Innate immunity in the edible ascidian Halocynthia roretzi developing soft tunic syndrome: Hemolymph can eliminate the causative flagellates and discriminate allogeneic hemocytes.

The infection of the kinetoplastid flagellate Azumiobodo hoyamushi causes soft tunic syndrome that often results in mass mortality in the aquaculture of the edible ascidian Halocynthia roretzi. In the diseased ascidian individuals, the flagellates are exclusively found in the tunic matrix that entirely cover the epidermis, and never invade into internal tissues, such as a mantle. The present study for the first time demonstrated that the ascidian blood plasma and hemolymph have an activity to agglutinate and disintegrate the flagellates, suggesting the innate immunity protects the internal tissue from the invasion of A. hoyamushi. This activity is indifferent between the healthy and the diseased individuals. Allo-specific recognition and cytotoxic reaction among ascidian hemocytes, so-called contact reaction, occur among the individuals of healthy-healthy, healthy-diseased, and diseased-diseased combination, and therefore, the hemocytes from diseased individuals still retain the allo-reactivity. Moreover, the allo-reactive combinations are not changed under the presence of the flagellates, indicating the flagellates neither suppress nor induce the effector system of the contact reaction. These results suggest that the infection of A. hoyamushi does not impair the innate immunity in the ascidian hemolymph.

Vector microbiota manipulation by host antibodies: the forgotten strategy to develop transmission-blocking vaccines.

Human and animal pathogens that are transmitted by arthropods are a global concern, particularly those vectored by ticks (e.g. Borrelia burgdorferi and tick-borne encephalitis virus) and mosquitoes (e.g. malaria and dengue virus). Breaking the circulation of pathogens in permanent foci by controlling vectors using acaricide-based approaches is threatened by the selection of acaricide resistance in vector populations, poor management practices and relaxing of control measures. Alternative strategies that can reduce vector populations and/or vector-mediated transmission are encouraged worldwide. In recent years, it has become clear that arthropod-associated microbiota are involved in many aspects of host physiology and vector competence, prompting research into vector microbiota manipulation. Here, we review how increased knowledge of microbial ecology and vector-host interactions is driving the emergence of new concepts and tools for vector and pathogen control. We focus on the immune functions of host antibodies taken in the blood meal as they can target pathogens and microbiota bacteria within hematophagous arthropods. Anti-microbiota vaccines are presented as a tool to manipulate the vector microbiota and interfere with the development of pathogens within their vectors. Since the importance of some bacterial taxa for colonization of vector-borne pathogens is well known, the disruption of the vector microbiota by host antibodies opens the possibility to develop novel transmission-blocking vaccines.

Segmental pairs of dermal secretory cells release proteins into the hemolymph at the larval-pupal molt.

At each molt of Manduca, the large dermal secretory cells expel the protein contents of their vacuoles into the hemocoel. The constellation of proteins expelled at the last larval-pupal molt, however, differs qualitatively from those proteins released at earlier larval-larval molts. Secretory cells at the two stages not only have different lectin staining properties but also have different proteins that separate on two-dimensional gels. Numerous physiological changes accompany the termination of the last larval instar, including increased chitin synthesis, diminished oxygen delivery, and reduced humoral immunity. Secretion of trehalase that is essential for chitin synthesis and the release of hypoxia up-regulated protein to ameliorate oxygen deprivation help ensure normal transition from larva to pupa. Proteins released by dermal secretory cells at this last molt could supplement the diminished immune defenses mediated by fat body and hemocytes at the end of larval life. Additional immune defenses provided by dermal secretory cells could help ensure a safe transition during a period of increased vulnerability for the newly molted pupa with its soft, thin cuticle and reduced mobility.

Impact of low temperatures on the immune system of honeybees.

