Pentachlorophenol impairs the antimicrobic capability of blood clam via undermining humoral immunity and disrupting humoral-cellular crosstalk.

Due to past massive usage and persistent nature, pentachlorophenol (PCP) residues are prevalent in environments, posing a potential threat to various organisms such as sessile filter-feeding bivalves. Although humoral immunity and its crosstalk with cellular one are crucial for the maintaining of robust antimicrobic capability, little is known about the impacts of PCP on these critical processes in bivalve mollusks. In this study, pathogenic bacterial challenge and plasma antimicrobic capability assays were carried out to assess the toxic effects of PCP on the immunity of a common bivalve species, blood clam (Tegillarca granosa). Moreover, the impacts of PCP-exposure on the capabilities of pathogen recognition, hemocyte recruitment, and pathogen degradation were analyzed as well. Furthermore, the activation status of downstream immune-related signalling pathways upon PCP exposure was also assessed. Data obtained illustrated that 28-day treatment with environmentally realistic levels of PCP resulted in evident declines in the survival rates of blood clam upon Vibrio challenge along with markedly weakened plasma antimicrobic capability. Additionally, the levels of lectin and peptidoglycan-recognition proteins (PGRPs) in plasma as well as the expression of pattern recognition receptors (PRRs) in hemocytes were found to be significantly inhibited by PCP-exposure. Moreover, along with the downregulation of immune-related signalling pathway, markedly fewer chemokines (interleukin 8 (IL-8), IL-17, and tumor necrosis factor α (TNF-α)) in plasma and significantly suppressed chemotactic activity of hemocytes were also observed in PCP-exposed blood clams. Furthermore, compared to that of the control, blood clams treated with PCP had markedly lower levels of antimicrobic active substances, lysozyme (LZM) and antimicrobial peptides (AMP), in their plasma. In general, the results of this study suggest that PCP exposure could significantly impair the antimicrobic capability of blood clam via undermining humoral immunity and disrupting humoral-cellular crosstalk.

E3 Ubiquitin Ligase CHIP Inhibits Haemocyte Proliferation and Differentiation via the Ubiquitination of Runx in the Pacific Oyster.

Mollusca first evolve primitive immune cells (namely, haemocytes), which assemble a notable complex innate immune system, which are continuously produced through proliferation and differentiation and infused in the haemolymph. As a typical E3 ligase, CHIP is critical for immune cell turnover and homeostasis in vertebrates. In this study, a CHIP homolog (CgCHIP) with a high expression in haemocytes was identified in oysters to investigate its role in the proliferation and differentiation of ancient innate immune cells. CgCHIP exhibited a widespread distribution across all haemocyte subpopulations, and the knockdown of CgCHIP altered the composition of haemocytes as examined by flow cytometry. Mechanistically screened with bioinformatics and immunoprecipitation, a key haematopoietic transcription factor CgRunx was identified as a substrate of CgCHIP. Moreover, amino acids in the interacted intervals of CgCHIP and CgRunx were determined by molecular docking. Experimental evidence from an in vitro culture model of an agranulocyte subpopulation and an in vivo oyster model revealed that the knockdown of CgCHIP and CgRunx had opposing effects on agranulocyte (precursor cells) differentiation and granulocyte (effector cells) proliferation. In summary, CgCHIP negatively regulated agranulocyte differentiation and granulocyte proliferation by mediating the ubiquitination and degradation of CgRunx in oysters. These results offer insight into the involvement of ubiquitylation in controlling haemocyte turnover in primitive invertebrates.

Enhancement of immunity and disease resistance in Litopenaeus vannamei through injection of tyramine formulated with polyethylene glycol.

