoult period so that overaged nymphs were detected. CONCLUSION: The results indicate that extracts and triterpenes from Manilkara subsericea act as potent growth inhibitors of phytophagous hemipteran nymphs. It is also concluded that the mixture of several different molecules in the extracts used produces synergic effects that do not occur after using the triterpenes (PFT) alone. These secondary metabolites in the M. subsericea extracts can potentially be used in integrated control programmes against crop pests. Copyright © 2012 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Abstract: The insect intercalary segment represents a small and appendage-less head segment that is homologous to the second antennal segment of Crustacea and the pedipalpal segment in Chelicerata, which are generally referred to as “tritocerebral segment.” In Drosophila, the gene collier (col) has an important role for the formation of the intercalary segment. Here we show that in the beetle Tribolium castaneum col is required for the activation of the segment polarity genes hedgehog (hh), engrailed (en) and wingless (wg) in the intercalary segment, and is a regulatory target of the intercalary segment specific Hox gene labial (lab). Loss of Tc col function leads to increased cell death in the intercalary segment. In the milkweed bug Oncopeltus fasciatus, the loss of col function has a more severe effect in lacking the intercalary segment and also affecting the adjacent mandibular and antennal segments. By contrast, col is not expressed early in the second antennal segment in the crustacean Parhyale hawaiensis or in the pedipalpal segment of the spider Achaearanea tepidariorum. This suggests that the early expression of col in a stripe and its role in tritocerebral segment development is insect-specific and might correlate with the appendage-less morphology of the intercalary segment. [ABSTRACT FROM AUTHOR]

Abstract: As extra-embryonic tissues, the amnion and serosa are not considered to contribute materially to the insect embryo, yet they must execute an array of morphogenetic movements before they are dispensable. In hemimetabolous insects, these movements have been known for over a century, but they have remained virtually unexamined. This study addresses late extraembryonic morphogenesis in the milkweed bug, Oncopeltus fasciatus. Cell shape changes and apoptosis profiles are used to characterize the membranes as they undergo a large repertoire of final reorganizational events that reposition the embryo (katatrepsis), and eliminate the membranes themselves in an ordered fashion (dorsal closure). A number of key features were identified. First, amnion–serosa “fusion” involves localized apoptosis in the amnion and the formation of a supracellular actin purse string at the amnion–serosa border. During katatrepsis, a ‘focus’ of serosal cells undergoes precocious columnarization and may serve as an anchor for contraction. Lastly, dorsal closure involves novel modifications of the amnion and embryonic flank that are without counterpart during the well-known process of dorsal closure in the fruit fly Drosophila melanogaster. These data also address the long-standing question of the final fate of the amnion: it undergoes apoptosis during dorsal closure and thus is likely to be solely extraembryonic. [Copyright &y& Elsevier]

Drosophila undergoes a form of development termed long germ segmentation, where all segments are specified nearly simultaneously so that by the blastoderm stage, the entire body plan has been determined. This mode of segmentation is evolutionarily derived. Most insects undergo short or intermediate germ segmentation, where only anterior segments are specified early, and posterior segments are sequentially specified during germband elongation. These embryological differences imply that anterior and posterior segments might rely upon different molecular mechanisms. In Drosophila, embryos mutant for giant show a gap in the anterior as well fusions of several abdominal segments. In Tribolium, a short germ beetle, giant is required for segmental identity, but not formation, in gnathal segments and also for segmentation of the entire abdomen. This raises the possibility that giant might not act as a gap gene in short and intermediate germ insects. Oncopeltus fasciatus is an intermediate germ insect that is an outgroup to the clade containing Drosophila and Tribolium. We cloned the Oncopeltus homolog of giant and determined its expression and function during segmentation. We find that Oncopeltus giant is a canonical gap gene in the maxillary and labial segments and also plays a gap-like role in the first four abdominal segments. Our results suggest that giant was a bona fide gap gene in the ancestor of these insects with this role being lost in the lineage leading towards Tribolium. This highlights the conservation of anterior patterning and evolutionary plasticity of the genetic regulation controlling posterior segmentation, even in short and intermediate germ insects. [ABSTRACT FROM AUTHOR]