Changes in temperature resulting from climate change can impact the distribution and survival of species, including bees, where temperature may also affect their immune system. Evaluation of immune system activity is often performed by the total count of circulating hemocytes in the hemolymph. However, there are few studies on bees examining the relationship between the amount of circulating hemocytes and temperature. This study evaluated changes of circulating hemocytes in Apis mellifera hemolymph at different temperatures and development stages. Total hemocytes of bees were determined at - 8, 16, 24, and 32 °C - and at different development stages - in vivo larvae, in vitro larvae, newly emerged, and forager bees. A. mellifera larvae had a greater number of circulating hemocytes compared to the other development stages (newly emerged and foragers). Additionally, temperature was an important factor explaining variation of circulating hemocytes in the hemolymph, according to principal component analyses (PCA), as the number of circulating hemocytes was greater at higher temperatures. Therefore, extreme events arising from climate change, such as variation in temperature, can directly impact the immune system of bees, both individually and at the colony level, threatening the distribution and survival of several species.

The Elevated Expressions of Anti-lipopolysaccharide Factors After Priming Stimulation Confer Lastingly Humoral Protection in Crab Eriocheir sinensis.

Evidence of immune memory in invertebrates (immune priming) has accumulated in various organisms, and both cellular and humoral immune reactions are speculated to be involved in immune priming. However, there is a lack of understanding of the molecular mechanisms involved. In the present study, the protective effect of primed haemolymph was further validated by the increased survival rate of naïve crabs receiving a transfusion of primed haemolymph. By proteomic analysis, there were 474 proteins identified from the primed haemolymph, and most of them were functionally annotated in transport and metabolism classes. A total of 70 proteins were found to be differentially expressed in haemolymph at 12 hours and 7 days after priming stimulation with Aeromonas hydrophila, among which anti-lipopolysaccharide factor 1 (EsALF-1) and 3 (EsALF-3) were identified as the most significant (p < 0.05). After being challenged with A. hydrophila, EsALF-1 and EsALF-3 were highly expressed at both mRNA (in haemocytes) and protein (in haemolymph) levels compared with blank crabs, and the mRNA expressions of components in the EsTLR1-EsMyd88-EsPelle-EsALF pathway also increased significantly (p < 0.05). The EsALF-3 and EsMyd88 were even significantly higher expressed in response to the second A. hydrophila challenge, but their expressions all decreased (p < 0.05) when EsTLR1 was knocked down by RNAi. After the naïve crabs received an injection with the recombinant protein of EsALF-1 (rEsALF-1) or EsALF-3 (rEsALF-3), their survival rate increased significantly (p < 0.05) upon A. hydrophila stimulation. In contrast, the survival rate of the primed crabs reduced significantly (p < 0.05) after they received an injection with the antibody of EsALF-1 or EsALF-3. The enhanced expressions of EsALF-1 and EsALF-3 after A. hydrophilap riming stimulation could sustain for four weeks. All the results suggested that the EsTLR1-mediated productions of EsALF-1 and EsALF-3 in haemolymph played an indispensable role in the month-long humoral immune protection induced by A. hydrophila, which provides solid evidence of immune priming in crabs and a valuable reference for further understanding immune memory in invertebrates.

Time- and tissue-specific antimicrobial activity of the common bed bug in response to blood feeding and immune activation by bacterial injection.

Unlike almost all hematophagous insects, common bed bugs, Cimex lectularius, are not known to transmit pathogens to humans. To help unravel the reasons for their lack of vector competence, we studied the time- and tissue-dependent expression of innate immune factors after blood feeding or immune activation through the intrathoracic injection of bacteria. We used minimum inhibitory concentration (MIC1) bioassays and the Kirby-Bauer protocol to evaluate antimicrobial peptide (AMP2) activity in tissue extracts from the midguts or 'rest of body' (RoB3) tissues (containing hemolymph and fat body AMPs) against Gram-positive and Gram-negative bacteria. We compared AMP activity between blood-fed female bed bugs and yellow fever mosquitoes, Aedes aegypti and determined how female and male bed bugs respond to immune challenges, and how long AMP gene expression remains elevated in bed bugs following a blood meal. Blood meal-induced AMP activity is 4-fold stronger in female bed bugs than in female mosquitoes. Male bed bugs have elevated AMP activity within 8 h of a blood meal or an intrathoracic injection with bacteria, with the strongest activity expressed in RoB tissue 24 h after the immune challenge. Female bed bugs have a stronger immune response than males within 24 h of a blood meal. The effects of blood meal-induced elevated AMP activity lasts longer against the Gram-positive bacterium, Bacillus subtilis, than against the Gram-negative bacterium Escherichia coli. Unravelling the specific immune pathways that are activated in the bed bugs' immune responses and identifying the bed bug-unique AMPs might help determine why these insects are not vectors of human parasites.

