Establishment of molecular genetic approaches to study gene expression and function in an invasive hemipteran, Halyomorpha halys

Lu, Yingzhi; Chen, Mengyao; Reding, Katie; Pick, Leslie

doi:10.1186/s13227-017-0078-6

Cited by 15 publications

(15 citation statements)

References 74 publications

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“…Post-transcriptional gene silencing also known as RNA interference (RNAi) was first elucidated in 1998 [5] and has since then been widely used as a tool in the study of gene function. In the hemipteran group, RNAi has been used to study the role of genes in insect development/reproduction of several species, such as Oncopeltus fasciatus [6,7], Nezara viridula ( [8], Diaphorina citri [9], Halyomorpha halys [10], among others. RNAi is a highly conserved mechanism among eukaryotic organisms, in which the messenger RNA (mRNA) is cleaved by the RNAi machinery, leading to the inactivation of gene expression [11].…”

Section: Introductionmentioning

confidence: 99%

RNAi and CRISPR/Cas9 as Functional Genomics Tools in the Neotropical Stink Bug, Euschistus heros

Cagliari

Smagghe

Zotti

et al. 2020

Insects

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The Neotropical brown stink bug, Euschistus heros, is one of the most important stink bug pests in leguminous plants in South America. RNAi and CRISPR/Cas9 are important and useful tools in functional genomics, as well as in the future development of new integrated pest management strategies. Here, we explore the use of these technologies as complementing functional genomic tools in E. heros. Three genes, abnormal wing disc (awd), tyrosine hydroxylase (th) and yellow (yel), known to be involved in wing development (awd) and the melanin pathway (th and yel) in other insects, were chosen to be evaluated using RNAi and CRISPR/Cas9 as tools. First, the genes were functionally characterized using RNAi knockdown technology. The expected phenotype of either deformed wing or lighter cuticle pigmentation/defects in cuticle sclerotization was observed for awd and th, respectively. However, for yel, no obvious phenotype was observed. Based on this, yel was selected as a target for the development of a CRISPR/Cas9 workflow to study gene knockout in E. heros. A total of 719 eggs were injected with the Cas9 nuclease (300 ng/µL) together with the sgRNA (300 ng/µL) targeting yel. A total of six insects successfully hatched from the injected eggs and one of the insects showed mutation in the target region, however, the phenotype was still not obvious. Overall, this study for the first time provides a useful CRISPR/Cas9 gene editing methodology to complement RNAi for functional genomic studies in one of the most important and economically relevant stink bug species.

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Section: Introductionmentioning

confidence: 99%

RNAi and CRISPR/Cas9 as Functional Genomics Tools in the Neotropical Stink Bug, Euschistus heros

Cagliari

Smagghe

Zotti

et al. 2020

Insects

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“…As such, this species meets all the basic requirements to serve as a model for molecular genetic analysis. Our lab previously tried to rear another pentatomid, the invasive brown marmorated stink bug Halyomorpha halys, and observed a collapse of our lab colony after three generations, possibly due to infection by a microsporidian [60,61]. We therefore subjected Murgantia to a 'trial period' of lab rearing before beginning experiments to ensure that we could sustain our lab Methods for examining gene expression in Murgantia were adapted from Oncopeltus protocols and can be performed readily with these modifications on a large number of samples.…”

Section: Discussionmentioning

confidence: 99%

“…Embryos were fixed as described above, except that fixation after chorion removal was done for 30 min only. After fixation, the protocol described by Lu et al [60] for Halyomorpha embryos was used except that post-fixation was not done after the staining reaction. If background staining was strong, embryos were washed in 50% methanol in PBST for 5 min and rinsed 3 times with PBST after the color reaction.…”

Section: Immunohistochemistrymentioning

confidence: 99%

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

Hernandez

Pick

Reding

2020

EvoDevo

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Background: Much 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. Results: Here we present the harlequin bug, Murgantia histrionica, as a new hemipteran model species. Murgantiaa 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. Conclusions: 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.

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“…Hemiptera genes have been successfully silenced through this method (Christiaens & Smagghe, 2014;Bansal et al, 2016;Ghosh et al, 2017;Lu et al, 2017;Mogilicherla et al, 2018, Riga et al, 2019.…”

Section: Discussionmentioning

confidence: 99%

Tyramine receptor drives olfactory response to (E)-2-decenal in the stink bugHalyomorpha halys

Finetti

Pezzi

Civolani

et al. 2020

Preprint

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In insects, the tyramine receptor 1 (TAR1) has been shown to control several physiological functions, including olfaction. We investigated the molecular and functional profile of the Halyomorpha halys type 1 tyramine receptor gene (HhTAR1) and its role in olfactory functions of this pest. Molecular and pharmacological analyses confirmed that the HhTAR1 gene codes for a true TAR1. The RT-qPCR analysis revealed that HhTAR1 is expressed mostly in adult brain and antennae as well as in early development stages (eggs, 1st and 2nd instar nymphs). In particular, among the antennomeres that compose a typical H. halys antenna, HhTAR1 was more expressed in flagellomeres. Scanning electron microscopy (SEM) investigation revealed the type and distribution of sensilla on adult H. halys antennae: both flagellomeres appear rich in trichoid and grooved sensilla, known to be associated with olfactory functions. Through a RNAi approach, topically delivered HhTAR1 dsRNA induced a 50 % gene downregulation after 24 h in H. halys 2nd instar nymphs. An innovative behavioral assay revealed that HhTAR1 RNAi-silenced 2nd instar nymphs were less susceptible to the alarm pheromone component (E)-2 decenal as compared to control. These results provide critical information concerning the TAR1 role in olfaction regulation, especially alarm pheromone reception, in H. halys. Furthermore, considering the emerging role of TAR1 as target of biopesticides, this work paves the way for further investigation on innovative methods for controlling H. halys.

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Establishment of molecular genetic approaches to study gene expression and function in an invasive hemipteran, Halyomorpha halys

Cited by 15 publications

References 74 publications

RNAi and CRISPR/Cas9 as Functional Genomics Tools in the Neotropical Stink Bug, Euschistus heros

RNAi and CRISPR/Cas9 as Functional Genomics Tools in the Neotropical Stink Bug, Euschistus heros

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

Tyramine receptor drives olfactory response to (E)-2-decenal in the stink bugHalyomorpha halys

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