Metamorphosis is one of the most common, yet dramatic of life history strategies. In insects, complete metamorphosis with morphologically distinct larval stages arose from hemimetabolous ancestors that were more direct developing. Over the past century, several ideas have emerged that suggest the holometabolous pupa is developmentally homologous to the embryonic stages of the hemimetabolous ancestor. Other theories consider the pupal stage to be a modification of a hemimetabolous nymph. To address this question, we have isolated an ortholog of the pupal determinant, broad ( br), from the hemimetabolous milkweed bug and examined its role during embryonic development. We show that Oncopeltus fasciatus br ( Of’br) is expressed in two phases. The first occurs during germ band invagination and segmentation when Of’br is expressed ubiquitously in the embryonic tissues. The second phase of Of’br expression appears during the pronymphal phase of embryogenesis and persists through nymphal differentiation to decline just before hatching. Knock-down of Of’br transcripts results in defects that range from posterior truncations in the least-affected phenotypes to completely fragmented embryonic tissues in the most severe cases. Analysis of the patterning genes engrailed and hunchback reveal loss of segments and a failure in neural differentiation after Of’br depletion. Finally, we show that br is constitutively expressed during embyrogenesis of the ametabolous firebrat, Thermobia domestica. This suggests that br expression is prominent during embryonic development of ametabolous and hemimetabolous insects but was lost with the emergence of the completely metamorphosing insects. [ABSTRACT FROM AUTHOR]

Abstract: Genetic studies of the fruit fly Drosophila have revealed a hierarchy of segmentation genes (maternal, gap, pair-rule and HOX) that subdivide the syncytial blastoderm into sequentially finer-scale coordinates. Within this hierarchy, the pair-rule genes translate gradients of information into periodic stripes of expression. How pair-rule genes function during the progressive mode of segmentation seen in short and intermediate-germ insects is an ongoing question. Here we report that the nuclear receptor Of''E75A is expressed with double segment periodicity in the head and thorax. In the abdomen, Of''E75A is expressed in a unique pattern during posterior elongation, and briefly resembles a sequence that is typical of pair-rule genes. Depletion of Of''E75A mRNA caused loss of a subset of odd-numbered parasegments, as well as parasegment 6. Because these parasegments straddle segment boundaries, we observe fusions between adjacent segments. Finally, expression of Of''E75A in the blastoderm requires even-skipped, which is a gap gene in Oncopeltus. These data show that the function of Of''E75A during embryogenesis shares many properties with canonical pair-rule genes in other insects. They further suggest that parasegment specification may occur through irregular and episodic pair-rule-like activity. [Copyright &y& Elsevier]

Abstract: Many insects undergo katatrepsis, essential reorganization by the extraembryonic membranes that repositions the embryo. Knockdown of the zen gene by RNA interference (RNAi) prevents katatrepsis in the milkweed bug Oncopeltus fasciatus. However, the precise morphogenetic defect has been uncertain, and katatrepsis itself has not been characterized in detail. The dynamics of wild type and zen RNAi eggs were analyzed from time-lapse movies, supplemented by analysis of fixed specimens. These investigations identify three zen RNAi defects. First, a reduced degree of tissue contraction implies a role for zen in baseline compression prior to katatrepsis. Subsequently, a characteristic ‘bouncing’ activity commences, leading to the initiation of katatrepsis in wild type eggs. The second zen RNAi defect is a delay in this activity, suggesting that a temporal window of opportunity is missed after zen knockdown. Ultimately, the extraembryonic membranes fail to rupture in zen RNAi eggs: the third defect. Nevertheless, the outer serosal membrane manages to contract, albeit in an aberrant fashion with additional phenotypic consequences for the embryo. These data identify a novel epithelial morphogenetic event – rupture of the ‘serosal window’ structure – as the ultimate site of defect. Overall, Oncopeltus zen seems to have a role in coordinating a number of pre-katatreptic events during mid embryogenesis. [Copyright &y& Elsevier]

