Cross-talk between immunity and behavior: insights from entomopathogenic fungi and their insect hosts.

Insects are one of the most successful animals in nature, and entomopathogenic fungi play a significant role in the natural epizootic control of insect populations in many ecosystems. The interaction between insects and entomopathogenic fungi has continuously coevolved over hundreds of millions of years. Many components of the insect innate immune responses against fungal infection are conserved across phyla. Additionally, behavioral responses, which include avoidance, grooming, and/or modulation of body temperature, have been recognized as important mechanisms for opposing fungal pathogens. In an effort to investigate possible cross-talk and mediating mechanisms between these fundamental biological processes, recent studies have integrated and/or explored immune and behavioral responses. Current information indicates that during discrete stages of fungal infection, several insect behavioral and immune responses are altered simultaneously, suggesting important connections between the two systems. This review synthesizes recent advances in our understanding of the physiological and molecular aspects influencing cross-talk between behavioral and innate immune antifungal reactions, including chemical perception and olfactory pathways.

A Simple Protocol for Isolating Hemolymph from Single Drosophila melanogaster Adult Flies.

Drosophila melanogaster is an excellent model for dissecting innate immune signaling and functions. Humoral and cellular immune mechanisms in the fly take place in the hemolymph, where host defense components are secreted and act in response to microbial invaders. Studying hemolymph factors is critical for understanding the regulation of the host's antimicrobial immune system. Therefore, methods for extracting the fly hemolymph efficiently and in sufficient quantities are essential for isolating and characterizing immune proteins and peptides. Here, we describe a novel and simple hemolymph isolation protocol for single D. melanogaster male and female adults. This procedure substantially improves the already used technique and allows fly immunologists to explore innate immune hemolymph activity in D. melanogaster individuals.

Functional role of the TGF-ß signaling in the Drosophila immune response.

TGF-ß signaling pathways are present in diverse animal species, which indicates their evolutionary importance in modulating several conserved biological processes and maintaining host homeostasis by adjusting the activity of innate immune mechanisms. Drosophila melanogaster utilizes two related but separable cascades of the canonical TGF-ß signaling pathway: The Bone Morphogenetic Protein and Activin branches. Recent studies have produced significant information on the immune role of TGF-ß signaling in the fruit fly model during response against certain bacterial pathogens. Results from further investigations have generated novel insights into the role of Drosophila TGF-ß signaling molecules as immune regulators opposing infection against nematode parasites and their mutualistic bacterial partners. This knowledge has revealed a previously unknown layer of the host innate immune system. Here we summarize these recent breakthroughs focusing on the participation of TGF-ß signaling factors in various Drosophila immune processes in relation to infection with potent bacteria and nematode parasites. The presented information provides important clues indicating directions for future research into the design of novel strategies for the effective control of infectious diseases caused by bacterial pathogens and parasitic nematodes.

The nematode parasite Steinernema hermaphroditum is pathogenic to Drosophila melanogaster larvae without activating their immune response.

Entomopathogenic nematodes are commonly used to control insect pest populations in the field. They also contribute substantially to understanding the molecular basis of nematode pathogenicity and insect anti-nematode immunity. Here, we tested the effect of the entomopathogenic nematode Steinernema hermaphroditum on the survival and immune signaling regulation of Drosophila melanogaster wild type larvae. Our results indicate that S. hermaphroditum infective juveniles are pathogenic toward D. melanogaster larvae, but they fail to activate certain immune pathway readout genes. These findings imply that S. hermaphroditum employs mechanisms that allow these parasitic nematodes to interfere with the D. melanogaster immune system.

The Drosophila melanogaster prophenoloxidase system participates in immunity against Zika virus infection.

