Oncopeltus Fasciatus Research Articles

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

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

Distinct Developmental Mechanisms Influence Sexual Dimorphisms in the Milkweed Bug Oncopeltus Fasciatus

Distinct Developmental Mechanisms Influence Sexual Dimorphisms in the Milkweed Bug Oncopeltus Fasciatus

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

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

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

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

Chromosome-level genome assembly of the bean bug Riptortus pedestris.

The bean bug (Riptortus pedestris) causes great economic losses of soybeans by piercing and sucking pods and seeds. Although R. pedestris has become the focus of numerous studies associated with insect-microbe interactions, plant-insect interactions, and pesticide resistance, a lack of genomic resources has limited deeper insights. Here, we report the first R. pedestris genome at the chromosomal level using PacBio, Illumina, and Hi-C technologies. The assembled genome was 1.080Gb in size with a contig N50 of 2.882Mb. More than 96.3% of the total genome bases were successfully anchored to six unique chromosomes. Genome resequencing of male and female individuals and chromosomic staining demonstrated that the sex chromosome system of R. pedestris is XO, and the shortest chromosome is the X chromosome. In total, 19,026 protein-coding genes were predicted, 18,745 of which were validated as being expressed. Temporospatial expression of R. pedestris genes in six tissues and 37 development stages revealed 4,657 and 7,793 genes mainly expressed in gonads and egg periods, respectively. Evolutionary analysis demonstrated that R. pedestris and Oncopeltus fasciatus formed a sister group and split ∼80million years ago (Mya). Additionally, a 5.04Mb complete genome of symbiotic Serratia marcescens Rip1 was assembled, and the virulence factors that account for successful colonization in the host midgut were identified. The high-quality R. pedestris genome provides a valuable resource for further research, as well as for the pest management of bug pests.

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

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

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

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

A conserved role for arrow in posterior axis patterning across Arthropoda

Segmentation is a key characteristic of Arthropoda that is linked to the evolutionary success of this lineage. It has previously been shown in both vertebrates and short germ insects that posterior segmentation requires canonical Wnt (cWnt) signaling, which maintains the expression of Caudal and the posterior growth zone; disruption of cWnt signaling incurs posterior truncations in these lineages due to the loss of the tail bud. However, comparable datasets for Wnt signaling are limited outside of holometabolous insects, due to incomparable phenotypic spectra and inefficacy of gene misexpression methods in certain model species. We applied RNA interference (RNAi) against the Wnt co-receptor arrow (arr), a key member of the cWnt signaling pathway in holometabolous insects and vertebrates, to examine posterior axis elongation of the cobweb spider Parasteatoda tepidariorum (short germ embryogenesis; one Wnt8 homolog), the cricket Gryllus bimaculatus (intermediate germ; one Wnt8 homolog), and the milkweed bug Oncopeltus fasciatus (short germ; two Wnt8 homologs). Knockdown of arr in insects resulted in posterior truncations affecting the gnathos through the abdomen in O. fasciatus, whereas posterior truncations only affected the T3 segment through the abdomen in G. bimaculatus. Spider embryos with disrupted arr expression exhibited defects along the entire axis, including segmentation defects throughout the germband. RNA-Seq-based differential gene expression analysis of severe Ptep-arr loss-of-function phenotypes at two developmental stages was used to confirm that knockdown of Ptep-arr results in systemic disruption of the Wnt pathway. Intriguingly, we found that knockdown of arr did not abrogate Wnt8 expression in any of the three species, with cad expression additionally retained in severe loss-of-function phenotypes in the cricket and the spider. Together with data from a holometabolous insect, our results suggest that cWnt signaling is not required for maintenance of Wnt8 expression across Arthropoda. These outcomes underscore the diagnostic power of differential gene expression analyses in characterizing catastrophic phenotypes in emerging model species.

