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Mosquito immune cells, or hemocytes, are integral components of the innate immune responses that define vector competence. To date, the characterization and functional classification of hemocytes has been hindered by the limited availability of genetic resources. Here, we map the composition of mosquito hemocytes by engineering five transgenic Anopheles gambiae lines that express fluorescent proteins under the control of candidate hemocyte promoters. We characterize these five transgenic lines through gene expression and microscopy-based approaches, and examine mosquito immune cell populations by leveraging advanced spectral imaging flow cytometry. We classify mosquito hemocytes into twelve distinct populations based on size, granularity, and ploidy, while defining these hemocyte subtypes based on their phagocytic capacity and the expression of genetic markers. By simultaneously analyzing these morphological and genetic properties, our work highlights the complexity and plasticity of mosquito hemocytes and provides the foundation for deeper investigations into their roles in immunity and pathogen transmission. Classification of mosquito hemocytes into subtypes has thus far relied on morphological properties. Here, the authors develop new genetic resources that facilitate the visualization and analysis of hemocyte subtypes in Anopheles gambiae . By combining the expression of hemocyte-specific genetic markers with the analysis of morphology, ploidy, and phagocytic capacity, the authors identify twelve distinct subtypes, revealing the complexity and plasticity of these immune cells with unprecedented resolution.

In present study, transmission electron microscopy and flow cytometry were utilized to investigate the classification, characterization and immune functions of hemocytes from horseshoe crab, Tachypleus tridentatus and Carcinoscorpius rotundicauda . Three types of hemocytes were distinguished respectively: the granular cell, the semi-granular cell and the hyaline cell by transmission electron microscopy, while three hemocyte subpopulations (Gate 1 cell, Gate 2 cell, Gate 3 cell) were classified by flow cytometry. Hyaline cell was the major cell type with the highest nuclear-cytoplasmic ratio and granular cell and semi-granular cell showed lower ratios. Immune parameters of hemocytes in horseshoe crabs were investigated by flow cytometry. Different hemocyte subpopulations respond for diverse functions. Lysosomal contents and hemocyte mortality in Gate 3 cell subpopulation were higher than that in other subpopulations, while reactive oxygen species, phagocytosis and non-specific esterase, in Gate 1 cell subpopulation, were higher than those in other subpopulations. The hemocyte types between the two species had no significant differences in staining or morphology.

Circulating hemocytes of the silkworm can be classified by fluorescence microscopy following staining with acridine orange and propidium iodide. Based on their fluorescence characteristics, three groups of circulating hemocytes can be distinguished. The first group, granulocytes and spherulocytes, is positive for acridine orange and contain bright green fluorescent granules when observed by fluorescence microscopy. In granulocytes, these green granules are heterogeneous and relatively small. In contrast, in spherulocytes, the green granules appear more homogenous and larger. The second group of hemocytes consists of prohemocytes and plasmatocytes. These cells appear faint green following staining with acridine orange and do not contain any green fluorescent granules in the cytoplasm. Prohemocytes are round, and their nuclei are dark and clear within a background of faint green fluorescence. Inside the nucleus there are one or two small bright green fluorescent bodies. Plasmatocytes are irregularly shaped and their nuclei are invisible. Oenocytoids belong to the third group, and their nuclei are positive for propidium iodide. Therefore, all five types of circulating hemocytes of the silkworm, including many peculiar ones that are difficult to identify by light microscopy, can now be easily classified by fluorescence microscopy following staining with acridine orange and propidium iodide. In addition, we show that hemocytes positive for acridine orange and propidium iodide are in fact living cells based on assays for hemocyte composition, phagocytosis, and mitochondrial enzyme activity.

In the present study, we carried out a detailed light microscopy investigation of the cytochemical properties of the haemocytes of the colonial ascidian Botryllus schlosseri, using new cytochemical stains and enzymatic markers, a panel of antibodies and lectins as probes to characterise Botryllus blood cells further. Results indicate that lymphocyte-like cells are circulating undifferentiated cells recognised by anti-CD34 antibody and there are at least two defined haemocyte differentiation pathways: i) phagocytes, represented by hyaline amoebocytes and macrophage-like cells, which share similar staining properties, the same hydrolytic enzyme content as well as the presence of detectable cytochrome-c-oxidase activity, recognition by anti-CD39 and Narcissus pseudonarcissus agglutinin; ii) cytotoxic cell line, represented by granular amoebocytes and morula cells which have vacuoles stained by Ehrlich's stain and Neutral Red; DOPA-containing protein are present inside morula cell vacuoles. Pigment cells and nephrocytes are involved in catabolite storage but their relationships with other cell types are less clear.