Immunological assays of hemocytes in the Northern Quahog Mercenaria mercenaria.

The northern quahog Mercenaria mercenaria (commonly named hard clam) is an important aquaculture and fishery species along the Atlantic west coast. Environmental stresses, such as heat shock, fluctuating salinity, and harmful algal blooms are major challenges for clam aquaculture. In response to environmental stresses, hemocytes would change dynamically for defense and immunity. The goal of this study was to characterize basic immunological assays of hemocytes in the northern quahog by use of flow cytometry. The objectives were to: 1) develop a non-lethal method for hemolymph collection and dilution; 2) verify the capability of flow cytometry for hemocyte count and type identification through comparison with microscopic observation; 3) validate hemocyte viability assay based on plasma membrane integrity, and 4) develop hemocyte phagocytosis assay by use of fluorescein labeled microbeads. A non-lethal hemocyte collection method was developed using needle insertion through the ligament. Osmolality measurement of serum was the same as that of culture seawater. The pH measurement of serum (7.2) was significantly different from that of culture seawater (8.4). By microscopic observation, three types of hemocytes were identified with granulocytes, the dominant cell type (70 ± 16%), agranulocyte (14 ± 4%), and blast-like cell (16 ± 4%), and no differences were found from the measurements by flow cytometer on FSC/SSC plot (cell size/granularity). The viability of hemocytes based on plasma membrane integrity was 88 ± 6% ranging from 70 to 97% (n = 60, three populations), and viability protocol was further validated with the pre-set expected viability (p ≥ 0.424). Phagocytosis assay of hemocytes with fluorescence beads showed a mean capacity of 10 ± 5% (n = 60, three populations). Incubation time (up to 6 h) or bead concentrations (2:1 or 5:1 to hemocytes) did not affect the phagocytosis measurement. Overall, this study reported the basic characteristics of hemolymph (serum and hemocytes) of northern quahogs. It is expected that the assay methodologies will be applied to evaluation of hemocyte responses to environmental stresses for clam aquaculture.

Probiotic, Lactobacillus pentosus BD6 boost the growth and health status of white shrimp, Litopenaeus vannamei via oral administration.

This study aims to assess and determine the oral-administration of probiotic, Lactobacillus pentosus BD6 on growth performance, immunity and disease resistance of white shrimp, Litopenaeus vannamei. Lac. pentosus BD6 effectively inhibited the growth of aquatic pathogens, which was used in the test. Shrimp were fed with the control diet (without probiotic supplement) for 60 days and the probiotic-containing diets at 107, 108, 109, and 1010 cfu kg-1, respectively. Shrimp fed with the diet containing probiotic at the doses of 109-10 cfu kg-1 showed significant increase in growth performance as well as feed efficiency than that of the control. After a challenge test with Vibrio alginolyticus, shrimp fed with a probiotic diet at a dose of 1010 cfu kg-1 showed a significantly lower mortality as compared to the control and that of shrimp fed the diet containing probiotic at the levels up to 107-8 cfu kg-1. In addition, a therapeutic potential of Lac. pentosus BD6 was discovered because the cumulative mortalities of shrimp fed with probiotic and pathogen V. parahaemolyticus simultaneously were significantly lower when compared to control shrimp. Probiotic in diet at a dose of 109-10 cfu kg-1 significantly increased PO activity of shrimp, while shrimp receiving probiotic at the doses of 108-10 cfu kg-1 showed significant increase in lysozyme activity and phagocytic activity. Shrimp fed with the diet containing probiotic at the level of 1010 cfu kg-1 also indicated higher gene expression of prophenoloxidase (proPO) I, but not proPO II, lipopolysaccharide and ß-1,3-glucan-binding protein and penaeidin 4. Analysis of the bacterial microbiota of the shrimp intestine revealed that oral administration of probiotic increased the relative abundance of beneficial bacteria and reduced the abundance of harmful pathogenic bacteria in the gut flora of shrimp. Despite no statistically significant difference, an analysis of microbial diversity recorded higher species richness, Shannon-Weaver diversity index and evenness in the probiotic group, compared to the control group. It was concluded that Lac. pentosus BD6 has great antibacterial ability against a wide range of pathogens and has therapeutic potential to reduce the mortality of shrimp infected with V. parahaemolyticus. Additionally, dietary Lac. pentosus BD6 at the level of 1010 cfu kg-1 was recommended to improve growth performance, immunity and disease resistance of shrimp against V. alginolyticus.

