Wolbachia endosymbionts in Drosophila regulate the resistance to Zika virus infection in a sex dependent manner.

Drosophila melanogaster has been used extensively for dissecting the genetic and functional bases of host innate antiviral immunity and virus-induced pathology. Previous studies have shown that the presence of Wolbachia endosymbionts in D. melanogaster confers resistance to infection by certain viral pathogens. Zika virus is an important vector-borne pathogen that has recently expanded its range due to the wide geographical distribution of the mosquito vector. Here, we describe the effect of Wolbachia on the immune response of D. melanogaster adult flies following Zika virus infection. First, we show that the presence of Wolbachia endosymbionts promotes the longevity of uninfected D. melanogaster wild type adults and increases the survival response of flies following Zika virus injection. We find that the latter effect is more pronounced in females rather than in males. Then, we show that the presence of Wolbachia regulates Zika virus replication during Zika virus infection of female flies. In addition, we demonstrate that the antimicrobial peptide-encoding gene Drosocin and the sole Jun N-terminal kinase-specific MAPK phosphatase Puckered are upregulated in female adult flies, whereas the immune and stress response gene TotM is upregulated in male individuals. Finally, we find that the activity of RNA interference and Toll signaling remain unaffected in Zika virus-infected female and male adults containing Wolbachia compared to flies lacking the endosymbionts. Our results reveal that Wolbachia endosymbionts in D. melanogaster affect innate immune signaling activity in a sex-specific manner, which in turn influences host resistance to Zika virus infection. This information contributes to a better understanding of the complex interrelationship between insects, their endosymbiotic bacteria, and viral infection. Interpreting these processes will help us design more effective approaches for controlling insect vectors of infectious disease.

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.

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.

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.

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.

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.

Virus Infection of the Brain: Lessons from Drosophila for Illuminating Virus Disease and Nervous System Function.

Recent studies using genomic and functional approaches in the fruit fly Drosophila melanogaster have revealed the effects of viral infection on nervous system homeostasis. An established connection between viral infection and brain function is critical due to its significant contribution to several areas of biomedical research, particularly the molecular pathogenesis of neurotropic viruses, the neurobiology of viral disease, and understanding the genetic basis and pathophysiology of viral tropism.

Drosophila melanogaster 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 and raise ethical issues. Unconventional models are notoriously cheap, easy to handle and culture, and do not take much space. They are genetically amenable and possess complete genome sequences, and their use presents no ethical considerations. The fruit fly Drosophila melanogaster, for instance, has provided great insights into a variety of behavior, development, metabolism, and immunity research. More specifically, D. melanogaster adult flies and larvae possess several innate defense reactions that are shared with vertebrate animals. The mechanisms regulating immune responses have been mostly revealed through genetic and molecular studies in the D. melanogaster model. Here a novel larval injection technique is provided, which will further promote investigations of innate immune processes in D. melanogaster larvae and explore the pathogenesis of a wide range of microbial infections.

JNK signaling in Drosophila immunity and homeostasis.

As members of the mitogen-activated protein kinase (MAPK) family, the c-Jun N-terminal kinases (JNKs) regulate cell responses to a wide range of extrinsic and intrinsic insults, including irradiation, reactive oxygen species (ROS), DNA damage, heat, bacterial antigens, and inflammatory cytokines. Particularly, JNK signaling regulates and promotes many important physiological processes that influence metabolic and tissue homeostasis, cell death/survival, and cell damage repair and ultimately impacts the lifespan of an organism. This diverse functionality causes a variety of tissue-specific and context-specific cellular responses, mediated by various cross talks between JNK and other cellular signaling pathways. Thus, highlighting its significance as a determinant of stress responses, JNK loss-of-function mutations have been implicated in a multitude of pathologies, including neurodegenerative diseases, diabetes, and cancer. Because JNK functions are specified in a context-dependent manner and can greatly vary, the underlying causes for these different outcomes remain largely unresolved despite the gained knowledge of many regulatory roles of JNK signaling during the past two decades. In Drosophila melanogaster, JNK signaling is conserved and required for immune responses, as well as the development for morphogenetic processes (embryonic dorsal closure and thorax closure). Therefore, Drosophila innate immunity provides the ideal model to understand the complex mechanisms underlying JNK activation and regulation. In the following, we review studies in Drosophila that highlight several mechanisms by which JNK signaling influences immunity and homeostasis.

Drosophila immunity against natural and nonnatural viral pathogens.

The fruit fly Drosophila melanogaster is extensively used as a model species for molecular biology and genetics. It is also widely studied for its innate immune system to expand our understanding of immune host defenses against numerous pathogens. More precisely, studies using both natural and nonnatural Drosophila pathogens have provided a better perspective of viral infection strategies and immunity processes than any other invertebrate. This has made significant advances in identifying and characterizing the innate immune mechanisms by which hosts can combat viral pathogens. However, in-depth studies on antiviral immunity are still lacking due in part to the narrow research focus on the evolution and conservation of antiviral strategies to combat infections caused by both natural and nonnatural viruses. In this review, we will cover three major areas. First, we will describe the well-characterized antiviral immune mechanisms in Drosophila. Second, we will survey the specific pathways induced by natural viruses that have been studied in Drosophila. Finally, we will discuss the pathways activated by nonnatural viruses, drawing comparisons to natural viruses and giving an unprecedented insight into the virus community of Drosophila that is necessary to understand the evolutionary and immune context needed to develop Drosophila as a model for virus research.