Biology subjects

Samantsidis, G.-R.

Publications and source records attributed to Samantsidis, G.-R..

4 recordsLinked to original sources

bioRxiv2024

Exploring new dimensions of immune cell biology in Anopheles gambiae through genetic immunophenotyping

Mosquito immune cells, or hemocytes, are integral components of the innate immune responses that define vector competence. However, the lack of genetic resources has limited their characterization and our understanding of their functional roles in immune signaling. To overcome these challenges, we engineered transgenic Anopheles gambiae that express fluorescent proteins under the control of candidate hemocyte promoters. Following the characterization of five transgenic constructs through gene expression and microscopy-based approaches, we examine mosquito immune cell populations by leveraging advanced spectral imaging flow cytometry. Our results comprehensively map the composition of mosquito hemocytes, classifying them into twelve distinct populations based on size, granularity, ploidy, phagocytic capacity, and the expression of PPO6, SPARC, and LRIM15 genetic markers. Together, our novel use of morphological properties and genetic markers provides increased resolution into our understanding of mosquito hemocytes, highlighting the complexity and plasticity of these immune cell populations, while providing the foundation for deeper investigations into their roles in immunity and pathogen transmission.

genetics↗

bioRxiv2024

A genome-wide CRISPR screen in Anopheles mosquito cells identifies essential genes and required components of clodronate liposome function

Anopheles mosquitoes are the sole vector of human malaria, the most burdensome vector-borne disease worldwide. Strategies aimed at reducing mosquito populations and limiting their ability to transmit disease show the most promise for disease control. Therefore, gaining an improved understanding of mosquito biology, and specifically that of the immune response, can aid efforts to develop new approaches that limit malaria transmission. Here, we use a genome-wide CRISPR screening approach for the first time in mosquito cells to identify essential genes in Anopheles and identify genes for which knockout confers resistance to clodronate liposomes, which have been widely used in mammals and arthropods to ablate immune cells. In the essential gene screen, we identified a set of 1280 Anopheles genes that are highly enriched for genes involved in fundamental cell processes. For the clodronate liposome screen, we identified several candidate resistance factors and confirm their roles in the uptake and processing of clodronate liposomes through in vivo validation in Anopheles gambiae, providing new mechanistic detail of phagolysosome formation and clodronate liposome function. In summary, we demonstrate the application of a genome-wide CRISPR knockout platform in a major malaria vector and the identification of genes that are important for fitness and immune-related processes.

genetics↗

bioRxiv2024

TNF signaling mediates cellular immune function and promotes malaria parasite killing in the mosquito Anopheles gambiae

Tumor Necrosis Factor- (TNF-) is a proinflammatory cytokine and a master regulator of immune cell function in vertebrates. While previous studies have implicated TNF signaling in invertebrate immunity, the roles of TNF in mosquito innate immunity and vector competence have yet to be explored. Herein, we confirm the identification of a conserved TNF- pathway in Anopheles gambiae consisting of the TNF- ligand, Eiger, and its cognate receptors Wengen and Grindelwald. Through gene expression analysis, RNAi, and in vivo injection of recombinant TNF-, we provide direct evidence for the requirement of TNF signaling in regulating mosquito immune cell function by promoting granulocyte midgut attachment, increased granulocyte abundance, and oenocytoid rupture. Moreover, our data demonstrate that TNF signaling is an integral component of anti-Plasmodium immunity that limits malaria parasite survival. Together, our data support the existence of a highly conserved TNF signaling pathway in mosquitoes that mediates cellular immunity and influences Plasmodium infection outcomes, offering potential new approaches to interfere with malaria transmission by targeting the mosquito host.

immunology↗

bioRxiv2022

Functional characterization of putative ecdysone transporters in lepidopteran pests

The insect steroid hormone ecdysone plays a critical role in insect development. Several recent studies have shown that ecdysone is transported through Organic Anion Transporting Polypeptides (OATPs) in insects such as flies and mosquitoes. However, the conservation of this mechanism across other arthropods and the role of this transporter in canonical ecdysone pathways are less well studied. Herein we functionally characterized the putative ecdysone transporter OATP74D from two major agricultural moth pests: Helicoverpa armigera (cotton bollworm) and Spodoptera frugiperda (fall armyworm). Phylogenetic analysis of OATP transporters across the superphylum Ecdysozoa revealed that Oatp74D is well represented among arthropod species and appeared only at the root of the arthropod lineage. Partial disruption of Oatp74D in S. frugiperda decreased embryo hatching rate and larval survival, suggesting that this gene is essential for development in vivo. Depletion and re-expression of OatP74D in the lepidoptera cell line RP-HzGUT-AW1(MG) confirmed the genes role in ecdysone import and demonstrated that OATP74D is essential for the transcriptional activation of ecdysone responsive genes including caspase-3, implicating this transporter in cell death pathways. Establishment of a simple and robust luciferase assay using the RP-HzGUT- AW1(MG) cell line demonstrated that both HaOATP74D and SfOATP74D are inhibited by rifampicin, a well-known organic anion transporter inhibitor. Overall, this work sheds more light on ecdysone uptake mechanisms across insect species and broadens our knowledge of the physiological roles of OATPs in the transportation of endogenous substrates. Author SummaryThe insect steroid hormone ecdysone is critical in regulating many aspects of insects life, including development and reproduction. A passive diffusion model was never functionally resolved, but was strongly supported until an organic anion transporting polypeptide was identified to mediate the transport of the hormone. The OATP74D, belonging to the Solute carrier superfamily, has been identified and functionally characterized for the first time in Drosophila melanogaster. Although phylogenetic analysis suggests that the Drosophila Oatp74D is probably conserved among several insect species, the theory for transporter mediated ecdysone uptake cannot be generalized to all insects without concrete proof. In here we provide functional evidence that the Oatp74D of two lepidopteran pest species: Helicoverpa armigera and Spodoptera frugiperda, is highly required for insect survival and development. Furthermore, we reveal that the OATP74D is necessary to regulate the expression of several ecdysone response genes, including caspase-3 which is involved in programmed cell death. In addition, we have developed a cell-based platform for screening chemical compounds against the lepidopteran orthologs of Oat74D and rifampicin was functionally shown to inhibit ecdysone uptake. Taken all together, our study reveals that Oatp74D is conserved among several arthropod species in the ecdysone pathway and given the high necessity for an effective control of these two lepidopteran species, we hypothesized that OATP74D could serve as a possible drug target in those two species.

genetics↗