Entomology
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Item Rapid transcriptome sequencing of an invasive pest, the brown marmorated stink bug Halyomorpha halys(Springer Nature, 2014-08-29) Ioannidis, Panagiotis; Lu, Yong; Kumar, Nikhil; Creasy, Todd; Daugherty, Sean; Chibucos, Marcus C; Orvis, Joshua; Shetty, Amol; Ott, Sandra; Flowers, Melissa; Sengamalay, Naomi; Tallon, Luke J; Pick, Leslie; Dunning Hotopp, Julie CHalyomorpha halys (Stål) (Insecta:Hemiptera;Pentatomidae), commonly known as the Brown Marmorated Stink Bug (BMSB), is an invasive pest of the mid-Atlantic region of the United States, causing economically important damage to a wide range of crops. Native to Asia, BMSB was first observed in Allentown, PA, USA, in 1996, and this pest is now well-established throughout the US mid-Atlantic region and beyond. In addition to the serious threat BMSB poses to agriculture, BMSB has become a nuisance to homeowners, invading home gardens and congregating in large numbers in human-made structures, including homes, to overwinter. Despite its significance as an agricultural pest with limited control options, only 100 bp of BMSB sequence data was available in public databases when this project began. Transcriptome sequencing was undertaken to provide a molecular resource to the research community to inform the development of pest control strategies and to provide molecular data for population genetics studies of BMSB. Using normalized, strand-specific libraries, we sequenced pools of all BMSB life stages on the Illumina HiSeq. Trinity was used to assemble 200,000 putative transcripts in >100,000 components. A novel bioinformatic method that analyzed the strand-specificity of the data reduced this to 53,071 putative transcripts from 18,573 components. By integrating multiple other data types, we narrowed this further to 13,211 representative transcripts. Bacterial endosymbiont genes were identified in this dataset, some of which have a copy number consistent with being lateral gene transfers between endosymbiont genomes and Hemiptera, including ankyrin-repeat related proteins, lysozyme, and mannanase. Such genes and endosymbionts may provide novel targets for BMSB-specific biocontrol. This study demonstrates the utility of strand-specific sequencing in generating shotgun transcriptomes and that rapid sequencing shotgun transcriptomes is possible without the need for extensive inbreeding to generate homozygous lines. Such sequencing can provide a rapid response to pest invasions similar to that already described for disease epidemiology.Item Dermestes maculatus: an intermediate-germ beetle model system for evo-devo(Springer Nature, 2015-10-16) Xiang, Jie; Forrest, Iain S.; Pick, LeslieUnderstanding how genes change during evolution to direct the development of diverse body plans is a major goal of the evo-devo field. Achieving this will require the establishment of new model systems that represent key points in phylogeny. These new model systems must be amenable to laboratory culture, and molecular and functional approaches should be feasible. To date, studies of insects have been best represented by the model system Drosophila melanogaster. Given the enormous diversity represented by insect taxa, comparative studies within this clade will provide a wealth of information about the evolutionary potential and trajectories of alternative developmental strategies. Here we established the beetle Dermestes maculatus, a member of the speciose clade Coleoptera, as a new insect model system. We have maintained a continuously breeding culture in the lab and documented Dermestes maculatus embryogenesis using nuclear and phalloidin staining. Anterior segments are specified during the blastoderm stage before gastrulation, and posterior segments are added sequentially during germ band elongation. We isolated and studied the expression and function of the pair-rule segmentation gene paired in Dermestes maculatus. In this species, paired is expressed in stripes during both blastoderm and germ band stages: four primary stripes arise prior to gastrulation, confirming an intermediate-germ mode of development for this species. As in other insects, these primary stripes then split into secondary stripes. To study gene function, we established both embryonic and parental RNAi. Knockdown of Dmac-paired with either method resulted in pair-rule-like segmentation defects, including loss of Engrailed expression in alternate stripes. These studies establish basic approaches necessary to use Dermestes maculatus as a model system. Methods are now available for use of this intermediate-germ insect for future studies of the evolution of regulatory networks controlling insect segmentation, as well as of other processes in development and homeostasis. Consistent with the role of paired in long-germ Drosophila and shorter-germ Tribolium, paired functions as a pair-rule segmentation gene in Dermestes maculatus. Thus, paired retains pair-rule function in insects with different modes of segment addition.Item Establishment of molecular genetic approaches to study gene expression and function in an invasive hemipteran, Halyomorpha halys(Springer Nature, 2017-10-18) Lu, Yong; Chen, Mengyao; Reding, Katie; Pick, LeslieHemiptera is a large clade of insects understudied in terms of developmental biology. Halyomorpha halys, the Brown Marmorated Stink Bug (BMSB, referred to throughout as H. halys), is an invasive hemipteran pest of the mid-Atlantic region of the USA