culturing-and-genetically-manipulating-entomopathogenic-nematodes

Culturing and Genetically Manipulating Entomopathogenic Nematodes

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic ...

The George Washington University

The common fruit fly, Drosophila melanogaster is an outstanding model to analyze the regulation of conserved signaling pathways. In this study, we examined whether signaling components in the Bone Morphogenic Protein (BMP) branch of the TGF-β signaling pathway are involved in the response to wounding caused by either sterile injury or infection by parasitic nematodes in D. melanogaster adult flies. We found that following sterile injury, the BMP pathway Type I receptor sax and intracellular transcription factor Mad were substantially upregulated. Also, inactivation of Mad or dpp promoted fly survival and increased antimicrobial peptide gene transcript levels upon sterile injury or H. bacteriophora nematode infection, respectively, but not against the bacterial pathogen Photorhabdus luminescens. Our findings indicate the roles of certain BMP signaling components in the regulation of the fly immune response against sterile injury or nematode infection. In conclusion, this study highlights the ability of D. melanogaster to activate the BMP branch of TGF-β signaling in order to modulate the response to injury in the absence or presence of pathogenic infection.

Wounding-induced upregulation of the Bone Morphogenic Protein signaling pathway in Drosophila promotes survival against parasitic nematode infection.

The common fruit fly Drosophila melanogaster is an exceptional model for dissecting innate immunity. However, our knowledge on responses to parasitic nematode infections still lags behind. Recent studies have demonstrated that the well-conserved TGF-β signaling pathway participates in immune processes of the fly, including the anti-nematode response. To elucidate the molecular basis of TGF-β anti-nematode activity, we performed a transcript level analysis of different TGF-β signaling components following infection of D. melanogaster larvae with the nematode parasite Heterorhabditis gerrardi. We found no significant changes in the transcript level of most extracellular ligands in both bone morphogenic protein (BMP) and activin branches of the TGF-β signaling pathway between nematode-infected larvae and uninfected controls. However, extracellular ligand, Scw, and Type I receptor, Sax, in the BMP pathway as well as the Type I receptor, Babo, in the activin pathway were substantially up-regulated following H. gerrardi infection. Our results suggest that receptor up-regulation leads to transcriptional up-regulation of the intracellular component Mad in response to H. gerrardi following changes in gene expression of intracellular receptors of both TGF-β signaling branches. These findings identify the involvement of certain TGF-β signaling pathway components in the immune signal transduction of D. melanogaster larvae against parasitic nematodes .

Transcriptional up-regulation of the TGF-β intracellular signaling transducer Mad of Drosophila larvae in response to parasitic nematode infection.

Drosophila melanogaster relies on an evolutionarily conserved innate immune system to protect itself from potentially deadly pathogens. One of the earliest pathways activated after injury or infection is the melanization pathway, which is responsible for synthesizing and depositing melanin at the site of injury, or onto invading microbes. Three genes, PPO1-3, encoding prophenoloxidase (PPO), an inactive precursor of phenoloxidase (PO), are responsible for the production of melanin after their activation via immune challenge. One pathogen capable of infecting D. melanogaster are entomopathogenic nematodes. Steinernema carpocapsae nematodes exist in a mutualistic relationship with Xenorhabdus nematophila bacteria and are an important biological control agent for controlling insect pests. The nematode-bacteria complex (symbiotic nematodes) can be separated, creating "axenic" nematodes, devoid of their associated bacteria, which are still capable of infecting and killing D. melanogaster. In order to investigate how the D. melanogaster melanization pathway contributes to the anti-nematode immune response, symbiotic and axenic S. carpocapsae were used to study D. melanogaster survival, PPO gene expression, and activation of PPO to PO. Our research suggests that the expression of all three D. melanogaster PPO genes contributes to survival, however only PPO1 or PPO3 appear to be up-regulated during axenic or symbiotic nematode infection. Additionally, we present data suggesting that a complex regulatory system exists between PPOs, potentially allowing for the compensation of PPOs by one another. Further, we found that axenic nematode infection leads to higher levels of PO, suggesting that X. nematophila suppresses this activation. We also report for the first time the differentiation of lamellocytes, a specialized type of hemocytes in D. melanogaster, in response to symbiotic S. carpocapsae nematode infection. Our results suggest an important role played by the melanization pathway in response to nematode infection, and demonstrate how this response can be manipulated by S. carpocapsae nematodes and their mutualistic X. nematophila bacteria.

