Cardenolides in the defensive fluid of adult large milkweed bugs have differential potency on vertebrate and invertebrate predator Na+/K+-ATPases.

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 < 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.

Polycomb group proteins confer robustness to aposematic coloration in the milkweed bug, Oncopeltus fasciatus.

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.

Comparative genomics reveals evolutionary drivers of the dietary shift in Hemiptera.

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.

A nutrition-defence trade-off drives diet choice in a toxic plant generalist.

Plant toxicity shapes the dietary choices of herbivores. Especially when herbivores sequester plant toxins, they may experience a trade-off between gaining protection from natural enemies and avoiding toxicity. The availability of toxins for sequestration may additionally trade off with the nutritional quality of a potential food source for sequestering herbivores. We hypothesized that diet mixing might allow a sequestering herbivore to balance nutrition and defence (via sequestration of plant toxins). Accordingly, here we address diet mixing and sequestration of large milkweed bugs (Oncopeltus fasciatus) when they have differential access to toxins (cardenolides) in their diet. In the absence of toxins from a preferred food (milkweed seeds), large milkweed bugs fed on nutritionally adequate non-toxic seeds, but supplemented their diet by feeding on nutritionally poor, but cardenolide-rich milkweed leaf and stem tissues. This dietary shift corresponded to reduced insect growth but facilitated sequestration of defensive toxins. Plant production of cardenolides was also substantially induced by bug feeding on leaf and stem tissues, perhaps benefitting this cardenolide-resistant herbivore. Thus, sequestration appears to drive diet mixing in this toxic plant generalist, even at the cost of feeding on nutritionally poor plant tissue.

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

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.

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, 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'.

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.

Shifting roles of Drosophila pair-rule gene orthologs: segmental expression and function in the milkweed bug Oncopeltus fasciatus.

The discovery of pair-rule genes (PRGs) in Drosophila revealed the existence of an underlying two-segment-wide prepattern directing embryogenesis. The milkweed bug Oncopeltus fasciatus, a hemimetabolous insect, is a more representative arthropod: most of its segments form sequentially after gastrulation. Here, we report the expression and function of orthologs of the complete set of nine Drosophila PRGs in Oncopeltus Seven Of-PRG-orthologs are expressed in stripes in the primordia of every segment, rather than every other segment; Of-runt is PR-like and several orthologs are also expressed in the segment addition zone. RNAi-mediated knockdown of Of-odd-skipped, paired and sloppy-paired impacted all segments, with no indication of PR-like register. We confirm that Of-E75A is expressed in PR-like stripes, although it is not expressed in this way in Drosophila, demonstrating the existence of an underlying PR-like prepattern in Oncopeltus These findings reveal that a switch occurred in regulatory circuits, leading to segment formation: while several holometabolous insects are 'Drosophila-like', using PRG orthologs for PR patterning, most Of-PRGs are expressed segmentally in Oncopeltus, a more basally branching insect. Thus, an evolutionarily stable phenotype - segment formation - is directed by alternate regulatory pathways in diverse species.

Robust reference gene design and validation for expression studies in the large milkweed bug, Oncopeltus fasciatus, upon cardiac glycoside stress.

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.

A study of the transit amplification divisions during spermatogenesis in Oncopetus fasciatus to assess plasticity in sperm numbers or sperm viability under different diets.

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.

The function and evolutionary significance of a triplicated Na,K-ATPase gene in a toxin-specialized insect.

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.

The Function and Evolution of Nuclear Receptors in Insect Embryonic Development.

Nuclear receptors are a family of transcription factors that are often responsive to small ligands, allowing for efficient gene expression-level responses to a stimulus. The average insect has 21 genes encoding nuclear receptors, whose functions are especially well studied in developmental transitions during the insect life cycle, such as metamorphosis and molting. However, their utility as well-controlled transcriptional regulators also lends them to important roles in embryogenesis, neurogenesis, metabolism, and organogenesis. Such developmental functions have been explored in depth in the model organism Drosophila melanogaster. More recently, advances in genomic resources and functional genomic methodologies have allowed for comparison of nuclear receptor function among a wider range of insect species. As has been the trend throughout the field of Evo-Devo, these new data sets reveal that many genes are shared, but the ways in which they are utilized in different lineages are more variable. In this chapter, we describe the suite of nuclear receptor genes found in Drosophila and their developmental functions. We then compare and contrast these genes and their functions in diverse insects.

Assessment of the effects of orally administered ferrous sulfate on Oncopeltus fasciatus (Heteroptera: Lygaeidae).

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.

Laboratory evaluation of Clusia fluminensis extracts and their isolated compounds against Dysdercus peruvianus and Oncopeltus fasciatus

ABSTRACT The effects of the hexanic extracts of the fruits and flowers of Clusia fluminensis Planch. & Triana, Clusiaceae, as well as their main constituents, the triterpene lanosterol and the benzophenone clusianone, were evaluated on hemipterans Dysdercus peruvianus and Oncopeltus fasciatus. The topical treatments of insects with the hexanic extracts significantly affected the survival of O. fasciatus, but not that of D. peruvianus. Concomitantly, extracts delayed the development of both hemipterans. Moreover, isolated lanosterol significantly reduced both the survival and development of O. fasciatus and D. peruvianus, while clusianone only reduce the survival of D. peruvianus and marginally inhibited the development of both insects. The results show the specific activity of lanosterol and clusianone against the two evaluated insect species and indicate the potential of compounds derived from C. fluminensis for the development of specific biopesticides for the control of agricultural pests. Subsequent work will examine the mode of action of lanosterol and clusianone isolates from C. fluminensis.

Susceptibility of Phytomonas serpens to calpain inhibitors in vitro: interference on the proliferation, ultrastructure, cysteine peptidase expression and interaction with the invertebrate host

A pleiotropic response to the calpain inhibitor MDL28170 was detected in the tomato parasite Phytomonas serpens. Ultrastructural studies revealed that MDL28170 caused mitochondrial swelling, shortening of flagellum and disruption of trans Golgi network. This effect was correlated to the inhibition in processing of cruzipain-like molecules, which presented an increase in expression paralleled by decreased proteolytic activity. Concomitantly, a calcium-dependent cysteine peptidase was detected in the parasite extract, the activity of which was repressed by pre-incubation of parasites with MDL28170. Flow cytometry and Western blotting analyses revealed the differential expression of calpain-like proteins (CALPs) in response to the pre-incubation of parasites with the MDL28170, and confocal fluorescence microscopy confirmed their surface location. The interaction of promastigotes with explanted salivary glands of the insect Oncopeltus fasciatus was reduced when parasites were pre-treated with MDL28170, which was correlated to reduced levels of surface cruzipain-like and gp63-like molecules. Treatment of parasites with anti-Drosophila melanogaster (Dm) calpain antibody also decreased the adhesion process. Additionally, parasites recovered from the interaction process presented higher levels of surface cruzipain-like and gp63-like molecules, with similar levels of CALPs cross-reactive to anti-Dm-calpain antibody. The results confirm the importance of exploring the use of calpain inhibitors in studying parasites’ physiology.

Gene duplications circumvent trade-offs in enzyme function: Insect adaptation to toxic host plants.

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.

The phenotypic plasticity of developmental modules.

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.

A Pathway Analysis of Melanin Patterning in a Hemimetabolous Insect.

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.

Evolution of epithelial morphogenesis: phenotypic integration across multiple levels of biological organization.

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.

Codon and Amino Acid Usage Are Shaped by Selection Across Divergent Model Organisms of the Pancrustacea.

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.