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Bacillus thuringiensis is a widely used bacterial entomopathogen producing insecticidal toxins, some of which are expressed in insect-resistant transgenic crops. Surprisingly, the killing mechanism of B. thuringiensis remains controversial. In particular, the importance of the septicemia induced by the host midgut microbiota is still debated as a result of the lack of experimental evidence obtained without drastic manipulation of the midgut and its content. Here this key issue is addressed by RNAi-mediated silencing of an immune gene in a lepidopteran host Spodoptera littoralis, leaving the midgut microbiota unaltered. The resulting cellular immunosuppression was characterized by a reduced nodulation response, which was associated with a significant enhancement of host larvae mortality triggered by B. thuringiensis and a Cry toxin. This was determined by an uncontrolled proliferation of midgut bacteria, after entering the body cavity through toxin-induced epithelial lesions. Consequently, the hemolymphatic microbiota dramatically changed upon treatment with Cry1Ca toxin, showing a remarkable predominance of Serratia and Clostridium species, which switched from asymptomatic gut symbionts to hemocoelic pathogens. These experimental results demonstrate the important contribution of host enteric flora in B. thuringiensis-killing activity and provide a sound foundation for developing new insect control strategies aimed at enhancing the impact of biocontrol agents by reducing the immunocompetence of the host.

The surface protein Pfs47 allows Plasmodium falciparum parasites to survive and be transmitted by making them “undetectable” to the mosquito immune system. P. falciparum parasites express Pfs47 haplotypes compatible with their sympatric vectors, while those with incompatible haplotypes are eliminated by the mosquito. We proposed that Pfs47 serves as a “key” that mediates immune evasion by interacting with a mosquito receptor “the lock,” which differs in evolutionarily divergent anopheline mosquitoes. Recombinant Pfs47 (rPfs47) was used to identify the mosquito Pfs47 receptor protein (P47Rec) using far-Western analysis. rPfs47 bound to a single 31-kDa band and the identity of this protein was determined by mass spectrometry. The mosquito P47Rec has two natterin-like domains and binds to Pfs47 with high affinity (17 to 32 nM). P47Rec is a highly conserved protein with submicrovillar localization in midgut cells. It has structural homology to a cytoskeletoninteracting protein and accumulates at the site of ookinete invasion. Silencing P47Rec expression reduced P. falciparum infection, indicating that the interaction of Pfs47 with the receptor is critical for parasite survival. The binding specificity of P47Rec from distant anophelines (Anopheles gambiae, Anopheles dirus, and Anopheles albimanus) with Pfs47-Africa (GB4) and Pfs47-South America (7G8) haplotypes was evaluated, and it is in agreement with the previously documented compatibility between P. falciparum parasites expressing different Pfs47 haplotypes and these three anopheline species. Our findings give further support to the role of Pfs47 in the adaptation of P. falciparum to different vectors.

We have developed genetically modified Ae. aegypti mosquitoes that activate the conserved antiviral JAK/STAT pathway in the fat body tissue, by overexpressing either the receptor Dome or the Janus kinase Hop by the blood feeding-induced vitellogenin (Vg) promoter. Transgene expression inhibits infection with several dengue virus (DENV) serotypes in the midgut as well as systemically and in the salivary glands. The impact of the transgenes Dome and Hop on mosquito longevity was minimal, but it resulted in a compromised fecundity when compared to wild-type mosquitoes. Overexpression of Dome and Hop resulted in profound transcriptome regulation in the fat body tissue as well as the midgut tissue, pinpointing several expression signatures that reflect mechanisms of DENV restriction. Our transcriptome studies and reverse genetic analyses suggested that enrichment of DENV restriction factor and depletion of DENV host factor transcripts likely accounts for the DENV inhibition, and they allowed us to identify novel factors that modulate infection. Interestingly, the fat body-specific activation of the JAK/STAT pathway did not result in any enhanced resistance to Zika virus (ZIKV) or chikungunya virus (CHIKV) infection, thereby indicating a possible specialization of the pathway's antiviral role.

Although West Nile virus (WNV) and other arthropod-borne viruses are a major public health problem, the mechanisms of antiviral immunity in mosquitoes are poorly understood. Dicer-2, responsible for the RNAi-mediated response through the C-terminal RNase-III domain, also contains an N-terminal DExD/H-box helicase domain similar to mammalian RIG-I/MDA5 which, in Drosophila , was found to be required for activation of an antiviral gene, Vago . Here we show that the Culex orthologue of Vago (Cx Vago) is up-regulated in response to WNV infection in a Dicer-2–dependent manner. Further, our data show that Cx Vago is a secreted peptide that restricts WNV infection by activation of the Jak-STAT pathway. Thus, Vago appears to function as an IFN-like antiviral cytokine in mosquitoes.