The prophenoloxidase system in Drosophila participates in the anti-nematode immune response.

The common fruit fly Drosophila melanogaster is a powerful model for studying signaling pathway regulation. Conserved signaling pathways underlying physiological processes signify evolutionary relationship between organisms and the nature of the mechanisms they control. This study explores the cross-talk between the well-characterized nuclear factor kappa B (NF-κB) innate immune signaling pathways and transforming growth factor beta (TGF-β) signaling pathway in response to parasitic nematode infection in Drosophila. To understand the link between signaling pathways, we followed on our previous studies by performing a transcript-level analysis of different TGF-β signaling components following infection of immune-compromised Drosophila adult flies with the nematode parasites Heterorhabditis gerrardi and H. bacteriophora. Our findings demonstrate the requirement of NF-κB transcription factors for activation of TGF-β signaling pathway in Drosophila in the context of parasitic nematode infection. We observe significant decrease in transcript level of glass bottom boat (gbb) and screw (scw), components of the bone morphogenic protein (BMP) branch, as well as Activinβ (actβ) which is a component of the Activin branch of the TGF-β signaling pathway. These results are observed only in H. gerrardi nematode-infected flies compared to uninfected control. Also, this significant decrease in transcript level is found only for extracellular ligands. Future research examining the mechanisms regulating the interaction of these signaling pathways could provide further insight into Drosophila anti-nematode immune function against infection with potent parasitic nematodes.

Transcript analysis reveals the involvement of NF-κB transcription factors for the activation of TGF-β signaling in nematode-infected Drosophila.

Lipid droplets (LDs) are lipid-carrying multifunctional organelles, which might also interact with pathogens and influence the host immune response. However, the exact nature of these interactions remains currently unexplored. Here we show that systemic infection of  adult flies with non-pathogenic , the extracellular bacterial pathogen  or the facultative intracellular pathogen  results in intestinal steatosis marked by lipid accumulation in the midgut. Accumulation of LDs in the midgut also correlates with increased whole-body lipid levels characterized by increased expression of genes regulating lipogenesis. The lipid-enriched midgut further displays reduced expression of the enteroendocrine-secreted hormone, Tachykinin. The observed lipid accumulation requires the Gram-negative cell wall pattern recognition molecule, PGRP-LC, but not PGRP-LE, for the humoral immune response. Altogether, our findings indicate that  LDs are inducible organelles, which can serve as markers for inflammation and, depending on the nature of the challenge, they can dictate the outcome of the infection.

Intestinal lipid droplets as novel mediators of host-pathogen interaction in .

Endosymbiotic bacteria that obligately associate with entomopathogenic nematodes as a complex are a unique model system to study competition. These nematodes seek an insect host and provide entry for their endosymbionts. Through their natural products, the endosymbionts nurture their nematodes by eliminating secondary infection, providing nutrients through bioconversion of the insect cadaver, and facilitating reproduction. On one hand, they cooperatively colonize the insect host and neutralize other opportunistic biotic threats. On the other hand, inside the insect cadaver as a fighting pit, they fiercely compete for the fittest partnership that will grant them the reproductive dominance. Here, we review the protective and nurturing nature of endosymbiotic bacteria for their nematodes and how their selective preference shapes the superior nematode-endosymbiont pairs as we know today.

Nematode endosymbiont competition: Fortune favors the fittest.

Entomopathogenic nematodes are parasitic organisms with an exceptional capacity to infect rapidly and efficiently a wide range of insect species. Their distinct pathogenic properties have established entomopathogenic nematodes as supreme biocontrol agents of insects as well as excellent models to simulate and dissect the molecular and physiological bases of conserved strategies employed by parasitic nematodes that cause infectious diseases in humans. The extreme infectivity of entomopathogenic nematodes is due in part to the presence of certain species of Gram-negative bacteria that live in mutualistic symbiosis during the infective juvenile stage, which forms the central part of the nematode life cycle. Both nematodes and their mutualistic bacteria are capable of interfering and undermining several aspects of the insect host innate immune system during the infection process. The mutualistic bacteria are also able to modulate other biological functions in their nematode host including growth, development, and reproduction. In this review, we will focus our attention on the mutualistic relationship between entomopathogenic nematodes and their associated bacteria to discuss the nature and distinct characteristics of the regulatory mechanisms, and their molecular as well as physiological components that control this specific biological partnership.