Oogenesis is a complex developmental process that involves spatiotemporally regulated coordination between the germline and supporting, somatic cell populations. This process has been modeled extensively using the Drosophila ovary. Although different ovarian cell types have been identified through traditional means, the large-scale expression profiles underlying each cell type remain unknown. Using single-cell RNA sequencing technology, we have built a transcriptomic data set for the adult Drosophila ovary and connected tissues. Using this data set, we identified the transcriptional trajectory of the entire follicle-cell population over the course of their development from stem cells to the oogenesis-to-ovulation transition. We further identify expression patterns during essential developmental events that take place in somatic and germline cell types such as differentiation, cell-cycle switching, migration, symmetry breaking, nurse-cell engulfment, egg-shell formation, and corpus luteum signaling. Extensive experimental validation of unique expression patterns in both ovarian and nearby, nonovarian cells also led to the identification of many new cell type-and stage-specific markers. The inclusion of several nearby tissue types in this data set also led to our identification of functional convergence in expression between distantly related cell types such as the immune-related genes that were similarly expressed in immune cells (hemocytes) and ovarian somatic cells (stretched cells) during their brief phagocytic role in nurse-cell engulfment. Taken together, these findings provide new insight into the temporal regulation of genes in a cell-type specific manner during oogenesis and begin to reveal the relatedness in expression between cell and tissues types.

Benzyl alcohol (E1519) is an aromatic alcohol used in the pharmaceutical and food industry. It is used to protect food products against microorganisms during storage, as a flavoring in the production of chocolate and confectionery products, as an important ingredient in fragrance, and as a preservative in medical products. However, little is known of its effect on insects. The main aim of this study was to determine the influence of benzyl alcohol on the defense systems of the wax moth Galleria mellonella, i.e., its cuticular lipid composition and critical elements of its immune system. A gas chromatography/mass spectrometry (GC/MS) analysis found benzyl alcohol treatment to elicit significant quantitative and qualitative differences in cuticular free fatty acid (FFA) profiles. Our findings indicate that benzyl alcohol treatment increased the levels of HSP70 and HSP90 and decreased those of HSF1, histamine, and cysteinyl leukotriene. Benzyl alcohol application also increased dismutase level in the hemolymph and lowered those of catalase and 8-OHdG. The treatment also had negative effects on G. mellonella hemocytes and a Sf9 cell line in vitro: 48-h treatment resulted in morphological changes, with the remaining cells being clearly spindle-shaped with numerous granules. The high insecticidal activity of compound and its lack of toxicity towards vertebrates suggest it could be an effective insecticide.

The evolution of TLR-mediated innate immunity is a fundamental question in immunology. Here, we report the characterization and functional analysis of four TLR members in the lophotrochozoans Crassostreagigas (CgTLRs). All CgTLRs bear a conserved domain organization and have a close relationship with TLRs in ancient non-vertebrate chordates. In HEK293 cells, every CgTLR could constitutively activate NF-κB responsive reporter, but none of the PAMPs tested could stimulate CgTLR-activated NF-κB induction. Subcellular localization showed that CgTLR members have similar and dual distribution on late endosomes and plasma membranes. Moreover, CgTLRs and CgMyD88 mRNA show a consistent response to multiple PAMP challenges in oyster hemocytes. As CgTLR-mediated NF-κB activation is dependent on CgMyD88, we designed a blocking peptide for CgTLR signaling that would inhibit CgTLR-CgMyD88 dependent NF-κB activation. This was used to demonstrate that a Vibrio parahaemolyticus infection-induced enhancement of degranulation and increase of cytokines TNF mRNA in hemocytes, could be inhibited by blocking CgTLR signaling. In summary, our study characterized the primitive TLRs in the lophotrocozoan C. gigas and demonstrated a fundamental role of TLR signaling in infection-induced hemocyte activation. This provides further evidence for an ancient origin of TLR-mediated innate immunity.