This study investigates the prolonged effect of immune disease resistance in Litopenaeus vannamei through the administration of tyramine (TA) formulated with polyethylene glycol (PEG). Facing the challenges of intensive farming, environmental stress, and global climate changes, innovative approaches to improve shrimp health are essential. The research focuses on the role of biogenic amines in stress response and immune regulation, demonstrating that TA, especially when combined with PEG, significantly prolongs immunity and resistance against Vibrio alginolyticus. The experimental design included administering TA, PEG, and TA-PEG, followed by evaluations of immunity, lactate and glucose levels, and immune-related gene expressions. Results showed notable prolonged effects in total hemocyte count, phenoloxidase activity, and phagocytic activity in the TA-PEG group, indicating enhanced immune activation period. Additionally, the expression of prophenoloxidase system-related genes was significantly upregulated in the TA-PEG group. Furthermore, the TA-PEG group exhibited a significantly higher survival rate in a susceptibility test against V. alginolyticus. The results of this study confirm that the combined use of PEG can effectively extend the immunostimulatory duration of TA.

Potential acetylcholine-based communication in honeybee haemocytes and its modulation by a neonicotinoid insecticide.

There is growing concern that some managed and wild insect pollinator populations are in decline, potentially threatening biodiversity and sustainable food production on a global scale. In recent years, there has been increasing evidence that sub-lethal exposure to neurotoxic, neonicotinoid pesticides can negatively affect pollinator immunocompetence and could amplify the effects of diseases, likely contributing to pollinator declines. However, a direct pathway connecting neonicotinoids and immune functions remains elusive. In this study we show that haemocytes and non-neural tissues of the honeybee Apis mellifera express the building blocks of the nicotinic acetylcholine receptors that are the target of neonicotinoids. In addition, we demonstrate that the haemocytes, which form the cellular arm of the innate immune system, actively express choline acetyltransferase, a key enzyme necessary to synthesize acetylcholine. In a last step, we show that the expression of this key enzyme is affected by field-realistic doses of clothianidin, a widely used neonicotinoid. These results support a potential mechanistic framework to explain the effects of sub-lethal doses of neonicotinoids on the immune function of pollinators.

Killing of xenogenous and virally infected homogenous target cells by shrimp lymphocyte-like haemocytes.

Haemocytes play a crucial role in the invertebrate's immune system. In our lab, five subpopulations of shrimp haemocytes were identified in the past: hyalinocytes, granulocytes, semi-granulocytes and two subpopulations of non-phagocytic cells. In the latter two subpopulations, their characteristics such as having small cytoplasmic rims and not adhering to plastic cell-culture plates are very similar to those of mammalian lymphocytes. Therefore, they were designated lymphocyte-like haemocytes. Although little is known about their function, we hypothesize, based on their morphology, that they may have a cytotoxic activity like natural killer cells, with the ability to recognize and kill target cells. In our study, K562 cells and Sf9 cells were used as xenogenous target cells to detect the cytotoxic activity of the shrimp non-adherent lymphocyte-like haemocytes. Non-adherent haemocytes were collected and mixed with K562 cells and Sf9 cells at a 5:1 ratio and the binding activity was examined under a microscope. The binding rate of non-adherent haemocytes to K562 cells and Sf9 cells reached 6.6 % and 2.4 % after 240 min of culture, respectively. Then, the killing activity of non-adherent haemocytes was detected by an EMA staining (fluorescence microscopy), which showed 3.75 % dead K562 cells and 1.025 % dead Sf9 cells, and by Sytox® blue staining (flow cytometry), which showed 4.97 % of dead K562 cells. Next, a killing assay was developed to visualize the killing activity of shrimp non-adherent haemocytes. Non-adherent haemocytes were pre-labeled in blue (CellTracker blue) and K562/Sf9 cells in green (CFSE); dead cells were differentially stained red with ethidium bromide. The cytotoxic activity increased and reached a level of 2.59 % in K562 cells and 0.925 % in Sf9 cells at 120 min after co-culture. Furthermore, in the co-cultures of non-adherent haemocytes with K562 cells and Sf9 cells, upregulation of the gene and protein expression of the cytotoxic molecules torso-like protein and granzyme B was observed by RT-qPCR at 240 min and western blotting at 180 min. Additionally, non-adherent haemocytes were co-cultured with WSSV-inoculated shrimp ovary and lymphoid organ cells to detect the cytotoxicity to homogenous target cells. The binding activity started at 60 min in both the ovary and lymphoid organ cultures and reached at 240 min 50.62 % and 40.7 %, respectively. The killing activity was detected by EMA staining and the percentage of dead ovary and lymphoid organ cells increased respectively from 10.84 % to 6.89 % at 0 min to 13.09 % and 8.37 % at 240 min. In conclusion, we demonstrated the existence of cytotoxic activity of shrimp lymphocyte-like haemocytes against xenogenous cells from mammals and insects and against WSSV-infected homogenous shrimp cells.