Proteolytic activation of phenoloxidase (PO) and the cytokine Spätzle during immune responses of insects is mediated by a network of hemolymph serine proteases (HPs) and noncatalytic serine protease homologs (SPHs) and inhibited by serpins. However, integration and conservation of the system and its control mechanisms are not fully understood. Here we present biochemical evidence that PO-catalyzed melanin formation, Spätzle-triggered Toll activation, and induced synthesis of antimicrobial peptides are stimulated via hemolymph (serine) protease 5 (HP5) in Manduca sexta. Previous studies have demonstrated a protease cascade pathway in which HP14 activates proHP21; HP21 activates proPAP2 and proPAP3, which then activate proPO in the presence of a complex of SPH1 and SPH2. We found that both HP21 and PAP3 activate proHP5 by cleavage at ESDR176*IIGG. HP5 then cleaves proHP6 at a unique site of LDLH112*ILGG. HP6, an ortholog of Drosophila Persephone, activates both proHP8 and proPAP1. HP8 activates proSpätzle-1, whereas PAP1 cleaves and activates proPO. HP5 is inhibited by Manduca sexta serpin-4, serpin-1A, and serpin-1J to regulate its activity. In summary, we have elucidated the physiological roles of HP5, a CLIPB with unique cleavage specificity (cutting after His) that coordinates immune responses in the caterpillar.

In the Afrotropical malaria vector Anopheles gambiae s.l. , the Imd pathway plays pleiotropic roles in immunity, including resistance to malaria parasites, that are mediated by its NF-κB transcription factor Rel2. Rel2 exists as a full-length form (Rel2-F) containing the Rel-homology domain (RHD) and the C-terminal inhibitory ankyrin (Ank) and death domains (DD), and a shorter alternatively spliced form (Rel2-S) proposed to encode a constitutively active protein containing only the RHD. Despite its important roles in immunity, there are still multiple uncertainties concerning the identity and function of key components of the pathway as well as its overall contribution to mosquito resistance to systemic bacterial infections. Here, we show that Rel2 is critical for limiting the burden of Gram-negative and Gram-positive bacterial proliferation in An. gambiae s.s. after systemic infections and this function is attributed to the endoproteolytic activation of Rel2-F in the fat body but not to Rel2-S. Interestingly, while Rel2-F activation in the fat body regulates Cecropin 1 and Defensin 1 expression, its activation in the midgut after oral infections is dispensable for their regulation. We provide direct evidence that PGRPLC1 is necessary and sufficient for Rel2-F activation in the fat body in response to infections with Gram-positive bacteria containing Lysine-type peptidoglycan, however sensing of Gram-negative bacteria and Gram-positive bacilli containing DAP-type peptidoglycan is more complex and may be mediated by various PGRPLC isoforms, indicating that the mosquito Imd pathway integrates distinct receptor modules to sense Gram-positive and Gram-negative bacterial infections.

Mosquito saliva is a very complex concoction of >100 proteins, many of which have unknown functions. The effects of mosquito saliva proteins injected into our skin during blood feeding have been studied mainly in mouse models of injection or biting, with many of these systems producing results that may not be relevant to human disease. Here, we describe the numerous effects that mosquito bites have on human immune cells in mice engrafted with human hematopoietic stem cells. We used flow cytometry and multiplex cytokine bead array assays, with detailed statistical analyses, to detect small but significant variations in immune cell functions after 4 mosquitoes fed on humanized mice footpads. After preliminary analyses, at different early times after biting, we focused on assessing innate immune and subsequent cellular responses at 6 hours, 24 hours and 7 days after mosquito bites. We detected both Th1 and Th2 human immune responses, and delayed effects on cytokine levels in the blood, and immune cell compositions in the skin and bone marrow, up to 7 days post-bites. These are the first measurements of this kind, with human immune responses in whole animals, bitten by living mosquitoes, versus previous studies using incomplete mouse models and salivary gland extracts or needle injected saliva. The results have major implications for the study of hematophagous insect saliva, its effects on the human immune system, with or without pathogen transmission, and the possibility of determining which of these proteins to target for vaccination, in attempts to block transmission of numerous tropical diseases.