Abstract: Hemimetabolous insects undergo an ancestral mode of development in which embryos hatch into first nymphs that resemble miniature adults. While recent studies have shown that homeotic (hox) genes establish segmental identity of first nymphs during embryogenesis, no information exists on the function of these genes during post-embryogenesis. To determine whether and to what degree hox genes influence the formation of adult morphologies, we performed a functional analysis of Sex combs reduced (Scr) during post-embryonic development in Oncopeltus fasciatus. The main effect was observed in prothorax of Scr-RNAi adults, and ranged from significant alterations in its size and shape to a near complete transformation of its posterior half toward a T2-like identity. Furthermore, while the consecutive application of Scr-RNAi at both of the final two post-embryonic stages (fourth and fifth) did result in formation of ectopic wings on T1, the individual applications at each of these stages did not. These experiments provide two new insights into evolution of wings. First, the role of Scr in wing repression appears to be conserved in both holo- and hemimetabolous insects. Second, the prolonged Scr-depletion (spanning at least two nymphal stages) is both necessary and sufficient to restart wing program. At the same time, other structures that were previously established during embryogenesis are either unaffected (T1 legs) or display only minor changes (labium) in adults. These observations reveal a temporal and spatial divergence of Scr roles during embryonic (main effect in labium) and post-embryonic (main effect in prothorax) development. [Copyright &y& Elsevier]

Abstract: Recently, the peptidomic analysis of neuropeptides from the retrocerebral complex and abdominal perisympathetic organs of polyphagous stinkbugs (Pentatomidae) revealed the group-specific sequences of pyrokinins, CAPA peptides (CAPA-periviscerokinins/PVKs and CAPA-pyrokinin), myosuppressin, corazonin, adipokinetic hormone, and short neuropeptide F. In this study, we used mass spectrometric profiling of nervous tissue from the species-rich taxon Hemiptera to identify products of two previously unobserved neuropeptide genes from these species, namely allatotropin-related peptide (ATRP) and tachykinin-related peptides (TKRPs). Since neither TKRPs nor allatotropin are accumulated in neurohemal organs, immunocytochemical data were analyzed to find potential accumulation sites within the central nervous system. By mass spectrometry, TKRPs were found to be accumulated in the antennal lobes, and ATRP was identified in the most posterior region of the abdominal ventral nerve cord and fourth abdominal nerves. In addition to neuropeptides from stink bugs, TKRPs and ATRP were also identified from the distantly related bugs Oncopeltus fasciatus (Lygaeidae) and Pyrrhocoris apterus (Pyrrhocoridae). In total, six TKRPs and one ATRP from each species could be elucidated by tandem mass spectrometry. The ATRP of all species is sequence-identical with Locusta migratoria accessory gland myotropin-1 (Lom-AG-MT-1), a member of the highly conserved insect allatotropin family. [Copyright &y& Elsevier]

From an Argentine collection of Annona cherimolia and a Bolivian collection of Annona montana (Annonaceae), the acetogenins, squamocin, molvizarin, itrabin, almuñequin, cherimolin-1, annonacin, annonacin-A, densicomacin-1, cis-annonacin-10-one and murihexocin-A, were obtained to study their toxicity against the cotton pest Oncopeltus fasciatus (Hemiptera: Lygaeidae). Topical ventral application of O. fasciatus nymphs with solutions of the mentioned acetogenins produced acute and delayed mortality. [ABSTRACT FROM AUTHOR]

Abstract: The milkweed bug, Oncopeltus fasciatus, is a plant feeding hemipteran. While there has been much research done on the neurohormonal control of the post-feeding diuresis in the blood-feeding hemipteran, Rhodnius prolixus, little is known about the control of the post-feeding diuresis in O. fasciatus. One of the neurohormones that may play a role in this rapid diuresis belongs to the calcitonin-like diuretic hormone (DH31) family of insect peptides. In this study we demonstrate the presence of DH31-like immunoreactivity in the central nervous system (CNS) and gut of O. fasciatus 5th instars. As well, DH31-like material was quantified and partially purified from the CNS of 5th instar O. fasciatus using reversed-phase liquid chromatography (RPLC) and monitored with an enzyme-linked immunosorbent assay (ELISA). When tested on O. fasciatus 5th instar Malpighian tubules, DH31-like peptides significantly increased the rate of secretion over saline controls. The results suggest that there is a DH31-like peptide(s) present in the CNS of O. fasciatus and that this peptide may play a role in the control of Malpighian tubule secretion. [Copyright &y& Elsevier]