A transcription factor ATF3 involves in the phagocytosis of granulocytes in oyster Crassostrea gigas.

Phagocytosis is a major cellular mechanism for mollusk granulocytes to eliminate nonself substances and dead cells, and thus to preserve the immune homeostasis. The knowledge of the regulatory mechanisms controlling phagocytic capacity is vital to understanding the immune system. In the present study, an ATF3 homolog (CgATF3) with a typical bZIP domain was identified in the Pacific oyster Crassostrea gigas. Its highly conserved bZIP domain consisted of two structural features, a basic region for DNA binding and a leucine zipper region for dimerization. Its transcript was found to be abundantly expressed in haemocytes, which was induced by Vibrio splendidus stimulation and recombinant CgTNF-2 treatment, along with an increase of its protein content in the nucleus. Moreover, CgATF3 showed a consistent and specific high expression in granulocytes, and CgATF3+ granulocytes were characterized morphologically by the largest diameter, smaller nucleus to cytoplasmic ratio, and abundant cytoplasmic granules, and functionally by a higher capacity for phagocytosis. When CgATF3 expression was inhibited by RNAi, the expression levels of CgRab1, CgRab33 and CgCathepsin L1, as well as the phagocytic rate and index of granulocytes all decreased after V. splendidus stimulation. These results together demonstrated the involvement of CgATF3 in regulating the expressions of Rabs and Cathepsin L1, as well as the phagocytosis of granulocytes in oyster C. gigas.

Physiological responses of the stingless bee Partamona helleri to oral exposure to three agrochemicals: impact on antioxidant enzymes and hemocyte count.

Agrochemicals pose significant threats to the survival of bees, yet the physiological impacts of sublethal doses on stingless bees remain poorly understood. This study investigated the effects of acute oral exposure to three commercial formulations of agrochemicals [CuSO4 (leaf fertilizer), glyphosate (herbicide), and spinosad (bioinsecticide)] on antioxidant enzymes, malondialdehyde content (MDA), nitric oxide (NO) levels, and total hemocyte count (THC) in the stingless bee Partamona helleri. Foragers were exposed to lethal concentrations aimed to kill 5% (LC5) of CuSO4 (120 µg mL-1) or spinosad (0.85 µg mL-1) over a 24-h period. Glyphosate-exposed bees received the recommended label concentration (7400 µg mL-1), as they exhibited 100% survival after exposure. Ingestion of CuSO4 or glyphosate-treated diets by bees was reduced. Levels of NO and catalase (CAT) remained unaffected at 0 h or 24 h post-exposure. Superoxide dismutase (SOD) activity was higher at 0 h compared to 24 h, although insignificantly so when compared to the control. Exposure to CuSO4 reduced glutathione S-transferase (GST) activity at 0 h but increased it after 24 h, for both CuSO4 and glyphosate. MDA levels decreased after 0 h exposure to CuSO4 or spinosad but increased after 24 h exposure to all tested agrochemicals. THC showed no difference among glyphosate or spinosad compared to the control or across time. However, CuSO4 exposure significantly increased THC. These findings shed light on the physiological responses of stingless bees to agrochemicals, crucial for understanding their overall health.

Yorkie negatively regulates the Crustin expression during molting in Chinese mitten crab, Eriocheir sinensis.

