drosophila-melanogaster-larva-injection-protocol

<em>Drosophila melanogaster</em> Larva Injection Protocol

The use of unconventional models to study innate immunity and pathogen virulence provides a valuable alternative to mammalian models, which can be costly ...

The George Washington University

Plant-parasitic nematodes are responsible for substantial damages within the agriculture industry every year, which is a challenge that has thus far gone largely unimpeded. Chemical nematicides have been employed with varying degrees of success, but their implementation can be cumbersome, and furthermore they could potentially be neutralising an otherwise positive effect from the entomopathogenic nematodes that coexist with plant-parasitic nematodes in soil environments and provide protection for plants against insect pests. Recent research has explored the potential of employing entomopathogenic nematodes to protect plants from plant-parasitic nematodes, while providing their standard degree of protection against insects. The interactions involved are highly complex, due to both the three-organism system and the assortment of variables present in a soil environment, but a strong collection of evidence has accumulated regarding the suppressive capacity of certain entomopathogenic nematodes and their mutualistic bacteria, in the context of limiting the infectivity of plant-parasitic nematodes. Specific factors produced by certain entomopathogenic nematode complexes during the process of insect infection appear to have a selectively nematicidal, or at least repellant, effect on plant-parasitic nematodes. Using this information, an opportunity has formed to adapt this relationship to large-scale, field conditions and potentially relieve the agricultural industry of one of its most substantial burdens. 

Entomopathogenic and plant pathogenic nematodes as opposing forces in agriculture.

Symbiotic interactions between microbes and animals are common in nature. Symbiotic organisms are particularly common in insects and, in some cases, they may protect their hosts from pathogenic infections. Wolbachia and Spiroplasma endosymbionts naturally inhabit various insects including Drosophila melanogaster fruit flies. Therefore, this symbiotic association is considered an excellent model to investigate whether endosymbiotic bacteria participate in host immune processes against certain pathogens. Here we have investigated whether the presence of Wolbachia alone or together with Spiroplasma endosymbionts in D. melanogaster adult flies affects the immune response against the virulent insect pathogen Photorhabdus luminescens and against non-pathogenic Escherichia coli bacteria.

Effects of co-occurring Wolbachia and Spiroplasma endosymbionts on the Drosophila immune response against insect pathogenic and non-pathogenic bacteria.

Molecular and genetic studies in model organisms have recently revealed a dynamic interplay between immunity and ageing mechanisms. In the fruit fly Drosophila melanogaster, inhibition of the insulin/insulin-like growth factor signaling pathway prolongs lifespan, and mutations in the insulin receptor substrate Chico extend the survival of mutant flies against certain bacterial pathogens. Here we investigated the immune phenotypes, immune signaling activation and immune function of chico mutant adult flies against the virulent insect pathogen Photorhabdus luminescens as well as to non-pathogenic Escherichia coli bacteria.

The insulin receptor substrate Chico regulates antibacterial immune function in Drosophila.

Endosymbiotic bacteria exist in many animals where they develop relationships that affect certain physiological processes in the host. Insects and their nematode parasites form great models for understanding the genetic and molecular basis of immune and parasitic processes. Both organisms contain endosymbionts that possess the ability to interfere with certain mechanisms of immune function and pathogenicity. This review summarizes recent information on the involvement of insect endosymbionts in the response to parasitic nematode infections, and the influence of nematode endosymbionts on specific aspects of the insect immune system. Analyzing this information will be particularly useful for devising endosymbiont-based strategies to intervene in insect immunity or nematode parasitism for the efficient management of noxious insects in the field.

Role of Endosymbionts in Insect-Parasitic Nematode Interactions.

Conference report: The 14th congress of the International Society of Developmental and Comparative Immunology.

