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Abstract Uncovering the complexity of systems in non-model organisms is critical for understanding arthropod immunology. Prior efforts have mostly focused on Dipteran insects, which only account for a subset of existing arthropod species in nature. Here we use and develop advanced techniques to describe immune cells (hemocytes) from the clinically relevant tick Ixodes scapularis at a single-cell resolution. We observe molecular alterations in hemocytes upon feeding and infection with either the Lyme disease spirochete Borrelia burgdorferi or the rickettsial agent Anaplasma phagocytophilum. We reveal hemocyte clusters exhibiting defined signatures related to immunity, metabolism, and proliferation. Depletion of phagocytic hemocytes affects hemocytin and astakine levels, two I. scapularis hemocyte markers, impacting blood-feeding, molting behavior, and bacterial acquisition. Mechanistically, astakine alters hemocyte proliferation, whereas hemocytin affects the c-Jun N-terminal kinase (JNK) signaling pathway in I. scapularis. Altogether, we discover a role for tick hemocytes in immunophysiology and provide a valuable resource for comparative biology in arthropods.

Forward genetic approaches such as CRISPR screens are powerful ways to identify essential genes and those that influence host-pathogen interactions. Here the authors design a bioinformatics portal for sgRNA design and a recombination-mediated cassette system for delivery into mosquito cell lines.

Culex mosquitoes are a global vector for insect-borne diseases, though progress with genetic tools lags behind other mosquito species. Here the authors present a Cas9-based toolkit and methods that could support future gene drive development in these mosquitoes.

Dengue fever (DF), caused by the dengue virus (DENV), is the most burdensome arboviral disease in the world, with an estimated 400 million infections each year. The Aedes aegypti mosquito is the main vector of DENV and transmits several other human pathogens, including Zika, yellow fever, and chikungunya viruses. Previous studies have shown that the pathogen infection of mosquitoes can alter reproductive fitness, revealing specific vector-pathogen interactions that are key determinants of vector competence. However, only a handful of studies have examined the effect of DENV infection in A. aegypti, showing a reduction in lifespan and fecundity over multiple blood meals. To provide a more comprehensive analysis of the impact of DENV infection on egg laying and fecundity, we assessed egg laying timing in DENV-2 blood-fed mosquitoes (infected group) compared to mock blood-fed mosquitoes (control group). We confirmed a significant decrease in fecundity during the first gonadotrophic cycle. To further investigate this phenotype and the underlying DENV-2 infection-dependent changes in gene expression, we conducted a transcriptomic analysis for differentially expressed genes in the ovaries of A. aegypti infected with DENV-2 vs. mock-infected mosquitoes. This analysis reveals several DENV-2-regulated genes; among them, we identified a group of 12 metabolic genes that we validated using reverse transcription-quantitative PCR (RT-qPCR). Interestingly, two genes found to be upregulated in DENV-infected mosquito ovaries exhibited an antiviral role for DENV-2 in an Aedes cell line. Altogether, this study offers useful insights into the virus-vector interface, highlighting the importance of gene expression changes in the mosquito’s ovary during DENV-2 infection in the first gonadotrophic cycle, triggering antiviral responses that may possibly interfere with mosquito reproduction. This information is extremely relevant for further investigation of A. aegypti’s ability to tolerate viruses since virally infected mosquitoes in nature constitute a powerful source of supporting viruses during intra-epidemic periods, causing a huge burden on the public health system.

Abstract Anopheles gambiae mosquitoes are the most important vectors of human malaria. The reproductive success of these mosquitoes relies on a single copulation event after which the majority of females become permanently refractory to further mating. This refractory behavior is at least partially mediated by the male-synthetized steroid hormone 20-hydroxyecdysone (20E), which is packaged together with other seminal secretions into a gelatinous mating plug and transferred to the female atrium during mating. In this study, we show that two 20E-regulated chymotrypsin-like serine proteases specifically expressed in the reproductive tract of An. gambiae females play an important role in modulating the female susceptibility to mating. Silencing these proteases by RNA interference impairs correct plug processing and slows down the release of the steroid hormone 20E from the mating plug. In turn, depleting one of these proteases, the Mating Regulated Atrial Protease 1 (MatRAP1), reduces female refractoriness to further copulation, so that a significant proportion of females mate again. Microscopy analysis reveals that MatRAP1 is localized on a previously undetected peritrophic matrix-like structure surrounding the mating plug. These data provide novel insight into the molecular mechanisms shaping the post-mating biology of these important malaria vectors.

Abstract Background Malaria parasites, transmitted by the bite of an anopheline mosquito, pose an immense public health burden on many tropical and subtropical regions. The most important malaria vectors in sub-Saharan Africa are mosquitoes of the Anopheles gambiae complex including An. gambiae (sensu stricto). Given the increasing rates of insecticide resistance in these mosquitoes, alternative control strategies based on the release of genetically modified males are being evaluated to stop transmission by these disease vectors. These strategies rely on the mating competitiveness of release males, however currently there is no method to determine male mating success without sacrificing the female. Interestingly, unlike other insects, during mating An. gambiae males transfer their male accessory glands (MAGs) seminal secretions as a coagulated mating plug which is deposited in the female atrium. Results Here we exploit this male reproductive feature and validate the use of a MAG-specific promoter to fluorescently label the mating plug and visualize the occurrence of insemination in vivo. We used the promoter region of the major mating plug protein, Plugin, to control the expression of a Plugin-tdTomato (PluTo) fusion protein, hypothesizing that this fusion protein could be incorporated into the plug for sexual transfer to the female. Anopheles gambiae PluTo transgenic males showed strong red fluorescence specifically in the MAGs and with a pattern closely matching endogenous Plugin expression. Moreover, the fusion protein was integrated into the mating plug and transferred to the female atrium during mating where it could be visualized microscopically in vivo without sacrificing the female. PluTo males were equally as competitive at mating as wild type males, and females mated to these males did not show any reduction in reproductive fitness. Conclusion The validation of the first MAG-specific promoter in transgenic An. gambiae facilitates the live detection of successful insemination hours after copulation has occurred. This provides a valuable tool for the assessment of male mating competitiveness not only in laboratory experiments but also in semi-field and field studies aimed at testing the feasibility of releasing genetically modified mosquitoes for disease control.