Drosophila melanogaster relies on an evolutionarily conserved innate immune system to protect itself from a wide range of pathogens, making it a convenient genetic model to study various human pathogenic viruses and host antiviral immunity. Here we explore for the first time the contribution of the Drosophila phenoloxidase (PO) system to host survival and defenses against Zika virus (ZIKV) infection by analyzing the role of mutations in the three prophenoloxidase (PPO) genes in female and male flies. We show that only PPO1 and PPO2 genes contribute to host survival and appear to be upregulated following ZIKV infection in Drosophila. Also, we present data suggesting that a complex regulatory system exists between Drosophila PPOs, potentially allowing for a sex-dependent compensation of PPOs by one another or other immune responses such as the Toll, Imd, and JAK/STAT pathways. Furthermore, we show that PPO1 and PPO2 are essential for melanization in the hemolymph and the wound site in flies upon ZIKV infection. Our results reveal an important role played by the melanization pathway in response to ZIKV infection, hence highlighting the importance of this pathway in insect host defense against viral pathogens and potential vector control strategies to alleviate ZIKV outbreaks.

Drosophila melanogaster larvae are tolerant to oral infection with the bacterial pathogen Photorhabdus luminescens.

The fruit fly Drosophila melanogaster is an excellent model for dissecting the molecular and functional bases of bacterial pathogenicity and host antibacterial immune response. The Gram-negative bacterium Photorhabdus luminescens is an insect-specific pathogen that forms a mutualistic relationship with the entomopathogenic nematode Heterorhabditis bacteriophora . Here we find that oral infection of D. melanogaster larvae with P. luminescens moderately reduces their survival ability while the bacteria replicate efficiently in the infected insects. This information will contribute towards understanding host gut immunity against potent bacterial pathogens.

The plant toxin 4-methylsulfinylbutyl isothiocyanate decreases herbivore performance and modulates cellular and humoral immunity.

Insect herbivores frequently encounter plant defense molecules, but the physiological and ecological consequences for their immune systems are not fully understood. The majority of studies attempting to relate levels of plant defensive chemistry to herbivore immune responses have used natural population or species-level variation in plant defensive chemistry. Yet, this potentially confounds the effects of plant defense chemistry with other potential plant trait differences that may affect the expression of herbivore immunity. We used an artificial diet containing known quantities of a plant toxin (4-methylsulfinylbutyl isothiocyanate; 4MSOB-ITC or ITC, a breakdown product of the glucosinolate glucoraphanin upon herbivory) to explicitly explore the effects of a plant toxin on the cellular and humoral immune responses of the generalist herbivore Trichoplusia ni (Lepidoptera: Noctuidae) that frequently feeds on glucosinolate-containing plants. Caterpillars feeding on diets with high concentrations of ITC experienced reduced survivorship and growth rates. High concentrations of ITC suppressed the appearance of several types of hemocytes and melanization activity, which are critical defenses against parasitic Hymenoptera and microbial pathogens. In terms of T. ni humoral immunity, only the antimicrobial peptide (AMP) genes lebocin and gallerimycin were significantly upregulated in caterpillars fed on diets containing high levels of ITC relative to caterpillars that were provided with ITC-free diet. Surprisingly, challenging caterpillars with a non-pathogenic strain of Escherichia coli resulted in the upregulation of the AMP gene cecropin. Feeding on high concentrations of plant toxins hindered caterpillar development, decreased cellular immunity, but conferred mixed effects on humoral immunity. Our findings provide novel insights into the effects of herbivore diet composition on insect performance demonstrating the role of specific plant defense toxins that shape herbivore immunity and trophic interactions.

Drosophila melanogaster Imd signaling interacts with insulin signaling and alters feeding rate upon parasitic nematode infection.

Significant progress has been made in recent years on exploring immunometabolism, a field that integrates two processes essential for maintaining tissue and organismal homeostasis, immunity and metabolism. The nematode parasite Heterorhabditis gerrardi, its mutualistic bacteria Photorhabdus asymbiotica, and the fruit fly Drosophila melanogaster constitute a unique system to investigate the molecular basis of host immunometabolic response to nematode-bacterial complexes. In this study, we explored the contribution of the two major immune signaling pathways, Toll and Imd, to sugar metabolism in D. melanogaster larvae during infection with H. gerrardi nematodes. We infected Toll or Imd signaling loss-of-function mutant larvae with H. gerrardi nematodes and assessed larval survival ability, feeding rate, and sugar metabolism. We found no significant differences in the survival ability or levels of sugar metabolites in any of the mutant larvae when responding to H. gerrardi infection. However, we found that the Imd mutant larvae have higher feeding rate than controls during the early stages of infection. In addition, feeding rates are lower in Imd mutants relative to the control larvae as the infection progresses. We further showed that Dilp2 and Dilp3 gene expression increases in Imd mutants compared to controls early in the infection, but their expression levels decrease at later times. These findings indicate that Imd signaling activity regulates the feeding rate and Dilp2 and Dilp3 expression in D. melanogaster larvae infected with H. gerrardi. Results from this study facilitate our understanding of the link between host innate immunity and sugar metabolism in the context of infectious diseases caused by parasitic nematodes.