Insecticidal activity of Pilocarpus spicatus Saint-Hilaire (Rutaceae) essential oil against the crop pest Dysdercus peruvianus (Guérin-Méneville, 1831) and Oncopeltus fasciatus (Dallas, 1852)

Studies were carried out to evaluate the insecticidal activity of Pilocarpus spicatus Saint-Hilaire (Rutaceae) essential oil (EO) on the development of the Hemiptera Dysdercus peruvianus (Guérin-Méneville, 1831) and Oncopeltus fasciatus (Dallas, 1852). Gas Chromatography/Mass spectrometry analysis revealed a chemical composition with sabinene (32.27%) and sylvestrene (27.26%) as major constituents. Topical and continuous treatment with the pure EO induced 100% of mortality while serial dilutions of the EO induced different levels of lethality in a dose response manner. Median lethal dose (LD50) and lethal dose 90% (LD90) were determined. Malformations in insects and permanent or supernumerary nymphs were often observed after treatments, and the use of scanning electron microscopy allowed the analysis of morphological changes. The different biological effects of P. spicatus EO point out its potential as a rich source of bioactive molecules to be used as an alternative control method against agricultural pest insects.

The multiple roles of caudal in early development of the milkweed bug Oncopeltus fasciatus

The homeobox transcription factor Caudal has conserved roles in all Bilateria in defining the posterior pole and in controlling posterior elongation. These roles are seemingly similar and are difficult to disentangle. We have carried out a detailed analysis of the expression, function and interactions of the caudal ortholog of the milkweed bug, Oncopeltus fasciatus, a hemimetabolous insect with a conservative early development process, in order to understand its different functions throughout development. In Oncopeltus, caudal is not maternally deposited, but has a sequence of roles in the posterior of the embryos throughout early development. It defines and maintains a posterior-anterior gradient in the blastoderm and modulates the activity of segmentation genes in simultaneous segmentation during the blastoderm stage. It later defines the invagination site and the posterior segment addition zone (SAZ) in the germband. It maintains the posterior SAZ cells in an undifferentiated proliferative state, while promoting dynamic expression of segmentation genes in the anterior SAZ. We show that rather than being a simple posterior determinant, Caudal is involved in several distinct regulatory networks, each with a distinct developmental role.

High-Efficiency CRISPR/Cas9 Mutagenesis of the white Gene in the Milkweed Bug Oncopeltus fasciatus.

In this manuscript, we report that clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 is highly efficient in the hemipteran Oncopeltus fasciatus. The white gene is well characterized in Drosophila where mutation causes loss of eye pigmentation; white is a reliable marker for transgenesis and other genetic manipulations. Accordingly, white has been targeted in a number of nonmodel insects to establish tools for genetic studies. Here, we generated mutations in the Of-white (Of-w) locus using CRISPR/Cas9. We found that Of-w is required for pigmentation throughout the body of Oncopeltus, not just the ommatidia. High rates of somatic mosaicism were observed in the injected generation, reflecting biallelic mutations, and a high rate of germline mutation was evidenced by the large proportion of heterozygous G1s. However, Of-w mutations are homozygous lethal; G2 homozygotes lacked pigment dispersion throughout the body and did not hatch, precluding the establishment of a stable mutant line. Embryonic and parental RNA interference (RNAi) were subsequently performed to rule out off-target mutations producing the observed phenotype and to evaluate the efficacy of RNAi in ablating gene function compared to a loss-of-function mutation. RNAi knockdowns phenocopied Of-w homozygotes, with an unusual accumulation of orange granules observed in unhatched embryos. This is, to our knowledge, the first CRISPR/Cas9-targeted mutation generated in Oncopeltus. While we were unable to establish white as a useful visible marker for Oncopeltus, these findings are instructive for the selection of visible markers in nonmodel species and reveal an unusual role for an ortholog of a classic Drosophila gene.