Exosomal miR-224 contributes to hemolymph microbiota homeostasis during bacterial infection in crustacean.

It is well known that exosomes could serve as anti-microbial immune factors in animals. However, despite growing evidences have shown that the homeostasis of the hemolymph microbiota was vital for immune regulation in crustaceans, the relationship between exosomes and hemolymph microbiota homeostasis during pathogenic bacteria infection has not been addressed. Here, we reported that exosomes released from Vibrio parahaemolyticus-infected mud crabs (Scylla paramamosain) could help to maintain the homeostasis of hemolymph microbiota and have a protective effect on the mortality of the host during the infection process. We further confirmed that miR-224 was densely packaged in these exosomes, resulting in the suppression of HSP70 and disruption of the HSP70-TRAF6 complex, then the released TRAF6 further interacted with Ecsit to regulate the production of mitochondrial ROS (mROS) and the expression of Anti-lipopolysaccharide factors (ALFs) in recipient hemocytes, which eventually affected hemolymph microbiota homeostasis in response to the pathogenic bacteria infection in mud crab. To the best of our knowledge, this is the first document that reports the role of exosome in the hemolymph microbiota homeostasis modulation during pathogen infection, which reveals the crosstalk between exosomal miRNAs and innate immune response in crustaceans.

The effects of ammonia-N stress on immune parameters, antioxidant capacity, digestive function, and intestinal microflora of Chinese mitten crab, Eriocheir sinensis, and the protective effect of dietary supplement of melatonin.

Ammonia nitrogen pollution seriously affects the economic benefits of Chinese mitten crab (Eriocheir sinensis) farming. In this study, we first evaluated the protective effects of melatonin (MT) on immune parameters, antioxidant capacity, and digestive enzymes of E. sinensis under acute ammonia nitrogen stress. The results showed that ammonia-N stress significantly decreased the antibacterial ability of crabs, nevertheless MT could significantly improve it under ammonia-N stress (P < 0.05). Ammonia-N group hemolymph antioxidant capacity indicators (T-AOC, T-SOD, GSH-Px) were significantly decreased than control (p < 0.05), while the MT ammonia-N group hemolymph T-SOD activity significantly increased than ammonia-N group (p < 0.05). For hepatopancreas, ammonia-N group GSH-PX activity significantly decreased than control group, but MT ammonia-N group was significant increased than ammonia-N (p < 0.05). Ammonia-N stress has significantly increased the content of MDA in hemolymph and hepatopancreas (p < 0.05), but MT ammonia-N treatment significantly decreased than ammonia-N group (p < 0.05). Compared with the control group, ammonia-N significantly reduced the activities of Trypsin in the intestine and hepatopancreas (p < 0.05), while MT ammonia-N group can significantly improve the intestinal trypsin activity than ammonia-N (p < 0.05). The intestinal microbiota of E. sinensis results showed that ammonia-N stress significantly decreased the relative abundance of Bacteroidetes (p < 0.05). Ammonia-N stress significantly decreased the Dysgonomonas and Rubellimicrobium, and the Citrobacter significantly increased. In summary, melatonin has a protective effect on E. sinensis under ammonia-N stress. Acute ammonia-N stress may lead to the decrease of probiotics and the increase of pathogenic bacteria, which may be closely related to the impairment of digestive function and immune function.