The role of Drosophila microbiota in gut homeostasis and immunity.

The gut epithelia of virtually all animals harbor complex microbial communities that play an important role in maintaining immune and cellular homeostasis. Gut microbiota have evolutionarily adapted to the host gut environment, serving as key regulators of intestinal stem cells to promote a healthy gut barrier and modulate epithelial self-renewal. Disruption of these populations has been associated with inflammatory disorders or cancerous lesions of the intestine. However, the molecular mechanisms controlling gut-microbe interactions are only partially understood due to the high diversity and biologically dynamic nature of these microorganisms. This article reviews the current knowledge on Drosophila gut microbiota and its role in signaling pathways that are crucial for the induction of distinct homeostatic and immune responses. Thanks to the genetic tractability of Drosophila and its cultivable and simple microbiota, this association model offers new efficient tools for investigating the crosstalk between a host and its microbiota while providing a framework for a better understanding of the ecological and evolutionary roles of the microbiome.

Temperature and sex shape Zika virus pathogenicity in the adult Brat cheesehead brain: A Drosophila model for virus-associated neurological diseases.

Severe neurological complications affecting brain growth and function have been well documented in newborn and adult patients infected by Zika virus (ZIKV), but the underlying mechanisms remain unknown. Here we use a Drosophila melanogaster mutant, cheesehead (chs), with a mutation in the brain tumor (brat) locus that exhibits both aberrant continued proliferation and progressive neurodegeneration in the adult brain. We report that temperature variability is a key driver of ZIKV pathogenesis, thereby altering host mortality and causing motor dysfunction in a sex-dependent manner. Furthermore, we show that ZIKV is largely localized to the brat chs brain and activates the RNAi and apoptotic immune responses. Our findings establish an in vivo model to study host innate immune responses and highlight the need of evaluating neurodegenerative deficits as a potential comorbidity in ZIKV-infected adults.

HETERORHABDITIS BACTERIOPHORA NEMATODES ARE SENSITIVE TO THE BACTERIAL PATHOGEN PHOTORHABDUS ASYMBIOTICA.

The entomopathogenic nematode (EPN) Heterorhabditis bacteriophora infects a wide range of insect hosts with the aid of its mutualistic bacteria Photorhabdus luminescens. While the mutualistic relationship between H. bacteriophora and P. luminescens and the infectivity of the nematode-bacteria complex have been characterized, how nematode fitness is affected by entomopathogenic bacteria existing in association with other EPN species remains poorly understood. In this study, the survival of H. bacteriophora infective juveniles containing or lacking P. luminescens was tested against the entomopathogenic bacteria Xenorhabdus nematophila and Photorhabdus asymbiotica as well as the non-pathogenic Escherichia coli. While X. nematophila and E. coli did not significantly affect the survival of H. bacteriophora, P. asymbiotica exerted a significant effect on nematode survival, particularly on those lacking P. luminescens. These results imply that P. asymbiotica encodes factors that are pathogenic to EPNs. Future efforts will focus on the identification of the bacterial molecular components that induce these effects. This study makes an important contribution to a growing body of research aimed at exploiting the full potential of nematode-bacterial complexes for eliminating noxious insect pests and treating infectious diseases caused by parasitic nematodes.

Functional role of thioester-containing proteins in the Drosophila anti-pathogen immune response.