that has rapidly spread to other regions in recent years, devastating a wide range of crops using a piercing and sucking mechanism. Its phylogenetic position, polyphagous habits, and rapid spread in the USA suggested that H. halys would be an ideal system to broaden our knowledge of developmental mechanisms in insects. We and others previously generated transcriptome sequences from different life stages of this insect. Here, we describe tools to examine gene expression patterns in whole-mount H. halys embryos and to test the response of H. halys to RNA interference (RNAi). We show that spatial and temporal patterns of gene expression in H. halys can be effectively monitored by both immunostaining and in situ hybridization. We also show that delivery of dsRNA to adult females knocks down gene function in offspring, using the homeotic gene Sex combs reduced (Scr). Knockdown of Hh-Scr resulted in dramatic malformations of the mouthparts, demonstrating for the first time that RNAi is effective in this species. Our results suggest that, despite difficulties with long-term laboratory culture of H. halys, this species shows promise as a developmental system.Item Oncopeltus-like gene expression patterns in Murgantia histrionica, a new hemipteran model system, suggest ancient regulatory network divergence(Springer Nature, 2020-04-22) Hernandez, Jessica; Pick, Leslie; Reding, KatieMuch 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. Here 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. The 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.Item Activation of Ftz-F1-Responsive Genes through Ftz/Ftz-F1 Dependent Enhancers(PLoS (Public Library of Science), 2016-10-10) Field, Amanda; Xiang, Jie; Anderson, W. Ray; Graham, Patricia; Pick, LeslieThe orphan nuclear receptor Ftz-F1 is expressed in all somatic nuclei in Drosophila embryos, but mutations result in a pair-rule phenotype. This was explained by the interaction of Ftz-F1 with the homeodomain protein Ftz that is expressed in stripes in the primordia of segments missing in either ftz-f1 or ftz mutants. Ftz-F1 and Ftz were shown to physically interact and coordinately activate the expression of ftz itself and engrailed by synergistic binding to composite Ftz-F1/Ftz binding sites. However, attempts to identify additional target genes on the basis of Ftz-F1/ Ftz binding alone has met with only limited success. To discern rules for Ftz-F1 target site selection in vivo and to identify additional target genes, a microarray analysis was performed comparing wildtype and ftz-f1 mutant embryos. Ftz-F1-responsive genes most highly regulated included engrailed and nine additional genes expressed in patterns dependent on both ftz and ftz-f1. Candidate enhancers for these genes were identified by combining BDTNP Ftz ChIP-chip data with a computational search for Ftz-F1 binding sites. Of eight enhancer reporter genes tested in transgenic embryos, six generated expression patterns similar to the corresponding endogenous gene and expression was lost in ftz mutants. These studies identified a new set of Ftz-F1 targets, all of which are co-regulated by Ftz. Comparative analysis of enhancers containing Ftz/Ftz-F1 binding sites that were or were not bona fide targets in vivo suggested that GAF negatively regulates enhancers that contain Ftz/Ftz-F1 binding sites but are not actually utilized. These targets include other regulatory factors as well as genes involved directly in morphogenesis, providing insight into how pair-rule genes establish the body pattern.Item Rapid isolation of gene homologs across taxa: Efficient identification and isolation of gene orthologs from non-model organism genomes, a technical report(2011-03-01) Heffer, Alison; Pick, LeslieBackground: Tremendous progress has been made in the field of evo-devo through comparisons of related genes from diverse taxa. While the vast number of species in nature precludes a complete analysis of the molecular evolution of even one single gene family, this would not be necessary to understand fundamental mechanisms underlying gene evolution if experiments could be designed to systematically sample representative points along the path of established phylogenies to trace changes in regulatory and coding gene sequence. This isolation of homologous genes from phylogenetically diverse, representative species can be challenging, especially if the gene is under weak selective pressure and evolving rapidly. Results: Here we present an approach - Rapid Isolation of Gene Homologs across Taxa (RIGHT) - to efficiently isolate specific members of gene families. RIGHT is based upon modification and a combination of degenerate polymerase chain reaction (PCR) and gene-specific amplified fragment length polymorphism (AFLP). It allows targeted isolation of specific gene family members from any organism, only requiring genomic DNA. We describe this approach and how we used it to isolate members of several different gene families from diverse arthropods spanning millions of years of evolution. Conclusions: RIGHT facilitates systematic isolation of one gene from large gene families. It allows for efficient gene isolation without whole genome sequencing, RNA extraction, or culturing of non-model organisms. RIGHT will be a generally useful method for isolation of orthologs from both distant and closely related species, increasing sample size and facilitating the tracking of molecular evolution of gene families and regulatory networks across the tree of life.