Molting is a key biological process of crustaceans, which is mainly regulated by 20-hydroxyecdyone (20E). The molting cycle could be divided into three main stages including pre-molt, post-molt and inter-molt stages. The mechanism of immune regulation during molting process still requires further exploration. Yorkie (Yki) is a pivotal transcription factor in the Hippo signaling pathway, and it plays an essential role in regulating cell growth and immune response. In the present study, a Yki gene was identified from Eriocheir sinensis (designed as EsYki), and the regulatory role of EsYki in controlling the expression of antimicrobial peptide genes throughout the molting process was investigated. The mRNA expression level of EsYki was higher at the pre-molt stage compared to the post-molt stage and inter-molt stage. Following the injection of 20E, there was a notable and consistent rise in the EsYki mRNA expression in haemocytes. The increase was observed from 3 h to 48 h with the maximum level at 12 h. And the phosphorylation of Yki in the haemocytes was also significantly up-regulated at 3 h post 20E injection. Moreover, the levels of EsYki mRNA expression at three molting stages were significantly increased post Aeromonas hydrophila stimulation. The maximum level was detected at post-molt stage following A. hydrophila stimulation, while the lowest level was observed at inter-molt stage. The expression pattern of EsCrus was in contrast to EsCrus. After EsYki mRNA transcripts were inhibited by Yki inhibitor (CA3), the mRNA expression levels of EsCrus1 and EsCrus2 following A. hydrophila stimulation were significantly elevated. Furthermore, the phosphorylation level of NF-κB was also increased following the inhibition of Yki. Collectively, our findings indicated that EsYki could be induced by 20E and has a suppressive effect on the expression of EsCrus via inhibiting NF-κB during molting process. This research contributes to the understanding of the immunological regulation mechanism during molting process in crustaceans.

Dragon fruit-derived oligosaccharides alter hemocyte-mediated immunity and expression of genes related to innate immunity and oxidative stress in Daphnia magna.

Dragon fruit oligosaccharide (DFO) is an indigestible prebiotic that enhances the growth and reproduction of Daphnia magna, increases the expression of genes involved in immunity, and reduces oxidative stress. This study investigated the effects of DFO on the expression of innate immunity- (Toll, Pelle, proPO, A2M, and CTL), oxidative stress- (Mn-SOD), and nitric oxide (NO) synthesis-related genes (NOS1, NOS2, and arginase) as well as NO localization and number of hemocytes in D. magna. For this ten-day-old D. magna were treated with 0 or 9 mg l-1 of DFO for 24 and 85 h. Gene expression levels, NO intensity and localization, and total hemocytes were evaluated. After 24 h, the expression of Toll and proPO increased significantly (p < 0.05), while that of C-type lectins (CTL) was reduced (p < 0.05). At 85 h, Mn-SOD and CTL expressions were markedly suppressed (p < 0.05). NO was mostly localized in the foregut, midgut, hindgut, and carapace. The expression of NOS1 was reduced after 24 h (p < 0.05). In addition, NO intensity at 24 h was insignificantly lower than the control (p > 0.05). At 85 h, the expression of NOS1, NOS2, and arginase was higher than control, but NO intensity did not differ significantly (p > 0.05). Furthermore, the total hemocyte count elevated remarkably at 85 h (p < 0.05). Our study suggested that 9 mg l-1 of DFO could alter the expression of the genes related to innate immunity, oxidative stress, and NO synthesis in D. magna and significantly stimulate hemocyte production.

Effects of a supplemented diet containing 7 probiotic strains (Honeybeeotic) on honeybee physiology and immune response: analysis of hemolymph cytology, phenoloxidase activity, and gut microbiome.