Thioester-containing proteins (TEPs) are present in many animal species ranging from deuterostomes to protostomes, which emphasizes their evolutionary conservation and importance in animal physiology. Phylogenetically, insect TEPs share sequence similarity with mammalian α2-macroglobulin. Drosophila melanogaster is specifically considered a superb model for teasing apart innate immune processes. Here we review recent discoveries on the involvement of Drosophila TEPs in the immune response against bacterial pathogens, nematode parasites, and parasitoid wasps. This information generates novel insights into the role of TEPs as regulators of homeostasis in Drosophila and supports the complexity of immune recognition and specificity in insects and more generally in invertebrates. These developments together with recent advances in gene editing and multi-omics will enable the fly immunity community to appreciate the molecular and mechanistic contributions of TEPs to the modulation of the host defense against infectious disease and possibly to translate this information into tangible therapeutic benefits.

An odorant binding protein is involved in counteracting detection-avoidance and Toll-pathway innate immunity.

INTRODUCTION: Odorant-binding proteins (OBPs) are a class of small molecular weight soluble proteins that exist as expanded gene families in all insects, acting as ligand carriers mediating olfaction and other physiological processes. During fungal infection, a subset of insect OBPs were shown to be differentially expressed. OBJECTIVES: We tested whether the altered expression of insect OBPs during pathogenic infection plays a role in behavioral or immune interactions between insect hosts and their pathogens. METHODS: A wide range of techniques including RNAi-directed knockdown, heterologous protein expression, electrophysiological/behavioral analyses, transcriptomics, gut microbiome analyses, coupled with tandem mass spectrometry ion monitoring, were used to characterize the function of a locust OBP in host behavioral and immune responses. RESULTS: The entomopathogenic fungus Metarhizium anisopliae produces the volatile compound phenylethyl alcohol (PEA) that causes behavioral avoidance in locusts. This is mediated by the locust odorant binding protein 11 (LmOBP11). Expression of LmOBP11 is induced by M. anisopliae infection and PEA treatment. LmOBP11 participates in insect detection of the fungal-produced PEA and avoidance of PEA-contaminated food, but the upregulation of LmOBP11 upon M. anisopliae infection negatively affects the insect immune responses to ultimately benefit successful mycosis by the pathogen. RNAi knockdown of LmOBP11 increases the production of antimicrobial peptides and enhances locust resistance to M. anisopliae infection, while reducing host antennal electrophysiological responses to PEA and locust avoidance of PEA treated food. Also, transcriptomic and gut microbiome analyses reveal microbiome dysbiosis and changes in host genes involved in behavior and immunity. These results are consistent with the elevated expression of LmOBP11 leading to enhanced volatile detection and suppression of immune responses. CONCLUSION: These findings suggest a crosstalk between olfaction and immunity, indicating manipulation of host OBPs as a novel target exploited by fungal pathogens to alter immune activation and thus promote the successful infection of the host.

A life-and-death struggle: interaction of insects with entomopathogenic fungi across various infection stages.

Insects constitute approximately 75% of the world's recognized fauna, with the majority of species considered as pests. Entomopathogenic fungi (EPF) are parasitic microorganisms capable of efficiently infecting insects, rendering them potent biopesticides. In response to infections, insects have evolved diverse defense mechanisms, prompting EPF to develop a variety of strategies to overcome or circumvent host defenses. While the interaction mechanisms between EPF and insects is well established, recent findings underscore that their interplay is more intricate than previously thought, especially evident across different stages of EPF infection. This review primarily focuses on the interplay between EPF and the insect defense strategies, centered around three infection stages: (1) Early infection stage: involving the pre-contact detection and avoidance behavior of EPF in insects, along with the induction of behavioral responses upon contact with the host cuticle; (2) Penetration and intra-hemolymph growth stage: involving the initiation of intricate cellular and humoral immune functions in insects, while symbiotic microbes can further contribute to host resistance; (3) Host insect's death stage: involving the ultimate confrontation between pathogens and insects. Infected insects strive to separate themselves from the healthy population, while pathogens rely on the infected insects to spread to new hosts. Also, we discuss a novel pest management strategy underlying the cooperation between EPF infection and disturbing the insect immune system. By enhancing our understanding of the intricate interplay between EPF and the insect, this review provides novel perspectives for EPF-mediated pest management and developing effective fungal insecticides.