Dicksonia sellowiana Hook. and Nepholepis cordifolia (L.) Lellinger extracts as potential green pesticides: insecticidal activity

Known as green insecticides, natural plant-based products have become a promising alternative to conventional insecticides. The primary objective of the present study was to analyze, under laboratory conditions, the insecticidal activity of crude ethanol extracts of the fern species Dicksonia sellowiana and Nephrolepis cordifolia against Oncopeltus fasciatus (Hemiptera). Fern leaves were collected from Itatiaia National Park (Brazil), dried and ground using 96% ethanol, with the extract concentrated in a rotary evaporator. The crude extract was used to prepare a 50 mg/mL solution, with acetone as solvent. Qualitative analysis of the terpenoid and phenolic substance profile in the extracts was performed by thin-layer chromatography (TLC). Fourth-instar insects were used, in groups of 10 animals per treatment, with three repetitions. Four treatments were used: D. sellowiana and N. cordifolia extracts, acetone control and water control. Then, 1 μL either of the respective solution was topically applied to the insect abdominal cuticle. After 21 days, all the insects in the water control group had reached adult. The N. cordifolia extract was responsible for 63% (p < 0.0001) of insect mortality around 16 days after treatment, whereas the D. sellowiana exhibited 50% (p < 0.0001) on the 21st day post-treatment. The extracts also caused delays in insect molting and metamorphosis. The D. sellowiana and N. cordifolia extracts exhibited 18% similarity in the terpenoid profile and 0% for phenolic substances. N. cordifolia and D. sellowiana show potential for research on selective biodegradable substances for use as green insecticides.

Oncopeltus-like gene expression patterns in Murgantia histrionica, a new hemipteran model system, suggest ancient regulatory network divergence

BackgroundMuch has been learned about basic biology from studies of insect model systems. The pre-eminent insect model system, Drosophila melanogaster, is a holometabolous insect with a derived mode of segment formation. While additional insect models have been pioneered in recent years, most of these fall within holometabolous lineages. In contrast, hemimetabolous insects have garnered less attention, although they include agricultural pests, vectors of human disease, and present numerous evolutionary novelties in form and function. The milkweed bug, Oncopeltus fasciatus (order: Hemiptera)—close outgroup to holometabolous insects—is an emerging model system. However, comparative studies within this order are limited as many phytophagous hemipterans are difficult to stably maintain in the lab due to their reliance on fresh plants, deposition of eggs within plant material, and long development time from embryo to adult.ResultsHere we present the harlequin bug, Murgantia histrionica, as a new hemipteran model species. Murgantia—a member of the stink bug family Pentatomidae which shares a common ancestor with Oncopeltus ~ 200 mya—is easy to rear in the lab, produces a large number of eggs, and is amenable to molecular genetic techniques. We use Murgantia to ask whether Pair-Rule Genes (PRGs) are deployed in ways similar to holometabolous insects or to Oncopeltus. Specifically, PRGs even-skipped, odd-skipped, paired and sloppy-paired are initially expressed in PR-stripes in Drosophila and a number of holometabolous insects but in segmental-stripes in Oncopeltus. We found that these genes are likewise expressed in segmental-stripes in Murgantia, while runt displays partial PR-character in both species. Also like Oncopeltus, E75A is expressed in a clear PR-pattern in blastoderm- and germband-stage Murgantia embryos, although it plays no role in segmentation in Drosophila. Thus, genes diagnostic of the split between holometabolous insects and Oncopeltus are expressed in an Oncopeltus-like fashion during Murgantia development.ConclusionsThe similarity in gene expression between Murgantia and Oncopeltus suggests that Oncopeltus is not a sole outlier species in failing to utilize orthologs of Drosophila PRGs for PR-patterning. Rather, strategies deployed for PR-patterning, including the use of E75A in the PRG-network, are likely conserved within Hemiptera, and possibly more broadly among hemimetabolous insects.