BACKGROUND: In this study, a probiotic mixture (Honeybeeotic) consisting of seven bacterial strains isolated from a unique population of honeybees (Apis mellifera ligustica) was used. That honeybee population was located in the Roti Abbey locality of the Marche Region in Italy, an area isolated from human activities, and genetic contamination from other honeybee populations. The aim was to investigate the effects of this probiotic mixture on the innate immunity and intestinal microbiome of healthy common honeybees in two hives of the same apiary. Hive A received a diet of 50% glucose syrup, while hive B received the same syrup supplemented with the probiotics, both administered daily for 1 month. To determine whether the probiotic altered the immune response, phenoloxidase activity and hemolymph cellular subtype count were investigated. Additionally, metagenomic approaches were used to analyze the effects on gut microbiota composition and function, considering the critical role the gut microbiota plays in modulating host physiology. RESULTS: The results revealed differences in hemocyte populations between the two hives, as hive A exhibited higher counts of oenocytoids and granulocytes. These findings indicated that the dietary supplementation with the probiotic mixture was safe and well-tolerated. Furthermore, phenoloxidase activity significantly decreased in hive B (1.75 ± 0.19 U/mg) compared to hive A (3.62 ± 0.44 U/mg, p < 0.005), suggesting an improved state of well-being in the honeybees, as they did not require activation of immune defense mechanisms. Regarding the microbiome composition, the probiotic modulated the gut microbiota in hive B compared to the control, retaining core microbiota components while causing both positive and negative variations. Notably, several genes, particularly KEGG genes involved in amino acid metabolism, carbohydrate metabolism, and branched-chain amino acid (BCAA) transport, were more abundant in the probiotic-fed group, suggesting an effective nutritional supplement for the host. CONCLUSIONS: This study advocated that feeding with this probiotic mixture induces beneficial immunological effects and promoted a balanced gut microbiota with enhanced metabolic activities related to digestion. The use of highly selected probiotics was shown to contribute to the overall well-being of the honeybees, improving their immune response and gut health.

Notch signaling promotes differentiation, cell death and autophagy in Drosophila hematopoietic system.

Notch signaling is a highly conserved pathway between mammals and Drosophila and plays a key role in various biological processes. Drosophila has emerged as a powerful model for studying hematopoiesis and leukemia. In exception to crystal cells, the strength of Notch signaling in Drosophila lymph gland cortical zone (CZ)/intermediate zone (IZ) cells is weak. However, the influence of Notch activation in the lymph gland CZ/IZ cells and circulating hemocytes on hematopoietic homeostasis maintenance is unclear. Here, we showed that Notch activation in lymph gland CZ/IZ cells induced overdifferentiation of progenitors. Moreover, Notch activation promoted lamellocyte generation via NFκB/Toll signaling activation and increased reactive oxygen species (ROS). In addition, we found that Notch activation in lymph gland CZ/IZ cells and circulating hemocytes caused caspase-independent and nonautophagic cell death. However, crystal cell autophagy was activated by upregulation of the expression of the target gene of the Hippo/Yki pathway Diap1. Moreover, we showed that Notch activation could alleviate cytokine storms and improve the survival of Rasv12 leukemia model flies. Our study revealed the various mechanisms of hematopoietic dysregulation induced by Notch activation in healthy flies and the therapeutic effect of Notch activation on leukemia model flies.

Galectins induced from hemocytes bridge phosphatidylserine and N-glycosylated Drpr/CED-1 receptor during dendrite pruning.

During neuronal pruning, phagocytes engulf shed cellular debris to avoid inflammation and maintain tissue homeostasis. How phagocytic receptors recognize degenerating neurites had been unclear. Here, we identify two glucosyltransferases Alg8 and Alg10 of the N-glycosylation pathway required for dendrite fragmentation and clearance through genetic screen. The scavenger receptor Draper (Drpr) is N-glycosylated with complex- or hybrid-type N-glycans that interact specifically with galectins. We also identify the galectins Crouching tiger (Ctg) and Hidden dragon (Hdg) that interact with N-glycosylated Drpr and function in dendrite pruning via the Drpr pathway. Ctg and Hdg are required in hemocytes for expression and function, and are induced during dendrite injury to localize to injured dendrites through specific interaction with exposed phosphatidylserine (PS) on the surface membrane of injured dendrites. Thus, the galectins Ctg and Hdg bridge the interaction between PS and N-glycosylated Drpr, leading to the activation of phagocytosis.

Differential toxic effects of nano-titanium dioxide on clams (Meretrix meretrix) with various individuality.