To gain a comprehensive view of the changes in host gene expression underlying Zika virus (ZIKV) pathogenesis, we performed whole-genome RNA sequencing (RNA-seq) of ZIKV-infected  adult flies. RNA-seq analysis revealed that ZIKV infection alters several and diverse biological processes, including stress, locomotion, lipid metabolism, imaginal disc morphogenesis and regulation of JAK/STAT signaling. To explore the interaction between ZIKV infection and JAK/STAT signaling regulation, we generated genetic constructs overexpressing ZIKV-specific non-structural proteins NS2A, NS2B, NS4A and NS4B. We found that ectopic expression of non-structural proteins in the developing  eye significantly restricts growth of the larval and adult eye and correlates with considerable repression of the  JAK/STAT reporter,  At the cellular level, eye growth defects are associated with reduced rate of proliferation without affecting the overall rate of apoptosis. In addition, ZIKV NS4A genetically interacts with the JAK/STAT signaling components; co-expression of  along with the dominant-negative form of  or  results in aggravated reduction in eye size, while co-expression of  in  (also known as  ) mutant background partially rescues the   -induced eye overgrowth phenotype. The function of ZIKV NS4A in regulating growth is maintained in the wing, where ZIKV  overexpression in the pouch domain results in reduced growth linked with diminished expression of Notch targets, Wingless (Wg) and Cut, and the Notch reporter,  Thus, our study provides evidence that ZIKV infection in  results in restricted growth of the developing eye and wing, wherein eye phenotype is induced through regulation of JAK/STAT signaling, whereas restricted wing growth is induced through regulation of Notch signaling. The interaction of ZIKV non-structural proteins with the conserved host signaling pathways further advance our understanding of ZIKV-induced pathogenesis.This article has an associated First Person interview with the first author of the paper.

Zika virus non-structural protein NS4A restricts eye growth in  through regulation of JAK/STAT signaling.

Insect pathogens have adopted an array of mechanisms to subvert the immune pathways of their respective hosts. Suppression may occur directly at the level of host-pathogen interactions, for instance phagocytic capacity or phenoloxidase activation, or at the upstream signaling pathways that regulate these immune effectors. Insect pathogens of the family Baculoviridae, for example, are known to produce a UDP-glycosyltransferase (UGT) that negatively regulates ecdysone signaling. Normally, ecdysone positively regulates both molting and antimicrobial peptide production, so the inactivation of ecdysone by glycosylation results in a failure of host larvae to molt, and probably a reduced antimicrobial response. Here, we examine a putative ecdysteroid glycosyltransferase, Hba_07292 (Hb-ugt-1), which was previously identified in the hemolymph-activated transcriptome of the entomopathogenic nematode Heterorhabditis bacteriophora. Injection of recombinant Hb-ugt-1 (rHb-ugt-1) into Drosophila melanogaster flies resulted in diminished upregulation of antimicrobial peptides associated with both the Toll and Immune deficiency pathways. Ecdysone was implicated in this suppression by a reduction in Broad Complex expression and reduced pupation rates in r Hb-ugt-1-injected larvae. In addition to the finding that H. bacteriophora excreted-secreted products contain glycosyltransferase activity, these results demonstrate that Hb-ugt-1 is an immunosuppressive factor and that its activity likely involves the inactivation of ecdysone.

A putative UDP-glycosyltransferase from Heterorhabditis bacteriophora suppresses antimicrobial peptide gene expression and factors related to ecdysone signaling.

Nematode virulence factors are of interest for a variety of applications including biocontrol against insect pests and the alleviation of autoimmune diseases with nematode-derived factors. In silico "omics" techniques have generated a wealth of candidate factors that may be important in the establishment of nematode infections, although the challenge of characterizing these individual factors in vivo remains. Here we provide a fundamental characterization of a putative lysozyme and serine carboxypeptidase from the host-induced transcriptome of Heterorhabditis bacteriophora. Both factors accelerated the mortality rate following Drosophila melanogaster infections with Photorhabdus luminescens, and both factors suppressed phenoloxidase activity in D. melanogaster hemolymph. Furthermore, the serine carboxypeptidase was lethal to a subpopulation of flies and suppressed the upregulation of antimicrobial peptides as well as phagocytosis. Together, our findings suggest that this serine carboxypeptidase possess both toxic and immunomodulatory properties while the lysozyme is likely to confer immunomodulatory, but not toxic effects.

A putative lysozyme and serine carboxypeptidase from Heterorhabditis bacteriophora show differential virulence capacities in Drosophila melanogaster.