Production of scFv, Fab, and IgG of CR3022 Antibodies Against SARS-CoV-2 Using Silkworm-Baculovirus Expression System.

COVID-19, caused by SARS-CoV-2, is currently spreading around the world and causing many casualties. Antibodies against such emerging infectious diseases are one of the important tools for basic viral research and the development of diagnostic and therapeutic agents. CR3022 is a monoclonal antibody against the receptor binding domain (RBD) of the spike protein (S protein) of SARS-CoV found in SARS patients, but it was also shown to have strong affinity for that of SARS-CoV-2. In this study, we produced large amounts of three formats of CR3022 antibodies (scFv, Fab and IgG) with high purity using a silkworm-baculovirus expression vector system. Furthermore, SPR measurements showed that the affinity of those silkworm-produced IgG antibodies to S protein was almost the same as that produced in mammalian expression system. These results indicate that the silkworm-baculovirus expression system is an excellent expression system for emerging infectious diseases that require urgent demand for diagnostic agents and therapeutic agents.

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

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.

Disruption of insect immunity using analogs of the pleiotropic insect peptide hormone Neb-colloostatin: a nanotech approach for pest control II.

This work continues our studies on the pleiotropic activity of the insect peptide Neb-colloostatin in insects. In vivo immunological bioassays demonstrated that hemocytotoxic analogs of Neb-colloostatin injected into Tenebrio molitor significantly reduced the number of hemocytes in the hemolymph and impaired phagocytosis, nodulation and phenoloxidase activities in the insects. Among the analogs tested, [Ala1]-,[Val1]-, [Hyp4]- and [Ach4]-colloostatin were particularly potent in disrupting cellular immunity in larvae, pupae and adult insects. This result suggests that the most effective analogs showed increases in the bioactivity period in the hemolymph of insects when compared to Neb-colloostatin. Recently, we demonstrated that it is possible to introduce Neb-colloostatin through the cuticle of an insect into the hemolymph when the peptide is coupled with nanodiamonds. In this study, we showed that [Ala1]-, [Val1]-, [Hyp4]- and [Ach4]-colloostatin, when complexed with nanodiamonds, may also pass through the cuticle into the hemolymph and induce long-term impairments of immunity in T. molitor at all developmental stages. Studies on the tissue selectivity and effectiveness of Neb-colloostatin analogs and efficient methods for their introduction into insects may contribute to the development of eco-friendly pest control methods based on bioactive peptidomimetics.

Isolation, Identification, and Bioinformatic Analysis of Antibacterial Proteins and Peptides from Immunized Hemolymph of Red Palm Weevil Rhynchophorus ferrugineus.

Red palm weevil (Rhynchophorus ferrugineus Olivier, 1791, Coleoptera: Curculionidae) is a destructive pest of palms, rapidly extending its native geographical range and causing large economic losses worldwide. The present work describes isolation, identification, and bioinformatic analysis of antibacterial proteins and peptides from the immunized hemolymph of this beetle. In total, 17 different bactericidal or bacteriostatic compounds were isolated via a series of high-pressure liquid chromatography steps, and their partial amino acid sequences were determined by N-terminal sequencing or by mass spectrometry. The bioinformatic analysis of the results facilitated identification and description of corresponding nucleotide coding sequences for each peptide and protein, based on the recently published R. ferrugineus transcriptome database. The identified compounds are represented by several well-known bactericidal factors: two peptides similar to defensins, one cecropin-A1-like peptide, and one attacin-B-like protein. Interestingly, we have also identified some unexpected compounds comprising five isoforms of pheromone-binding proteins as well as seven isoforms of odorant-binding proteins. The particular role of these factors in insect response to bacterial infection needs further investigation.