Nano-TiO2 is inevitably released into aquatic environment with increasing of nanotechnology industries. Study pointed that different individuality showed divergent behavioral and physiological response when facing environmental stress. However, the effects of nano-TiO2 on tolerance of bivalves with different individualities remain unknown. In the study, clams were divided into two types of individuality - proactive and reactive by post-stress recovery method. It turned out that proactive individuals had quicker shell opening level, stronger burrowing behavior, faster feeding recovery, higher standard metabolic rate and more rapid ammonia excretion ability than reactive individuals after exposed to air. Then, the survival rate, hemocytes response and oxidase activity of classified clams were evaluated after nano-TiO2 exposure. Results showed that after 30 d exposure, proactive individuals accelerated burrowing behavior with higher survival rate. Moreover, proactive clams had better adaptability and less hemocytes response and oxidative damage than reactive clams. The study highlights the individualities of marine shell fish determine individual capacity to adapt to environmental changes, play important roles in aquaculture and coastal ecosystem health.

Exploring the role of RNASET2 in the immune response of black soldier fly larvae.

T2 RNases are transferase-type enzymes distributed across phyla, crucial for breaking down single-stranded RNA molecules. In addition to their canonical function, several T2 enzymes exhibit pleiotropic roles, contributing to various biological processes, such as the immune response in invertebrates and vertebrates. This study aims at characterizing RNASET2 in the larvae of black soldier fly (BSF), Hermetia illucens, which are used for organic waste reduction and the production of valuable insect biomolecules for feed formulation and other applications. Given the exposure of BSF larvae to pathogens present in the feeding substrate, it is likely that the mechanisms of their immune response have undergone significant evolution and increased complexity. After in silico characterization of HiRNASET2, demonstrating the high conservation of this T2 homolog, we investigated the expression pattern of the enzyme in the fat body and hemocytes, two districts mainly involved in the insect immune response, in larvae challenged with bacterial infection. While no variation in HiRNASET2 expression was observed in the fat body following infection, a significant upregulation of HiRNASET2 synthesis occurred in hemocytes shortly after the injection of bacteria in the larva. The intracellular localization of HiRNASET2 in lysosomes of plasmatocytes, its extracellular association with bacteria, and the presence of a putative antimicrobial domain in the molecule, suggest its potential role in RNA clean-up and as an alarm molecule promoting phagocytosis activation by hemocytes. These insights contribute to the characterization of the immune response of Hermetia illucens larvae and may facilitate the development of animal feedstuff enriched with highly valuable BSF bioactive compounds.

Transcriptomic and functional analysis of the antiviral response of mussels after a poly I:C stimulation.

The study of mussels (Mytilus galloprovincialis) has grown in importance in recent years due to their high economic value and resistance to pathogens. Because of the biological characteristics revealed by mussel genome sequencing, this species is a valuable research model. The high genomic variability and diversity, particularly in immune genes, may be responsible for their resistance to pathogens found in seawater and continuously filtered and internalized by them. These facts, combined with the lack of proven mussel susceptibility to viruses in comparison to other bivalves such as oysters, result in a lack of studies on mussel antiviral response. We used RNA-seq to examine the genomic response of mussel hemocytes after they were exposed to poly I:C, simulating immune cell contact with viral dsRNA. Apoptosis and the molecular axis IRFs/STING-IFI44/IRGC1 were identified as the two main pathways in charge of the response but we also found a modulation of lncRNAs. Finally, in order to obtain new information about the response of mussels to putative natural challenges, we used VHSV virus (Viral Hemorrhagic Septicemia Virus) to run some functional analysis and confirm poly I:C's activity as an immunomodulator in a VHSV waterborne stimulation. Both, poly I:C as well as an injury stimulus (filtered sea water injection) accelerated the viral clearance by hemocytes and altered the expression of several immune genes, including IL-17, IRF1 and viperin.

CgIκB2 negatively regulates the expression of interferon-like protein by Rel/NF-κB signal in Crassostrea gigas.