Much of the available knowledge of entomopathogenic virulence factors has been gleaned from studies in the nematode parasite Steinernema carpocapsae, but there is good reason to complement this knowledge with similar studies in Heterorhabditis bacteriophora. Three candidate virulence factors from H. bacteriophora have recently been characterised, and each was demonstrated to contribute to infection. This information can be used not only to advance efforts in the biocontrol of insect pests, but also to make inferences about the emergence of parasitism among Clade V nematodes.

Secreted virulence factors from Heterorhabditis bacteriophora highlight its utility as a model parasite among Clade V nematodes.

Parasitic nematodes constitute one of the major threats to human health, causing diseases of major socioeconomic importance worldwide. Recent estimates indicate that more than 1 billion people are infected with parasitic nematodes around the world. Current measures to combat parasitic nematode infections include anthelmintic drugs. However, heavy exposure to anthelmintics has selected populations of livestock parasitic nematodes that are no longer susceptible to the drugs, rendering several anthelmintics useless for parasitic nematode control in many areas of the world. The rapidity with which anthelmintic resistance developed in response to these drugs suggests that increasing the selective pressure on human parasitic nematodes will also rapidly generate resistant worm populations. Therefore, development of new anthelmintics is of major importance before resistance becomes widespread in human parasitic nematode populations. G-Protein Coupled Receptors (GPCRs) represent an important target for many pharmacological interventions due to their ubiquitous expression in various cell types. GPCRs contribute to numerous physiological processes, and their ligand binding sites located on cell surfaces make them accessible targets and attractive substrates in terms of druggability. In fact, ∼35 % of Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved drugs target GPCRs and their associated proteins, with over 300 additional drugs targeting GPCRs at the clinical trial stage. Nematode Chemosensory GPCRs (NemChRs) are unique to nematodes, and therefore represent ideal substrates for target-based drug discovery. Here we set out to identify NemChRs that are transcriptionally active inside the host, and to use these NemChRs in a reverse pharmacological screen to impede parasitic development. Our data identified several NemChRs, and we focused on one that was expressed in neuronal cells and exhibited the highest fold change in transcription after host activation. Next, we performed homology modelling and molecular dynamics simulations of this NemChR in order to conduct a virtual screening campaign to identify candidate drug targets which were ranked and selected for experimental testing in bioassays. Taken together, our results identify and characterize a candidate NemChR drug target, and provide a chemogenomic pipeline for identifying nematicide substrates.

Article	Year
Entomopathogenic and plant pathogenic nematodes as opposing forces in agriculture. International journal for parasitology\| PubMed ID: 26527129	2016
Effects of co-occurring Wolbachia and Spiroplasma endosymbionts on the Drosophila immune response against insect pathogenic and non-pathogenic bacteria. BMC microbiology\| PubMed ID: 26862076	2016
The insulin receptor substrate Chico regulates antibacterial immune function in Drosophila. Immunity & ageing : I & A\| PubMed ID: 27134635	2016
Role of Endosymbionts in Insect-Parasitic Nematode Interactions. Trends in parasitology\| PubMed ID: 29150386	2018
Conference report: The 14th congress of the International Society of Developmental and Comparative Immunology. Developmental and comparative immunology\| PubMed ID: 30851292	2019
Zika virus non-structural protein NS4A restricts eye growth in through regulation of JAK/STAT signaling. Disease models & mechanisms\| PubMed ID: 32152180	2020
A putative UDP-glycosyltransferase from Heterorhabditis bacteriophora suppresses antimicrobial peptide gene expression and factors related to ecdysone signaling. Scientific reports\| PubMed ID: 32704134	2020
A putative lysozyme and serine carboxypeptidase from Heterorhabditis bacteriophora show differential virulence capacities in Drosophila melanogaster. Developmental and comparative immunology\| PubMed ID: 32791175	2021
Secreted virulence factors from Heterorhabditis bacteriophora highlight its utility as a model parasite among Clade V nematodes. International journal for parasitology\| PubMed ID: 33421438	2021
Chemogenomic approach to identifying nematode chemoreceptor drug targets in the entomopathogenic nematode Heterorhabditis bacteriophora. Computational biology and chemistry\| PubMed ID: 33667976	2021

Eric Kenney

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