The antiviral immunity of ticks against transmitted viral pathogens.

Ticks, being obligate hematophagous arthropods, are exposed to various blood-borne pathogens, including arboviruses. Consequently, their feeding behavior can readily transmit economically important viral pathogens to humans and animals. With this tightly knit vector and pathogen interaction, the replication and transmission of tick-borne viruses (TBVs) must be highly regulated by their respective tick vectors to avoid any adverse effect on the ticks' biological development and viability. Knowledge about the tick-virus interface, although gaining relevant advances in recent years, is advancing at a slower pace than the scientific developments related to mosquito-virus interactions. The unique and complicated feeding behavior of ticks, compared to that of other blood-feeding arthropods, also limits the studies that would further elaborate the antiviral immunity of ticks against TBVs. Hence, knowledge of molecular and cellular immune mechanisms at the tick-virus interface, will further elucidate the successful viral replication of TBVs in ticks and their effective transmission to human and animal hosts.

The stress-immunity axis in shellfish.

It is a difficult task to describe what constitutes a 'healthy' shellfish (e.g., crustacean, bivalve). Visible defects such as discolouration, missing limbs or spines, fouling, lesions, and exoskeletal fractures can be indicative of underlying issues, senescence, or a 'stressed' animal. The absence of such symptoms is not evidence of a disease-free or a stress-free state. Now, more than ever, aquatic invertebrates must cope with acute and chronic environmental perturbations, such as, heatwaves and cold shocks, xenobiotic contaminants, intoxication events, and promiscuous pathogens expanding their host and geographic ranges. With that in mind, how does one determine the extent to which shellfish become stressed in situ (natural) or in cultured (artificial) settings to enhance disease susceptibility? Many biomarkers - predominantly biochemical and cellular measures of shellfish blood (haemolymph) - are considered to gauge immunosuppression and immunocompetence. Such measures range from immune cell (haemocyte) counts to enzymic activities and metabolite quantitation. Stressed invertebrates often reflect degraded conditions of their ecosystems, referred to as environmental indicators. We audit briefly the broad immune functions of shellfish, how they are modulated by known and emerging stressors, and discuss these concepts with respect to neuroendocrinology and immunotoxicology. We assert that chronic stress, alone or in combination with microbial, chemical and abiotic factors, increases the risk of infectious disease in shellfish, exacerbates idiopathic morbidity, and reduces the likelihood of recovery. Acute stress events can lead to immunomodulation, but these effects are largely transient. Enhancing our understanding of shellfish health and immunity is imperative for tackling losses at each stage of the aquatic food cycle and disease outbreaks in the wild.

Heptabladed ß-propeller lectins PLL2 and PHL from Photorhabdus spp. recognize O-methylated sugars and influence the host immune system.

O-methylation is an unusual sugar modification with a function that is not fully understood. Given its occurrence and recognition by lectins involved in the immune response, methylated sugars were proposed to represent a conserved pathogen-associated molecular pattern. We describe the interaction of O-methylated saccharides with two ß-propeller lectins, the newly described PLL2 from the entomopathogenic bacterium Photorhabdus laumondii, and its homologue PHL from the related human pathogen Photorhabdus asymbiotica. The crystal structures of PLL2 and PHL revealed up to 10 out of 14 potential binding sites per protein subunit to be occupied with O-methylated structures. The avidity effect strengthens the interaction by 4 orders of magnitude. PLL2 and PHL also interfere with the early immune response by modulating the production of reactive oxygen species and phenoloxidase activity. Since bacteria from Photorhabdus spp. have a complex life cycle involving pathogenicity towards different hosts, the involvement of PLL2 and PHL might contribute to the pathogen overcoming insect and human immune system defences in the early stages of infection. DATABASES: Structural data are available in PDB database under the accession numbers 6RG2, 6RGG, 6RFZ, 6RG1, 6RGU, 6RGW, 6RGJ, and 6RGR.