Inhibitors of NF-κB (IκBs) have been implicated as major components of the Rel/NF-κB signaling pathway, playing an important negative regulatory role in host antiviral immunity such as in the activation of interferon (IFN) in vertebrates. In the present study, the immunomodulatory effect of IκB (CgIκB2) on the expression of interferon-like protein (CgIFNLP) was evaluated in Pacific oyster (Crassostrea gigas). After poly (I:C) stimulation, the mRNA expression level of CgIκB2 in haemocytes was significantly down-regulated at 3-12 h while up-regulated at 48-72 h. The mRNA expression of CgIκB2 in haemocytes was significantly up-regulated at 3 h after rCgIFNLP stimulation. In the CgIκB2-RNAi oysters, the mRNA expression of CgIFNLP, interferon regulatory factor-8 (CgIRF8) and NF-κB subunit (CgRel), the abundance of CgIFNLP and CgIRF8 protein in haemocytes, as well as the abundance of CgRel protein in nucleus were significantly increased after poly (I:C) stimulation. Immunofluorescence assay showed that nuclear translocation of CgIRF8 and CgRel protein was promoted in CgIκB2-RNAi oysters compared with that in EGFP-RNAi group. In the CgRel-RNAi oysters, the mRNA and protein expression level of CgIFNLP significantly down-regulated after poly (I:C) stimulation. The collective results indicated that CgIκB2 plays an important role in regulating CgIFNLP expression through its effects on Rel/NF-κB and IRF signaling pathways.

Polyunsaturated fatty acids stimulate immunity and eicosanoid production in Drosophila melanogaster.

Eicosanoids are a class of molecules derived from C20 polyunsaturated fatty acids (PUFAs) that play a vital role in mammalian and insect biological systems, including development, reproduction, and immunity. Recent research has shown that insects have significant but lower levels of C20 PUFAs in circulation in comparison to C18 PUFAs. It has been previously hypothesized in insects that eicosanoids are synthesized from C18 precursors, such as linoleic acid (LA), to produce downstream eicosanoids. In this study, we show that introduction of arachidonic acid (AA) stimulates production of cyclooxygenase, lipoxygenase, and cytochrome P450-derived eicosanoids. Downstream immune readouts showed that LA stimulates phagocytosis by hemocytes, while both LA and AA stimulate increased antimicrobial peptide production when D. melanogaster is exposed to a heat-killed bacterial pathogen. In totality, this work identifies PUFAs that are involved in insect immunity and adds evidence to the notion that Drosophila utilizes immunostimulatory lipid signaling to mitigate bacterial infections. Our understanding of immune signaling in the fly and its analogies to mammalian systems will increase the power and value of Drosophila as a model organism in immune studies.

Participation of shrimp pva-miR-166 in hemocyte homeostasis by modulating apoptosis-related gene PvProsaposin during white spot syndrome virus infection.

Tiny controllers referred to as microRNAs (miRNAs) impede the expression of genes to modulate biological processes. In invertebrates, particularly in shrimp as a model organism, it has been demonstrated that miRNAs play a crucial role in modulating innate immune responses against viral infection. By analyzing small RNAs, we identified 60 differentially expressed miRNAs (DEMs) in Penaues vannamei hemocytes following infection with white spot syndrome virus (WSSV). We predicted the target genes of WSSV-responsive miRNAs, shedding light on their participation in diverse biological pathways. We are particularly intrigued by pva-miR-166, which is the most notably elevated miRNA among 60 DEMs. At 24 h post-infection (hpi), the negative correlation between the expression of pva-miR-166 and its target gene, PvProsaposin, was evident and their interaction was confirmed by a reduction in luciferase activity in vitro. Suppression of PvProsaposin in unchallenged shrimp led to decreased survival rates, reduced total hemocyte count (THC), and increased caspase 3/7 activity, suggesting its significant role in maintaining hemocyte homeostasis. In WSSV-infected shrimp, a lower number of hemocytes corresponded to a lower WSSV load, but higher shrimp mortality was observed when PvProsaposin was suppressed. Conformingly, the introduction of the pva-miR-166 mimic to WSSV-infected shrimp resulted in decreased levels of PvProsaposin transcripts, a significant loss of THC, and an increase in the hemocyte apoptosis. Taken together, we propose that pva-miR-166 modulates hemocyte homeostasis during WSSV infection by suppressing the PvProsaposin, an anti-apoptotic gene. PvProsaposin inhibition disrupts hemocyte homeostasis, rendering the shrimp's inability to withstand WSSV invasion.IMPORTANCEGene regulation by microRNAs (miRNAs) has been reported during viral infection. Furthermore, hemocytes serve a dual role, not only producing various immune-related molecules to combat viral infections but also acting as a viral replication site. Maintaining hemocyte homeostasis is pivotal for the shrimp's survival during infection. The upregulated miRNA pva-miR-166 could repress PvProsaposin expression in shrimp hemocytes infected with WSSV. The significance of PvProsaposin in maintaining hemocyte homeostasis via apoptosis led to reduced survival rate, decreased total hemocyte numbers, and elevated caspase 3/7 activity in PvProsaposin-silenced shrimp. Additionally, the inhibitory ability of pva-miR-166-mimic and dsRNA-PvProsaposin on the expression of PvProsaposin also lowered the THC, increases the hemocyte apoptosis, resulting in a lower WSSV copy number. Ultimately, the dysregulation of the anti-apoptotic gene PvProsaposin by pva-miR-166 during WSSV infection disrupts hemocyte homeostasis, leading to an immunocompromised state in shrimp, rendering them incapable of surviving WSSV invasion.

