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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.

Global loss of honey bee colonies is threatening the human food supply. Diverse pathogens reduce honey bee hardiness needed to sustain colonies, especially in winter. We isolated a free-living Gram negative bacillus from hemolymph of worker honey bees (Apis mellifera) found separated from winter clusters. In some hives, greater than 90% of the dying bees detached from the winter cluster were found to contain this bacterium in their hemolymph. Throughout the year, the same organism was rarely found in bees engaged in normal hive activities, but was detected in about half of Varroa destructor mites obtained from colonies that housed the septic bees. Flow cytometry of hemolymph from septic bees showed a significant reduction of plasmatocytes and other types of hemocytes. Interpretation of the16S rRNA sequence of the bacterium indicated that it belongs to the Serratia genus of Gram-negative Gammaproteobacteria, which has not previously been implicated as a pathogen of adult honey bees. Complete genome sequence analysis of the bacterium supported its classification as a novel strain of Serratia marcescens, which was designated as S. marcescens strain sicaria (Ss1). When compared with other strains of S. marcescens, Ss1 demonstrated several phenotypic and genetic differences, including 65 genes not previously found in other Serratia genomes. Some of the unique genes we identified in Ss1 were related to those from bacterial insect pathogens and commensals. Recovery of this organism extends a complex pathosphere of agents which may contribute to failure of honey bee colonies.

Iron sequestration is a recognized innate immune mechanism against invading pathogens mediated by iron-binding proteins called transferrins. Despite many studies on antimicrobial activity of transferrins in vitro, their specific in vivo functions are poorly understood. Here we use Drosophila melanogaster as an in vivo model to investigate the role of transferrins in host defense. We find that systemic infections with a variety of pathogens trigger a hypoferremic response in flies, namely, iron withdrawal from the hemolymph and accumulation in the fat body. Notably, this hypoferremia to infection requires Drosophila nuclear factor κB (NF-κB) immune pathways, Toll and Imd, revealing that these pathways also mediate nutritional immunity in flies. Next, we show that the iron transporter Tsf1 is induced by infections downstream of the Toll and Imd pathways and is necessary for iron relocation from the hemolymph to the fat body. Consistent with elevated iron levels in the hemolymph, Tsf1 mutants exhibited increased susceptibility to Pseudomonas bacteria and Mucorales fungi, which could be rescued by chemical chelation of iron. Furthermore, using siderophore-deficient Pseudomonas aeruginosa, we discover that the siderophore pyoverdine is necessary for pathogenesis in wild-type flies, but it becomes dispensable in Tsf1 mutants due to excessive iron present in the hemolymph of these flies. As such, our study reveals that, similar to mammals, Drosophila uses iron limitation as an immune defense mechanism mediated by conserved iron-transporting proteins transferrins. Our in vivo work, together with accumulating in vitro studies, supports the immune role of insect transferrins against infections via an iron withholding strategy.

Crustaceans and mollusks have major economic importance and are also key players in aquatic biogeochemical cycles. However, disease outbreaks, temperature fluctuations, pollutants, and other stressors have severely threatened their global production. Invertebrates generally rely on their innate immune system as the primary defense mechanism, operating at cellular and humoral levels to protect against pathogens. The hemolymph plays a vital role in immune responses, containing microbial communities that interact with the host’s immune processes. Significant advances in molecular methods such as metagenomics, metatranscriptomics, metaproteomics, and metabolomics have revealed the presence of a resident hemolymph microbiome and delineated its potentially vital role in immune homeostasis and overall host health. Accordingly, understanding the composition and role of the hemolymph microbiota, alongside innate immune responses, has become a key focus in recent research aimed at unraveling disease resistance mechanisms and supporting sustainable aquaculture practices. Here, we summarize the latest advancements in understanding the host and environmental factors that shape hemolymph microbiota diversity in various crustacean and mollusk species. We also consider the innate immune responses of the hosts, as these modulate interactions between hosts, microbes, and environments. Interactions within the hemolymph microbiome significantly affect host health, providing critical insights for advancing sustainable aquaculture.