A single ortholog of teashirt and tiptop regulates larval pigmentation and adult appendage patterning in Bombyx mori

Two paralogous genes, teashirt (tsh) and tiptop (tio), encode zinc-finger transcription factors and play important roles in insect growth and development. In the fruit fly, Drosophila melanogaster, tsh promotes trunk segmental identities and contributes to the patterning of other tissues during the embryonic stage. During the adult stage, tsh contributes to the specification and patterning of appendages, including the leg, wing and eye. While tio acts redundantly with tsh, flies lacking tio function are viable without deleterious phenotypes. This gene pair is present in the genomes of all Drosophila species but only as a single homologue in several other insect species. In Oncopeltus fasciatus and Tribolium castaneum, tsh/tio has been functionally characterized as specifying the identity of the leg during the adult stage. However, in lepidopteran insects which include large numbers of pests in agriculture and forestry, as well as the major silk producer silkworm Bombyx mori, the biological functions of tsh/tio are still poorly understood. In the current study, we performed functional analysis of tsh/tio by using both CRISPR/Cas9-mediated mutagenesis and transposon-mediated ectopic expression in B. mori. The results show that loss of tsh/tio function affected pigmentation during the larval stage and appendage pattering during the adult stage. RNA-seq analysis and subsequent q-RT-PCR analysis revealed that depletion of tsh/tio significantly elevated the expression of the kynurenine 3-monooxygenase gene, as well as melanin synthase-related genes during the larval stage. Furthermore, ubiquitous ectopic expression of tsh/tio induces developmental retardation and eventually larval lethality. These data reveal evolutionarily conserved functions of tsh/tio in controlling adult appendage patterning, as well as the novel function of regulating larval pigmentation in B. mori, providing novel insights into how tsh/tio regulates insect growth and development.

A hemipteran insect reveals new genetic mechanisms and evolutionary insights into tracheal system development

The diversity in the organization of the tracheal system is one of the drivers of insect evolutionary success; however, the genetic mechanisms responsible are yet to be elucidated. Here, we highlight the advantages of utilizing hemimetabolous insects, such as the milkweed bug Oncopeltus fasciatus, in which the final adult tracheal patterning can be directly inferred by examining its blueprint in embryos. By reporting the expression patterns, functions, and Hox gene regulation of trachealess (trh), ventral veinless (vvl), and cut (ct), key genes involved in tracheal development, this study provides important insights. First, Hox genes function as activators, modifiers, and suppressors of trh expression, which in turn results in a difference between the thoracic and abdominal tracheal organization. Second, spiracle morphogenesis requires the input of both trh and ct, where ct is positively regulated by trh As Hox genes regulate trh, we can now mechanistically explain the previous observations of their effects on spiracle formation. Third, the default state of vvl expression in the thorax, in the absence of Hox gene expression, features three lateral cell clusters connected to ducts. Fourth, the exocrine scent glands express vvl and are regulated by Hox genes. These results extend previous findings [Sánchez-Higueras et al., 2014], suggesting that the exocrine glands, similar to the endocrine, develop from the same primordia that give rise to the trachea. The presence of such versatile primordia in the miracrustacean ancestor could account for the similar gene networks found in the glandular and respiratory organs of both insects and crustaceans.

More Than DNA Methylation: Does Pleiotropy Drive the Complex Pattern of Evolution of Dnmt1?

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.

The POU factor Ventral veins lacking regulates ecdysone and juvenile hormone biosynthesis during development and reproduction of the milkweed bug, Oncopeltus fasciatus