Extracts of 121 medicinal and ornamental plants were screened for insecticidal and growth regulating activity to milkweed bug, Oncopeltus fasciatus Dallas. The most effective extracts stemmed from Inula helenium L., Rumex crispus L., R. acetosa L., Asarum europaeum L., and Calendula officinalis L. All these extracts exerted growth inhibiting activities and moderate or low acute toxicity. Most promising were extracts of dried roots of I. helenium. Besides a moderate chronic toxicity which reduced the fitness of the milkweed bug, the treatment produced overaged nymphs, supernumerary nymphs, disturbances of the moulting process, morphological defects, delay of adult maturation and reduction of fecundity. In addition, I. helenium extracts had a strong antifeedant effect when topically applied and very strong repellent effect when applied to the food (peeled sunflower kernels). It is obvious that the extract contains several active constituents. Such a cocktail may reduce the threat of rapid resistance development. [ABSTRACT FROM AUTHOR]

The article discusses the method for obtaining and counting hemocytes in milkweed bugs. One method relies on estimates of overall hemocyte volume after centrifugation of hemolymph in hematocrit tubes. Electronic particle counters have been used to count marine arthropod hemocytes. Liquid nitrogen fixation has been used to compare the numbers of hemocytes in tissue sections to counts made of hemolymph from severed appendages.

Abstract: The heavy metal Cd has recently been shown to have endocrine disrupting activity in different vertebrate species. In the insect Oncopeltus fasciatus, Cd exposure delays ovarian maturation and inhibits vitellogenesis, probably through a reduction in vitellogenin (Vg) polypeptide synthesis. An O. fasciatus strain was selected in the laboratory for Cd resistance with gradually increasing Cd concentrations for 50 generations. Cd-selected (OCdR) insects were 2.7–2.3 times more resistant to chronic exposure to Cd than susceptible (OS) insects. OCdR females showed increased (up to 90%) Vg polypeptide levels in hemolymph. Vg polypeptide synthesis was equally decreased in OCdR and OS females after acute Cd exposure, indicating that resistance in the OCdR strain was not due to a reduced sensitivity of the target of Cd. Vg polypeptide concentrations and ovarian maturation in acutely exposed OCdR females, however, were equal to those of control OS females. OCdR females chronically exposed to Cd showed a 30% decrease in Vg polypeptide concentrations, relative to control OS females. It is concluded that increased Vg polypeptide levels play an important role in resistance to the toxic effects of Cd on reproduction in the OCdR strain, and that this novel Cd resistance mechanism has the potential to evolve in vertebrate species in which Cd inhibits Vg synthesis by disruption of the endocrine system. [Copyright &y& Elsevier]

Abstract: Newly moulted females of the insect Oncopeltus fasciatus were exposed to cadmium (Cd) dissolved in the drinking water (50–400mgl−1 Cd) for 5 days. Cd exposure delayed ovarian maturation and inhibited egg production. Exposure to Cd, moreover, decreased hemolymph levels of the two major vitellogenin polypeptides of O. fasciatus, VG1 and VG2, in a concentration-dependent way, probably by a reduction in their synthesis. The ovarian levels of VG1 and VG2 were also decreased in Cd-exposed females. It was next investigated whether Cd effects might be a consequence of the endocrine disruption of vitellogenin synthesis, which is controlled by juvenile hormone (JH). JH replacement therapy did not restore VG1 or VG2 levels in Cd-exposed females, but did so in starved females. Our results do not therefore support a disturbance of JH production or a reduction in feeding as the cause of the reduced vitellogenin polypeptide levels, but rather point to the site of action of JH, the JH receptor, as the target of Cd effects. [Copyright &y& Elsevier]

In insects, selector genes are thought to modify the development of a default, or 'ground state', appendage into a tagma-specific appendage such as a mouthpart, antenna or leg. In the best described example, Drosophila melanogaster, the primary determination of leg identity is thought to result from regulatory interactions between the Hox genes and the antennal-specifying gene homothorax. Based on RNA-interference, a functional analysis of the selector gene tiptop and the Hox gene Antennapedia in Oncopeltus fasciatus embryogenesis is presented. It is shown that, in O. fasciatus, tiptop is required for the segmentation of distal leg segments and is required to specify the identity of the leg. The distal portions of legs with reduced tiptop develop like antennae. Thus, tiptop can act as a regulatory switch that chooses between antennal and leg identity. By contrast, Antennapedia does not act as a switch between leg and antennal identity. This observation suggests a significant difference in the mechanism of leg specification between O. fasciatus and D. melanogaster. These observations also suggest a significant plasticity in the mechanism of leg specification during insect evolution that is greater than would have been expected based on strictly morphological or molecular comparisons. Finally, it is proposed that a tiptop-like activity is a likely component of an ancestral leg specification mechanism. Incorporating a tiptop-like activity into a model of the leg-specification mechanism explains several mutant phenotypes, previously described in D. melanogaster, and suggests a mechanism for the evolution of legs from a ground state. [ABSTRACT FROM AUTHOR]