Molecular Regulators of Entomopathogenic Nematode-Bacterial Symbiosis.

Insects in nature interact with a wide variety of microbial enemies including nematodes. These include entomopathogenic nematodes that contain mutualistic bacteria and together are able to infect a broad range of insects in order to complete their life cycle and multiply, filarial nematodes which are vectored by mosquitoes, and other parasitic nematodes. Entomopathogenic nematodes are commonly used in biological control practices and they form excellent research tools for understanding the genetic and functional bases of nematode pathogenicity and insect anti-nematode immunity. In addition, clarifying the mechanism of transmission of filarial nematodes by mosquitoes is critical for devising strategies to reduce disease transmission in humans. In all cases and in order to achieve these goals, it is vital to determine the number and type of insect host genes which are differentially regulated during infection and encode factors with anti-nematode properties. In this respect, the use of transcriptomic approaches has proven a key step for the identification of insect molecules with anti-nematode activity. Here, we review the progress in the field of transcriptomics that deals with the insect response to nematode infection. This information is important because it will expose conserved pathways of anti-nematode immunity in humans.

Transcriptomic Insights into the Insect Immune Response to Nematode Infection.

Insects possess an immune system that protects them from attacks by various pathogenic microorganisms that would otherwise threaten their survival. Immune mechanisms may deal directly with the pathogens by eliminating them from the host organism or disarm them by suppressing the synthesis of toxins and virulence factors that promote the invasion and destructive action of the intruder within the host. Insects have been established as outstanding models for studying immune system regulation because innate immunity can be explored as an integrated system at the level of the whole organism. Innate immunity in insects consists of basal immunity that controls the constitutive synthesis of effector molecules such as antimicrobial peptides, and inducible immunity that is activated after detection of a microbe or its product(s). Activation and coordination of innate immune defenses in insects involve evolutionary conserved immune factors. Previous research in insects has led to the identification and characterization of distinct immune signalling pathways that modulate the response to microbial infections. This work has not only advanced the field of insect immunology, but it has also rekindled interest in the innate immune system of mammals. Here we review the current knowledge on key molecular components of insect immunity and discuss the opportunities they present for confronting infectious diseases in humans.

Regulators and signalling in insect antimicrobial innate immunity: Functional molecules and cellular pathways.

The host defence of insects includes a combination of cellular and humoral responses. The cellular arm of the insect innate immune system includes mechanisms that are directly mediated by haemocytes (e.g., phagocytosis, nodulation and encapsulation). In addition, melanization accompanying coagulation, clot formation and wound healing, nodulation and encapsulation processes leads to the formation of cytotoxic redox-cycling melanin precursors and reactive oxygen and nitrogen species. However, demarcation between cellular and humoral immune reactions as two distinct categories is not straightforward. This is because many humoral factors affect haemocyte functions and haemocytes themselves are an important source of many humoral molecules. There is also a considerable overlap between cellular and humoral immune functions that span from recognition of foreign intruders to clot formation. Here, we review these immune reactions starting with the cellular mechanisms that limit haemolymph loss and participate in wound healing and clot formation and advancing to cellular functions that are critical in restricting pathogen movement and replication. This information is important because it highlights that insect cellular immunity is controlled by a multilayered system, different components of which are activated by different pathogens or during the different stages of the infection.

Haemocyte-mediated immunity in insects: Cells, processes and associated components in the fight against pathogens and parasites.

The innate immune response of insects provides a robust line of defense against pathogenic microbes and eukaryotic parasites. It consists of two types of overlapping immune responses, named humoral and cellular, which share protective molecules and regulatory mechanisms that closely coordinate to prevent the spread and replication of pathogens within the compromised insect hemocoel. The major feature of the humoral part of the insect immune system involves the production and secretion of antimicrobial peptides from the fat body, which is considered analogous to adipose tissue and liver in vertebrates. Previous research has identified and characterized the nature of antimicrobial peptides that are directed against various targets during the different stages of infection. Here we review this information focusing mostly on the diversity and mode of action of these host defense components, and their critical contribution to maintaining host homeostasis. Extending this knowledge is paramount for understanding the evolution of innate immune function and the physiological balance required to provide sufficient protection to the host against external enemies while avoiding overactivation signaling events that would severely undermine physiological stability.

Diversity of insect antimicrobial peptides and proteins - A functional perspective: A review.

