Molecular Evolutionary Trends and Feeding Ecology Diversification in the Hemiptera, Anchored by the Milkweed Bug Genome

Background: The 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. Results: The 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. Conclusions: With 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

The genome of the water strider Gerris buenoi reveals expansions of gene repertoires associated with adaptations to life on the water.

BACKGROUND: Having conquered water surfaces worldwide, the semi-aquatic bugs occupy ponds, streams, lakes, mangroves, and even open oceans. The diversity of this group has inspired a range of scientific studies from ecology and evolution to developmental genetics and hydrodynamics of fluid locomotion. However, the lack of a representative water strider genome hinders our ability to more thoroughly investigate the molecular mechanisms underlying the processes of adaptation and diversification within this group. RESULTS: Here we report the sequencing and manual annotation of the Gerris buenoi (G. buenoi) genome; the first water strider genome to be sequenced thus far. The size of the G. buenoi genome is approximately 1,000 Mb, and this sequencing effort has recovered 20,949 predicted protein-coding genes. Manual annotation uncovered a number of local (tandem and proximal) gene duplications and expansions of gene families known for their importance in a variety of processes associated with morphological and physiological adaptations to a water surface lifestyle. These expansions may affect key processes associated with growth, vision, desiccation resistance, detoxification, olfaction and epigenetic regulation. Strikingly, the G. buenoi genome contains three insulin receptors, suggesting key changes in the rewiring and function of the insulin pathway. Other genomic changes affecting with opsin genes may be associated with wavelength sensitivity shifts in opsins, which is likely to be key in facilitating specific adaptations in vision for diverse water habitats. CONCLUSIONS: Our findings suggest that local gene duplications might have played an important role during the evolution of water striders. Along with these findings, the sequencing of the G. buenoi genome now provides us the opportunity to pursue exciting research opportunities to further understand the genomic underpinnings of traits associated with the extreme body plan and life history of water striders

The genome of the water strider Gerris buenoi reveals expansions of gene repertoires associated with adaptations to life on the water

Background: Having conquered water surfaces worldwide, the semi-aquatic bugs occupy ponds, streams, lakes, mangroves, and even open oceans. The diversity of this group has inspired a range of scientific studies from ecology and evolution to developmental genetics and hydrodynamics of fluid locomotion. However, the lack of a representative water strider genome hinders our ability to more thoroughly investigate the molecular mechanisms underlying the processes of adaptation and diversification within this group. Results: Here we report the sequencing and manual annotation of the Gerris buenoi (G. buenoi) genome; the first water strider genome to be sequenced thus far. The size of the G. buenoi genome is approximately 1,000Mb, and this sequencing effort has recovered 20,949 predicted protein-coding genes. Manual annotation uncovered a number of local (tandem and proximal) gene duplications and expansions of gene families known for their importance in a variety of processes associated with morphological and physiological adaptations to a water surface lifestyle. These expansions may affect key processes associated with growth, vision, desiccation resistance, detoxification, olfaction and epigenetic regulation. Strikingly, the G. buenoi genome contains three insulin receptors, suggesting key changes in the rewiring and function of the insulin pathway. Other genomic changes affecting with opsin genes may be associated with wavelength sensitivity shifts in opsins, which is likely to be key in facilitating specific adaptations in vision for diverse water habitats. Conclusions: Our findings suggest that local gene duplications might have played an important role during the evolution of water striders. Along with these findings, the sequencing of the G. buenoi genome now provides us the opportunity to pursue exciting research opportunities to further understand the genomic underpinnings of traits associated with the extreme body plan and life history of water striders

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

BACKGROUND: The 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. RESULTS: The 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. CONCLUSIONS: With 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

Expression levels of select genes involved in lactation and autophagy from <i>Glossina</i> in fat body and milk gland during late pregnancy and involution.