Excreted secreted products from the parasitic nematode Steinernema carpocapsae manipulate the Drosophila melanogaster immune response.

Steinernema carpocapsae is an entomopathogenic nematode (EPN) that rapidly infects and kills a wide range of insect hosts and has been linked to host immunosuppression during the initial stages of infection. The lethal nature of S. carpocapsae infections has previously been credited to its symbiotic bacteria; however, it has become evident that the nematodes are able to effectively kill their hosts independently through their excretion/secretion products (ESPs). Here we examined how the adult Drosophila melanogaster immune system is modulated in response to S. carpocapsae ESPs in an attempt to ascertain individual pathogenic contributions of the isolated compound. We found that the S. carpocapsae ESPs decrease the survival of D. melanogaster adult flies, they induce the expression of certain antimicrobial peptide-encoding genes, and they cause significant reduction in phenoloxidase enzyme activity and delay in the melanization response in males flies. We also report that S. carpocapsae ESPs affect hemocyte numbers in both male and female individuals. Our results indicate the manipulative role of EPN ESPs and reveal sex-specific differences in the host response against nematode infection factors. These findings are beneficial as they promote our understanding of the molecular basis of nematode pathogenicity and the parasite components that influence nematode-host interactions.

Zika Virus Induces Sex-Dependent Metabolic Changes in Drosophila melanogaster to Promote Viral Replication.

Zika is a member of the Flaviviridae virus family that poses some of the most significant global health risks, causing neurologic complications that range from sensory neuropathy and seizures to congenital Zika syndrome (microcephaly) in infants born to mothers infected during pregnancy. The recent outbreak of Zika virus (ZIKV) and its serious health threats calls for the characterization and understanding of Zika pathogenesis, as well as host antiviral immune functions. Although ZIKV has been associated with activating the RNA interference (RNAi) immune pathway and altering host metabolism, in-depth studies are still required to uncover the specifics of the complex host-virus interactions and provide additional insights into the molecular components that determine the outcome of this disease. Previous research establishes the fruit fly Drosophila melanogaster as a reliable model for studying viral pathogens, as it shares significant similarities with that of vertebrate animal systems. Here, we have developed an in vivo Drosophila model to investigate ZIKV-mediated perturbed metabolism in correlation to the RNAi central mediator Dicer-2. We report that ZIKV infection reprograms glucose and glycogen metabolism in Dicer-2 mutants to maintain efficient replication and successful propagation. Flies that exhibit these metabolic effects also show reduced food intake, which highlights the complicated neurological defects associated with ZIKV. We show that ZIKV infection significantly reduces insulin gene expression in Dicer-2 mutants, suggesting an insulin antiviral role against ZIKV and a direct connection to RNAi immunity. Moreover, we find that the insulin receptor substrate chico is crucial to the survival of ZIKV-infected flies. These observations are remarkably more severe in adult female flies compared to males, indicating possible sex differences in the rates of infection and susceptibility to the development of disease. Such findings not only demonstrate that metabolic alterations can be potentially exploited for developing immune therapeutic strategies but also that preventive measures for disease development may require sex-specific approaches. Therefore, further studies are urgently needed to explore the molecular factors that could be considered as targets to inhibit ZIKV manipulation of host cell metabolism in females and males.

Pathogen infection routes and host innate immunity: Lessons from insects.

Recent advances in insect-pathogen interactions have started to reveal the role of insect tissues and organs as natural infection routes for parasites and microbial pathogens. Here we summarize this information highlighting the micro- and macro-parasites that enter insects through distinct infection routes and link them to innate immune activity. We also examine whether the infection route determines the insect immune response and if the resulting immunological and physiological processes underpinning these different routes of infection are clearly distinct. Understanding how the infection route is associated with the robustness in insect host defense will help us identify conserved evolutionary and ecological patterns in order to design novel strategies for the management of destructive agricultural pests and disease vectors.