Dorsal, a member of the nuclear factor-kappa B (NF-κB) family of transcription factors, is a critical downstream component of the Toll pathway that regulates the expression of antimicrobial peptides (AMPs) against pathogen invasion. In this study, the full-length ORF of Dorsal was identified from the RNA-seq database of the mealworm beetle Tenebrio molitor ( TmDorX2 ). The ORF of TmDorX2 was 1,482 bp in length, encoding a polypeptide of 493 amino acid residues. Tm DorX2 contains a conserved Rel homology domain (RHD) and an immunoglobulin-like, plexins, and transcription factors (IPT) domain. TmDorX2 mRNA was detected in all developmental stages, with the highest levels observed in 3-day-old adults. TmDorX2 transcripts were highly expressed in the adult Malpighian tubules (MT) and the larval fat body and MT tissues. After challenging the larvae with Staphylococcus aureus and Escherichia coli , the TmDorX2 mRNA levels were upregulated 6 and 9 h post infection in the whole body, fat body, and hemocytes. Upon Candida albicans challenge, the TmDorX2 mRNA expression were found highest at 9 h post-infection in the fat body. In addition, TmDorX2 -knockdown larvae exposed to E. coli , S. aureus , or C. albicans challenge showed a significantly increased mortality rate. Furthermore, the expression of 11 AMP genes was downregulated in the gut and fat body of ds TmDorX2 -injected larvae upon E. coli challenge. After C . albicans and S. aureus challenge of ds TmDorX2 -injected larvae, the expression of 11 and 10 AMPs was downregulated in the gut and fat body, respectively. Intriguingly, the expression of antifungal transcripts TmTenecin-3 and TmThaumatin-like protein-1 and -2 was greatly decreased in TmDorX2 -silenced larvae in response to C. albicans challenge, suggesting that TmDorX2 regulates antifungal AMPs in the gut in response to C. albicans infection. The AMP expression profiles in the fat body, hemocytes, gut, and MTs suggest that TmDorX2 might have an important role in promoting the survival of T. molitor larvae against all mentioned pathogens.

Insect bite hypersensitivity (IBH) is an IgE-mediated dermatitis in horses caused by bites of spp. The allergens are salivary gland proteins from these insects, and nine major allergens from have been identified and expressed in . However, proteins expressed in procaryotic systems have limitations in cellular assays, particularly in functional assays assessing the allergen-induced release of mediators , such as sulphidoleukotrienes (sLT) from basophils. The aims of the study were to produce functional allergens in insect cells, to assess their allergenicity using a sLT release assay, and to relate the sLT release with IgE sensitization to the respective allergens using ELISA. Eight major allergens (Cul o 1P, Cul o 2P, Cul o 3, Cul o 5, Cul o 7, Cul o 8, Cul o 9, and Cul o 11) were expressed in insect cells and purified. sLT release from peripheral blood leukocytes (PBL) following stimulation with the eight allergens was measured in 28 IBH-affected and 24 healthy control horses. Allergen-specific serum IgE levels was determined by ELISA. The eight major allergens were successfully expressed in insect cells and purified. All allergens induced a significantly higher sLT release from PBL of IBH-affected horses compared to healthy controls. There was a high correlation and substantial to excellent agreement between sLT release and serum IgE levels for six allergens, while for two, the agreement was moderate. Positivity rates in IBH horses were usually higher in IgE serology, but more false-positive results were obtained. The allergens performing best in both assays were Cul o 3, Cul o 8, and Cul o 9, with very high specificity and good sensitivity. Insect-cell-expressed recombinant allergens are functionally relevant and will open new opportunities for the study of hypersensitivity not only in horses but also potentially in human patients or other species. They will also greatly improve IBH diagnostics using cellular assays and IgE serology.

Serine protease cascades regulate important insect immune responses namely melanization and Toll pathway activation. An important component of these cascades are clip-domain serine protease homologs (cSPHs), which are non-catalytic, but essential for activating the enzyme prophenoloxidase (PPO) in the melanization response during septic infections. The activation of cSPHs requires their proteolytic cleavage, yet factors that control their activation and the complexity of their interactions within these cascades remain unclear. Here, we report the identification of CLIPA28 as a novel immune-related cSPH in the malaria vector Anopheles gambiae. Functional genetic analysis using RNA interference (RNAi) revealed that CLIPA28 is essential for the melanization of Plasmodium berghei parasites in refractory mosquitoes, and for mosquito resistance to fungal infections. We further show, using combined biochemical and genetic approaches, that CLIPA28 is member of a network of at least four cSPHs, whereby members are activated in a hierarchical manner following septic infections. Depletion of the complement-like protein TEP1 abolished the activation of this network after septic infections, whereas, depletion of the serine protease inhibitor 2 (SRPN2) triggered enhanced network activation, even in naïve mosquitoes, culminating in a dramatic reduction in cSPHs hemolymph levels, which paralleled that of PPO. Our data suggest that cSPHs are engaged in complex and multilayered interactions within serine protease cascades that regulate melanization, and identify TEP1 and SRPN2 as two master regulators of the cSPH network.