<p>Transcript abundance was determined by qRT-PCR analysis. The data were analyzed with CFX Manager software version 3.1 (Bio-Rad). A-F are specific genes examined. Data represent the mean ± SE for four samples normalized to <i>tubulin</i>. One-way ANOVA with a post-hoc Tukey test was employed to evaluate statistical differences. Gray lines above the bars indicate significantly higher values than those indicated with a black line. G. <i>In situ</i> hybridization of <i>atg8</i> 36–48 h before and 0–4 h after birth.</p

Rapid autophagic regression of the milk gland during involution is critical for maximizing tsetse viviparous reproductive output

<div><p>Tsetse flies are important vectors of human and animal trypanosomiasis. Ability to reduce tsetse populations is an effective means of disease control. Lactation is an essential component of tsetse’s viviparous reproductive physiology and requires a dramatic increase in the expression and synthesis of milk proteins by the milk gland organ in order to nurture larval growth. In between each gonotrophic cycle, tsetse ceases milk production and milk gland tubules undergo a nearly two-fold reduction in width (involution). In this study, we examined the role autophagy plays during tsetse fly milk gland involution and reproductive output. Autophagy genes show elevated expression in tissues associated with lactation, immediately before or within two hours post-parturition, and decline at 24-48h post-parturition. This expression pattern is inversely correlated with that of the <i>milk gland proteins</i> (lactation-specific protein coding genes) and the autophagy inhibitor <i>fk506-bp1</i>. Increased expression of <i>Drosophila inhibitor of apoptosis 1</i>, <i>diap1</i>, was also observed in the milk gland during involution, when it likely prevents apoptosis of milk gland cells. RNAi-mediated knockdown of <i>autophagy related gene 8a</i> (<i>atg8a</i>) prevented rapid milk gland autophagy during involution, prolonging gestation, and reducing fecundity in the subsequent gonotrophic cycle. The resultant inhibition of autophagy reduced the recovery of stored lipids during the dry (non-lactating) periods by 15–20%. Ecdysone application, similar to levels that occur immediately before birth, induced autophagy, and increased milk gland involution even before abortion. This suggests that the ecdysteroid peak immediately preceding parturition likely triggers milk gland autophagy. Population modeling reveals that a delay in involution would yield a negative population growth rate. This study indicates that milk gland autophagy during involution is critical to restore nutrient reserves and allow efficient transition between pregnancy cycles. Targeting post-birth phases of reproduction could be utilized as a novel mechanism to suppress tsetse populations and reduce trypanosomiasis.</p></div

Reproductive output following suppression of <i>atg8a</i>.

<p>A. Cumulative pupae deposited over the course of 58 days from thirty knockdown or thirty control flies (Two independent replicates) B. Average number of pupae per female and C. duration of pregnancy over 58 days from thirty knockdown or thirty control flies (Two independent replicates). Average duration of pregnancy for each female, over the course of 58 days (1^st pregnancy omitted due to longer duration, typically 17–20 days).</p

Phenotypic changes following suppression of <i>atg8a</i>.

<p>A. Transcript and protein (inset) levels following injection of PBS (control), siGFP (control) and siATG8a. Data for qPCR represents the means ± SE for four biological replicates. B. Changes in the milk gland width following injection of siGFP (control) or siATG8a. Data represents the means ± SE for six biological replicates measured in five locations at each time point. C. Expression of <i>mgp</i> 24 hours after parturition, as determined by qPCR. Data represents the means ± SE for four biological replicates. D. Expression of <i>mgp7</i> days after parturition, as determined by qPCR. Data represents the means ± SE for four biological replicates. One- and two-way ANOVA with a post-hoc Tukey test was employed to evaluate statistical differences. a (low) and b (high), indicates significance at P < 0.05 compared to the PBS-treated control. Line before day 9 denoted parturition.</p

Structural changes in the milk gland during lactation and following involution.

<p>A. Relative changes in specific cell components before, during and immediately after lactation. Each bar represents at least 10 separate counts of images from Hecker and Moloo [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006204#pntd.0006204.ref014" target="_blank">14</a>] and Denlinger et al. [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006204#pntd.0006204.ref011" target="_blank">11</a>]. B. Width of milk gland throughout the pregnancy cycle. Each point represents the width measured from 8 individuals. One-way ANOVA with a post-hoc Tukey test was employed to measure statistical differences. *, indicates significance at P < 0.05.</p

Expression levels of select genes involved in lactation and autophagy from <i>Glossina</i> in fat body and milk gland during late pregnancy and involution.

<p>Transcript abundance was determined by qRT-PCR analysis. The data were analyzed with CFX Manager software version 3.1 (Bio-Rad). A-F are specific genes examined. Data represent the mean ± SE for four samples normalized to <i>tubulin</i>. One-way ANOVA with a post-hoc Tukey test was employed to evaluate statistical differences. Gray lines above the bars indicate significantly higher values than those indicated with a black line. G. <i>In situ</i> hybridization of <i>atg8</i> 36–48 h before and 0–4 h after birth.</p