The Route of Infection Influences the Contribution of Key Immunity Genes to Antibacterial Defense in Anopheles gambiae.

Insect systemic immune responses to bacterial infections have been mainly studied using microinjections, whereby the microbe is directly injected into the hemocoel. While this methodology has been instrumental in defining immune signaling pathways and enzymatic cascades in the hemolymph, it remains unclear whether and to what extent the contribution of systemic immune defenses to host microbial resistance varies if bacteria invade the hemolymph after crossing the midgut epithelium subsequent to an oral infection. Here, we address this question using the pathogenic Serratia marcescens (Sm) DB11 strain to establish systemic infections of the malaria vector Anopheles gambiae, either by septic Sm injections or by midgut crossing after feeding on Sm. Using functional genetic studies by RNAi, we report that the two humoral immune factors, thioester-containing protein 1 and C-type lectin 4, which play key roles in defense against Gram-negative bacterial infections, are essential for defense against systemic Sm infections established through injection, but they become dispensable when Sm infects the hemolymph following oral infection. Similar results were observed for the mosquito Rel2 pathway. Surprisingly, blocking phagocytosis by cytochalasin D treatment did not affect mosquito susceptibility to Sm infections established through either route. Transcriptomic analysis of mosquito midguts and abdomens by RNA-seq revealed that the transcriptional response in these tissues is more pronounced in response to feeding on Sm. Functional classification of differentially expressed transcripts identified metabolic genes as the most represented class in response to both routes of infection, while immune genes were poorly regulated in both routes. We also report that Sm oral infections are associated with significant downregulation of several immune genes belonging to different families, specifically the clip-domain serine protease family. In sum, our findings reveal that the route of infection not only alters the contribution of key immunity genes to host antimicrobial defense but is also associated with different transcriptional responses in midguts and abdomens, possibly reflecting different adaptive strategies of the host.

Host Defense Effectors Expressed by Hemocytes Shape the Bacterial Microbiota From the Scallop Hemolymph.

The interaction between host immune response and the associated microbiota has recently become a fundamental aspect of vertebrate and invertebrate animal health. This interaction allows the specific association of microbial communities, which participate in a variety of processes in the host including protection against pathogens. Marine aquatic invertebrates such as scallops are also colonized by diverse microbial communities. Scallops remain healthy most of the time, and in general, only a few species are fatally affected on adult stage by viral and bacterial pathogens. Still, high mortalities at larval stages are widely reported and they are associated with pathogenic Vibrio. Thus, to give new insights into the interaction between scallop immune response and its associated microbiota, we assessed the involvement of two host antimicrobial effectors in shaping the abundances of bacterial communities present in the scallop Argopecten purpuratus hemolymph. To do this, we first characterized the microbiota composition in the hemolymph from non-stimulated scallops, finding both common and distinct bacterial communities dominated by the Proteobacteria, Spirochaetes and Bacteroidetes phyla. Next, we identified dynamic shifts of certain bacterial communities in the scallop hemolymph along immune response progression, where host antimicrobial effectors were expressed at basal level and early induced after a bacterial challenge. Finally, the transcript silencing of the antimicrobial peptide big defensin ApBD1 and the bactericidal/permeability-increasing protein ApLBP/BPI1 by RNA interference led to an imbalance of target bacterial groups from scallop hemolymph. Specifically, a significant increase in the class Gammaproteobacteria and the proliferation of Vibrio spp. was observed in scallops silenced for each antimicrobial. Overall, our results strongly suggest that scallop antimicrobial peptides and proteins are implicated in the maintenance of microbial homeostasis and are key molecules in orchestrating host-microbiota interactions. This new evidence depicts the delicate balance that exists between the immune response of A. purpuratus and the hemolymph microbiota.

Hemolymph protease-5 links the melanization and Toll immune pathways in the tobacco hornworm, Manduca sexta.

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.