Myeloid differentiation factor 88 (MyD88) is a universal and essential signaling protein in Toll-like receptor/interleukin-1 receptor-induced activation of nuclear factor-kappa B. In this study, two MyD88 protein variants (LvMyD88 and LvMyD88-1) were identified in Litopenaeus vannamei. The LvMyD88 cDNA is 1,848 bp in length and contains an open reading frame (ORF) of 1,428 bp, whereas the LvMyD88-1 cDNA is 1,719 bp in length and has an ORF of 1,299 bp. Both variants encode proteins with death and Toll/interleukin-1 receptor domains and share 91% sequence identity. In healthy L. vannamei, the LvMyD88 genes were highly expressed in hemocytes but at a low level in the hepatopancreas. The LvMyD88s expression was induced in hemocytes after challenge with lipopolysaccharide, CpG-ODN2006, Vibrio parahaemolyticus, Staphyloccocus aureus, and white spot syndrome virus, but not by poly I∶C. Overexpression of LvMyD88 and LvMyD88-1 in Drosophila Schneider 2 cells led to activation of antimicrobial peptide genes and wsv069 (ie1), wsv303, and wsv371. These results suggested that LvMyD88 may play a role in antibacterial and antiviral response in L. vannamei. To our knowledge, this is the first report on MyD88 in shrimp and a variant of MyD88 gene in invertebrates.

Hemocytes (insect blood cells) consist of several morphological types and perform a variety of physiological processes, including immune responses. However, we do not know how many cell types are functionally differentiated in hemocytes or how they perform independent physiological processes. To address this fundamental question, we analyzed hemocyte transcripts with a single-cell RNA-sequencing (scRNA-Seq) technique. The hemocytes were collected from larvae of a lepidopteran insect, Spodoptera exigua, in which four different hemocyte types were morphologically recognized. scRNA-Seq discriminated 24 hemocyte clusters based on the transcripts of each cell. The clusters were separated into seven functional groups predicted from the top three highly expressed and annotated genes in each cluster: active protein synthesis (12 clusters), apoptosis (5 clusters), melanization (2 clusters), modulating cell shape (6 clusters), antimicrobial peptide production (9 clusters), calcium homeostasis (8 clusters), and cell repairing (1 cluster). Signal components of Toll/IMD immune pathways were variably expressed among the clusters. Biosynthetic genes associated with oxylipin immune mediators were specifically expressed among the clusters. Immune effectors such as melanization and apoptosis were expressed in specific hemocyte clusters. Specifically expressed genes that discriminate hemocyte types were used to develop fluorescence in situ hybridization (FISH) markers. In addition, five new hemocyte groups, which were not among the four known hemocyte types in the transcript profile, were identified and discriminated with their specific FISH markers. The hemocyte clusters underwent dynamic changes upon immune challenge. A trajectory analysis using the transcriptome suggests at least three different hemocyte differentiation pathways. These results indicate that the hemocytes of S. exigua are functionally highly differentiated and exhibit a dynamic transition in response to environmental changes.

Background Mitochondria participate in various cellular processes including energy metabolism, apoptosis, autophagy, production of reactive oxygen species, stress responses, inflammation and immunity. However, the role of mitochondrial metabolism in immune cells and tissues shaping the innate immune responses are not yet fully understood. We investigated the effects of tissue-specific mitochondrial perturbation on the immune responses at the organismal level. Genes for oxidative phosphorylation (OXPHOS) complexes cI-cV were knocked down in the fruit fly Drosophila melanogaster , targeting the two main immune tissues, the fat body and the immune cells (hemocytes). Results While OXPHOS perturbation in the fat body was detrimental, hemocyte-specific perturbation led to an enhanced immunocompetence. This was accompanied by the formation of melanized hemocyte aggregates (melanotic nodules), a sign of activation of cell-mediated innate immunity. Furthermore, the hemocyte-specific OXPHOS perturbation induced immune activation of hemocytes, resulting in an infection-like hemocyte profile and an enhanced immune response against parasitoid wasp infection. In addition, OXPHOS perturbation in hemocytes resulted in mitochondrial membrane depolarization and upregulation of genes associated with the mitochondrial unfolded protein response. Conclusions Overall, we show that while the effects of mitochondrial perturbation on immune responses are highly tissue-specific, mild mitochondrial dysfunction can be beneficial in immune-challenged individuals and contributes to variation in infection outcomes among individuals.