Recent studies have demonstrated endocrine roles for the POU domain transcription factor Ventral veins lacking (Vvl) during larval development of holometabolous insects - insects that undergo complete metamorphosis. In this study, the role of Vvl was examined in the milkweed bug, Oncopeltus fasciatus, a hemimetabolous insect. In the embryos, vvl was found to be expressed in the presumptive prothoracic glands. When vvl expression was knocked down using RNA interference (RNAi), embryos arrested their development after dorsal closure. Vvl double-stranded RNA (dsRNA)-injected nymphs failed to molt and had reduced expression of the ecdysone response gene, hormone receptor 3 (HR3), the ecdysone biosynthesis genes, disembodied and spook, and the juvenile hormone (JH) response gene, Krüppel homolog 1 (Kr-h1). Injection of 20-hydroxyecdysone rescued the molting phenotype and HR3 expression in vvl knockdown nymphs. In adults, vvl RNAi inhibited egg laying and suppressed the expression of Kr-h1 and vitellogenin in the fat body. Application of JH III or methoprene restored oviposition in vvl knockdown adults, indicating that Vvl regulates JH biosynthesis during reproduction. Thus, Vvl functions as a critical regulator of hormone biosynthesis throughout all developmental stages of O. fasciatus. Our study demonstrates that Vvl is a critical transcription factor involved in JH and ecdysteroid biosynthesis in both hemimetabolous and holometabolous insects.

Natural infection by the protozoan Leptomonas wallacei impacts the morphology, physiology, reproduction, and lifespan of the insect Oncopeltus fasciatus

Trypanosomatids are protozoan parasites that infect thousands of globally dispersed hosts, potentially affecting their physiology. Several species of trypanosomatids are commonly found in phytophagous insects. Leptomonas wallacei is a gut-restricted insect trypanosomatid only retrieved from Oncopeltus fasciatus. The insects get infected by coprophagy and transovum transmission of L. wallacei cysts. The main goal of the present study was to investigate the effects of a natural infection by L. wallacei on the hemipteran insect O. fasciatus, by comparing infected and uninfected individuals in a controlled environment. The L. wallacei-infected individuals showed reduced lifespan and morphological alterations. Also, we demonstrated a higher infection burden in females than in males. The infection caused by L. wallacei reduced host reproductive fitness by negatively impacting egg load, oviposition, and eclosion, and promoting an increase in egg reabsorption. Moreover, we associated the egg reabsorption observed in infected females, with a decrease in the intersex gene expression. Finally, we suggest alterations in population dynamics induced by L. wallacei infection using a mathematical model. Collectively, our findings demonstrated that L. wallacei infection negatively affected the physiology of O. fasciatus, which suggests that L. wallacei potentially has a vast ecological impact on host population growth.

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.

Molecular evolutionary trends and feeding ecology diversification in the Hemiptera, anchored by the milkweed bug genome

BackgroundThe Hemiptera (aphids, cicadas, and true bugs) are a key insect order, with high diversity for feeding ecology and excellent experimental tractability for molecular genetics. Building upon recent sequencing of hemipteran pests such as phloem-feeding aphids and blood-feeding bed bugs, we present the genome sequence and comparative analyses centered on the milkweed bug Oncopeltus fasciatus, a seed feeder of the family Lygaeidae.ResultsThe 926-Mb Oncopeltus genome is well represented by the current assembly and official gene set. We use our genomic and RNA-seq data not only to characterize the protein-coding gene repertoire and perform isoform-specific RNAi, but also to elucidate patterns of molecular evolution and physiology. We find ongoing, lineage-specific expansion and diversification of repressive C2H2 zinc finger proteins. The discovery of intron gain and turnover specific to the Hemiptera also prompted the evaluation of lineage and genome size as predictors of gene structure evolution. Furthermore, we identify enzymatic gains and losses that correlate with feeding biology, particularly for reductions associated with derived, fluid nutrition feeding.ConclusionsWith the milkweed bug, we now have a critical mass of sequenced species for a hemimetabolous insect order and close outgroup to the Holometabola, substantially improving the diversity of insect genomics. We thereby define commonalities among the Hemiptera and delve into how hemipteran genomes reflect distinct feeding ecologies. Given Oncopeltus’s strength as an experimental model, these new sequence resources bolster the foundation for molecular research and highlight technical considerations for the analysis of medium-sized invertebrate genomes.