The skin microbiome maintains healthy human skin, and disruption of the microbiome balance leads to inflammatory skin diseases such as folliculitis and atopic dermatitis. Staphylococcus aureus and Cutibacterium acnes are pathogenic bacteria that simultaneously inhabit the skin and cause inflammatory diseases of the skin through the activation of innate immune responses. Silkworms are useful invertebrate animal models for evaluating innate immune responses. In silkworms, phenoloxidase generates melanin as an indicator of innate immune activation upon the recognition of bacterial or fungal components. We hypothesized that S . aureus and C . acnes interact to increase the innate immunity-activating properties of S . aureus . In the present study, we showed that acidification is involved in the activation of silkworm hemolymph melanization by S . aureus . Autoclaved-killed S . aureus ( S . aureus [AC]) alone does not greatly activate silkworm hemolymph melanization. On the other hand, applying S . aureus [AC] treated with C . acnes culture supernatant increased the silkworm hemolymph melanization. Adding C . acnes culture supernatant to the medium decreased the pH. S . aureus [AC] treated with propionic acid, acetic acid, or lactic acid induced higher silkworm hemolymph melanization activity than untreated S . aureus [AC]. S . aureus [AC] treated with hydrochloric acid also induced silkworm hemolymph melanization. The silkworm hemolymph melanization activity of S . aureus [AC] treated with hydrochloric acid was inhibited by protease treatment of S . aureus [AC]. These results suggest that acid treatment of S . aureus induces innate immune activation in silkworms and that S . aureus proteins are involved in the induction of innate immunity in silkworms.

Metarhizium anisopliae infects mosquitoes through the cuticle and proliferates in the hemolymph. To allow M. anisopliae to combat malaria in mosquitoes with advanced malaria infections, we produced recombinant strains expressing molecules that target sporozoites as they travel through the hemolymph to the salivary glands. Eleven days after a Plasmodium-infected blood meal, mosquitoes were treated with M. anisopliae expressing salivary gland and midgut peptide 1 (SM1), which blocks attachment of sporozoites to salivary glands; a single-chain antibody that agglutinates sporozoites; or scorpine, which is an antimicrobial toxin. These reduced sporozoite counts by 71%, 85%, and 90%, respectively. M. anisopliae expressing scorpine and an [SM1]₈:scorpine fusion protein reduced sporozoite counts by 98%, suggesting that Metarhizium-mediated inhibition of Plasmodium development could be a powerful weapon for combating malaria.

Background The entomopathogenic fungus (EPF) Ophiocordyceps sinensis has a long-term coexistence with its host insect, Thitarodes xiaojinensis , making it a unique model for host–pathogen interactions. Hemolymph, a critical component in insects, plays an essential role in maintaining both nutritional and immune homeostasis. However, the mechanism of the host’s immune response remains unclear when O. sinensis proliferates in the hemolymph. Results O. sinensis caused damage to the insect’s intestinal barrier, facilitating the translocation of gut bacteria into the hemocoel. Subsequently, the presence of O. sinensis and opportunistic pathogenic bacteria from the gut disrupted the homeostasis of the hemolymph microbiota, resulting in an increase in bacterial diversity. This disruption triggered a series of physiological responses in the host, including elevated levels of endocrine hormones specifically 20-hydroxyecdysone (20E) and juvenile hormone 3 (JH3). Additionally, there was an enhancement of antioxidant capacity, as indicated by increased total antioxidant capacity and glutathione S-transferase activity, along with the production of antimicrobial peptides (AMPs) as part of the immune defense. Notably, the rise in 20E levels during O. sinensis infection might have significantly contributed to the increased production of AMPs. Conclusions O. sinensis infection significantly alters T. xiaojinensis physiology. Humoral immunity in infected hosts is primarily in response to hemolymph microbial homeostasis due to intestinal translocation. Among them, 20E upregulates AMP-related genes, suggesting a key immune strategy for managing microbial imbalances while tolerating fungal pathogens.