Newly hatched nymphs of the insect Oncopeltus fasciatus were exposed to various concentrations of CdCl2 administered in drinking water until the end of adult life. Significant nymphal mortalities were observed at concentrations above 30 mg Cd/l (corresponding to the LC50). The duration of the nymphal stages increased in proportion to the Cd concentration; at the lowest Cd concentration of 10 mg Cd/l, the median duration was significantly prolonged by one day, while at the highest concentration of 100 mg Cd/l it was increased by 10 days over the control group. The weight of newly emerged adults lineally decreased with Cd concentration, being reduced to half the weight of controls at 100 mg Cd/l. In addition, a proportionality between delay in development and weight reduction was found in Cd-treated adults. Survival of adult females was decreased at concentrations higher than 10 mg Cd/l, while males were only affected at 30 mg Cd/l or higher doses. Reproduction was the most affected parameter. Oviposition rate, fecundity and fertility of females exposed to 10 mg Cd/l were significantly lower than controls (73%, 58% and 55% relative to controls, respectively). Hatchability of the eggs laid by treated females was also reduced. These results show that development and reproduction of O. fasciatus are seriously impaired at sublethal Cd concentrations. [Copyright &y& Elsevier]

While investigating the distribution of Leptomonas wallacei in the intestine of the insect host Oncopeltus fasciatus, promastigotes and cyst-like forms of L. wallacei were observed only in the midgut ventricles V3 and V4 and the hindgut. In video-microscopy, once contact had occurred, the parasites remained attached to the midgut epithelium. Scanning electron microscopy revealed the adhesion of flagellates and cyst-like forms to the midgut wall and to the rectal pads of the hindgut. Using transmission electron microscopy, we observed that adhesion occurred mainly between the flagellum and the perimicrovillar membranes secreted by the midgut epithelium. No modifications were observed either in the parasite or in the epithelial cells. In the hindgut, adhesion to the superficial wax layer of the epithelial cells of the rectal pads was via flagellum. Host cell morphology appeared unaffected by L. wallacei. [Copyright &y& Elsevier]

Homologues of the Drosophila segment polarity gene engrailed have been cloned from many insect species, as well as other arthropods and non-arthropods. We have cloned partial cDNAs of two engrailed homologues, which we call engrailed-related genes, from the phylogenetically basal insect, Thermobia domestica (Order Thysanura) and possibly as many as four engrailed-related genes from the phylogenetically intermediate insect, Oncopeltus fasciatus (Order Hemiptera). Previous to our findings, only single engrailed-related homologues had been found in phylogenetically intermediate insect species ( Tribolium and Schistocerca) and in the crustacean Artemia, while two engrailed-related homologues have been found in more derived orders (Hymenoptera and the engrailed and invected genes of lepidopterans and dipterans). Consequently, we performed a phylogenetic analysis of insect engrailed-related genes to determine whether insects ancestrally had one or two engrailed-related genes. We have found evidence of concerted evolution among engrailed-related paralogues, however, that masks the true phylogenetic history of these genes; the phylogeny may only be decipherable, therefore, by examining the presence or absence of engrailed-specific and invected-specific motifs, which will require cloning the full length cDNAs from more species. In addition, we examined the embryonic expression pattern of the two Thermobia engrailed-related genes; like Drosophila engrailed and invected, they are expressed in very similar patterns, but show one temporal difference in pregnathal segments that correlates with the tentative phylogenetic placement of the genes. Thermobia engrailed-related expression also confirms that the dorsal ridge is an ancient structure in insects. [ABSTRACT FROM AUTHOR]

The dynamics of the 'A' cells of the parsintercerebralis of Oncopeltus fasciatus over the first eight days of adult life was studied by microspectrophotometry of sections stained either with aldehyde fuchsin or alcian blue 8 GX. The data show that the two stains differ in their selectivity as they record different events in the history of the cells. A hypothesis is proposed that the aldehyde fuchsin is more sensitive to the presence of a 'carrier' protein in the cell, whereas alcian blue 8 GX is more sensitive to the presence of the 'active principle' in the cell. [ABSTRACT FROM AUTHOR]