As the genetic basis of natural and domesticated variation has been described in recent years, a number of hotspot genes have been repeatedly identified as the targets of selection, Heliconius butterflies display a spectacular diversity of pattern variants in the wild and the genetic basis of these patterns has been well-described. Here we sought to identify the mechanism behind an unusual pattern variant that is instead found in captivity, the ivory mutant, in which all scales on both the wings and body become white or yellow. Using a combination of autozygosity mapping and coverage analysis from 37 captive individuals, we identify a 78kb deletion at the cortex wing patterning locus, a gene which has been associated with wing pattern evolution in H. melpomene and 10 divergent lepidopteran species. This deletion is undetected among 458 wild Heliconius genomes samples, and its dosage explains both homozygous and heterozygous ivory phenotypes found in captivity. The deletion spans a large 5' region of the cortex gene that includes a facultative 5'UTR exon detected in larval wing disk transcriptomes. CRISPR mutagenesis of this exon replicates the wing phenotypes from coding knock-outs of cortex, consistent with a functional role of ivory-deleted elements in establishing scale color fate. Population demographics reveal that the stock giving rise to the ivory mutant has a mixed origin from across the wild range of H. melpomene, and supports a scenario where the ivory mutation occurred after the introduction of cortex haplotypes from Ecuador. Homozygotes for the ivory deletion are inviable while heterozygotes are the targets of artificial selection, joining 40 other examples of allelic variants that provide heterozygous advantage in animal populations under artificial selection by fanciers and breeders. Finally, our results highlight the promise of autozygosity and association mapping for identifying the genetic basis of aberrant mutations in captive insect populations.

A large deletion at the cortex locus eliminates butterfly wing patterning.

The pantry moth  is a worldwide pest of stored food products and a promising laboratory model system for lepidopteran functional genomics. Here we describe efficient methods for precise genome editing in this insect. A spontaneous recessive white-eyed phenotype maps to a frameshift deletion (.) in the  gene. CRISPR NHEJ mutagenesis of  replicates this phenotype with high rates of somatic biallelic knockout. G individuals with mutant clones on both eyes produced 100% mutant progeny, making  an ideal marker for co-conversion when targeting other genes. CRISPR HDR experiments corrected . and reverted white eyes to a pigmented state in 37% of G mosaic adults. These repaired alleles showed practical rates of germline transmission in backcrosses, demonstrating the potential of the technique for precise genome editing.  offers a promising avenue for research in this taxon because of its lab-ready features, egg injectability, and editability.

Article	Year
Wounding-induced upregulation of the Bone Morphogenic Protein signaling pathway in Drosophila promotes survival against parasitic nematode infection. Gene\| PubMed ID: 29920363	2018
Transcriptional up-regulation of the TGF-β intracellular signaling transducer Mad of Drosophila larvae in response to parasitic nematode infection. Innate immunity\| PubMed ID: 30049242	2018
The prophenoloxidase system in Drosophila participates in the anti-nematode immune response. Molecular immunology\| PubMed ID: 30909122	2019
Transcript analysis reveals the involvement of NF-κB transcription factors for the activation of TGF-β signaling in nematode-infected Drosophila. Immunogenetics\| PubMed ID: 31147740	2019
Intestinal lipid droplets as novel mediators of host-pathogen interaction in . Biology open\| PubMed ID: 31278163	2019
Nematode endosymbiont competition: Fortune favors the fittest. Molecular and biochemical parasitology\| PubMed ID: 32621939	2020
Molecular Regulators of Entomopathogenic Nematode-Bacterial Symbiosis. Results and problems in cell differentiation\| PubMed ID: 33263883	2020
Transcriptomic Insights into the Insect Immune Response to Nematode Infection. Genes\| PubMed ID: 33573306	2021
Regulators and signalling in insect antimicrobial innate immunity: Functional molecules and cellular pathways. Cellular signalling\| PubMed ID: 33836260	2021
Haemocyte-mediated immunity in insects: Cells, processes and associated components in the fight against pathogens and parasites. Immunology\| PubMed ID: 34233014	2021
Diversity of insect antimicrobial peptides and proteins - A functional perspective: A review. International journal of biological macromolecules\| PubMed ID: 34543628	2021
A large deletion at the cortex locus eliminates butterfly wing patterning. G3 (Bethesda, Md.)\| PubMed ID: 35099556	2022
Mapping and CRISPR homology-directed repair of a recessive white eye mutation in moths. iScience\| PubMed ID: 35243245	2022

Christa Heryanto

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