Biological responses of zebra mussel Dreissena polymorpha are investigated to assess the impact of contaminants on aquatic organisms and ecosystems. In addition to concentrate chemical contaminants in their tissues, zebra mussels accumulate several microorganisms such as viruses, protozoa and bacteria. In order to understand the molecular mechanisms involved in the defence against microorganisms this study aims at identifying immune proteins from D. polymorpha hemolymph involved in defence against protozoa and viruses. For this purpose, hemolymph were exposed ex vivo to Cryptosporidium parvum and RNA poly I:C. Differential proteomics on both hemocytes and plasma revealed immune proteins modulated under exposures. Different patterns of response were observed after C. parvum and RNA poly I:C exposures. The number of modulated proteins per hemolymphatic compartments suggest that C. parvum is managed in cells while RNA poly I:C is managed in plasma after 4 h exposure. BLAST annotation and GO terms enrichment analysis revealed further characteristics of immune mechanisms. Results showed that many proteins involved in the recognition and destruction of microorganisms were modulated in both exposure conditions, while proteins related to phagocytosis and apoptosis were exclusively modulated by C. parvum. This differential proteomic analysis highlights in zebra mussels modulated proteins involved in the response to microorganisms, which reflect a broad range of immune mechanisms such as recognition, internalization and destruction of microorganisms. This study paves the way for the identification of new markers of immune processes that can be used to assess the impact of both chemical and biological contaminations on the health status of aquatic organisms.

Introduction: Vibrio species are widespread in coastal environments, where they are being increasingly associated with mortality episodes of farmed bivalves. This is the case for Vibrio ostreicida strain r172 isolated from a mortality event of adult mussels Mytilus galloprovincialis in Spain in 2022. Methods and Results: In this study, the infection dynamics and immune responses of adult M. galloprovincialis challenged with V. ostreicida r172 were investigated using different in vivo experimental approaches. First, a virulence assay by injection (1 x 108 CFU/100 µL) was performed at different temperatures (12, 18 and 24 °C). The results showed that mussel mortality (about 50 % within 8 days) was independent of increasing temperatures. Subsequently, an injection/cohabitation experiment was carried out placing together in the same tank V. ostreicida-injected (Donors, 108 CFU/mL) with un-injected mussels (Recipients). The time course of infection was then followed by evaluating positivity to V. ostreicida by PCR, responses of haemolymph components (haemocyte lysosomal membrane stability and serum lysozyme activity) and tissue histopathology (gills and digestive gland). The results showed a partial horizontal transfer of V. ostreicida from infected to uninfected mussels, with transient effects on haemolymph responses and histopathological lesions in both groups. Finally, in order to mimic more realistic environmental conditions, a bath infection experiment was carried out, exposing mussels to V. ostreicida in seawater (105 CFU/mL). This condition resulted in lower stress in haemocytes; moreover, no lysozyme release or histopathological alterations were observed. Discussion: Overall, the results show that M. galloprovincialis is able to cope with challenge with V. ostreicida, indicating that this Vibrio species is moderately pathogenic to adult mussels under the established experimental conditions.

Neonicotinoids, including imidacloprid (IM), cause harm to Apis mellifera in a number of ways, among others by impairing body maintenance, resistance and immunity. Energy resources are important to preventing this, as we hypothesized, not only in the hemolymph but particularly in the fat body, the insufficiently investigated, as yet, equivalent of the mammalian liver and pancreas. Both suppression and hormesis (diaphasic stressor response) of energy supply was reported in the energy-dependent traits of bees exposed to sublethal doses of imidacloprid. Therefore, our goal was to answer which of these two phenomena occurs in the hemolymph/fat body and at what doses of imidacloprid. concentrations/activities of respiratory/citric cycle compounds (acetyl-CoA, IDH-2, AKG, succinate, fumarate, NADH2, COX, UQRC, and ATP) were compared in the hemolymph and fat bodies of nurse workerbees sampled from honeybee colonies fed with diets containing 200 ppb (IM-200), 5 ppb (IM-5; sublethal), and 0 ppb of IM in a field experiment. the assayed compounds had higher values in the fat body than in the hemolymph, whereas their variability was higher in the hemolymph. The pattern of response to IM was the same in both tissues, but markedly differed between IM-200 and IM-5. The concentrations of the strongly correlated NADH2, ATP and acetyl-CoA decreased both in IM-200 and IM-5, whereas the levels of the other compounds decreased in IM-200 but increased in IM-5. decreased ATP and acetyl-CoA levels both in IM-5 and IM-200 show that the pesticide impairs the hemolymph and fat-body energy metabolism in spite of hormesis in six of the nine respiratory and citric cycle compounds even in low, residual doses. This finding better explains how residual doses of neonicotinoids may disturb the fat body functions, and therefore suppress the apian resistance, which expands our knowledge about honeybee colony losses.