Octanoic acid kills Lucilia sericata (Diptera: Calliphoridae) by affecting two major defence systems: cuticular free fatty acids and immunocompetent cells.

This work examines the insecticidal activity of octanoic acid (C8:0), a short-chain fatty acid detected in entomopathogenic fungus - Conidiobolus coronatus medium, against Lucilia sericata larvae and adults. The LD50 value was calculated as 3.04±0.26 µg/mg (3040 mg/kg) of insect body mass, which places the compound in category 5 of acute toxicity (slightly hazardous). The presented research also describes its probable mechanism, with a particular focus on changes in two main insect defense mechanisms: (1) the composition of the cuticle (GC-MS analysis) and (2) immunocompetent cells (microscopic analysis of cultured hemocytes). More precisely, octanoic acid application resulted in changes in cuticular free fatty acid (FFA) profiles in both adults and larvae; generally, treatment increased short-chain FFAs, and a decrease of middle- and long-chain FFAs. Both in vivo and in vitro applications of octanoic acid resulted in vacuolisation, disintegration, and destruction of nets formed by plasmatocytes. As the compound has also previously been found to be toxic against Galleria mellonella, it appears to have lethal potential against insects in both the Orders Diptera and Lepidoptera, indicating it may have strong entomopathogenic potential. It is worth noting that octanoic acid is approved as a food additive with well-documented insecticidal activity, and hence may be a valuable component in the design of new insecticides that are safe for both humans and the environment.

Professional phagocytes are recruited for the clearance of obsolete nonprofessional phagocytes in the Drosophila ovary.

Cell death is an important process in the body, as it occurs throughout every tissue during development, disease, and tissue regeneration. Phagocytes are responsible for clearing away dying cells and are typically characterized as either professional or nonprofessional phagocytes. Professional phagocytes, such as macrophages, are found in nearly every part of the body while nonprofessional phagocytes, such as epithelial cells, are found in every tissue type. However, there are organs that are considered "immune-privileged" as they have little to no immune surveillance and rely on nonprofessional phagocytes to engulf dying cells. These organs are surrounded by barriers to protect the tissue from viruses, bacteria, and perhaps even immune cells. The Drosophila ovary is considered immune-privileged, however the presence of hemocytes, the macrophages of Drosophila, around the ovary suggests they may have a potential function. Here we analyze hemocyte localization and potential functions in response to starvation-induced cell death in the ovary. Hemocytes were found to accumulate in the oviduct in the vicinity of mature eggs and follicle cell debris. Genetic ablation of hemocytes revealed that the presence of hemocytes affects oogenesis and that they phagocytose ovarian cell debris and in their absence fecundity decreases. Unpaired3, an IL-6 like cytokine, was found to be required for the recruitment of hemocytes to the oviduct to clear away obsolete follicle cells. These findings demonstrate a role for hemocytes in the ovary, providing a more thorough understanding of phagocyte communication and cell clearance in a previously thought immune-privileged organ.