The ASH1-PEX16 regulatory pathway controls peroxisome biogenesis for appressorium-mediated insect infection by a fungal pathogen.

Entomopathogenic fungi infect insects by penetrating through the cuticle into the host body. To breach the host cuticle, some fungal pathogens produce specialized infection cells called appressoria, which develop enormous turgor pressure to allow cuticle penetration. However, regulatory mechanisms underlying appressorium turgor generation are poorly understood. Here, we show that the histone lysine methyltransferase ASH1 in the insecticidal fungus Metarhizium robertsii, which is strongly induced during infection of the mosquito cuticle, regulates appressorium turgor generation and cuticle penetration by activating the peroxin gene Mrpex16 via H3K36 dimethylation. MrPEX16 is required for the biogenesis of peroxisomes that participate in lipid catabolism and further promotes the hydrolysis of triacylglycerols stored in lipid droplets to produce glycerol for turgor generation, facilitating appressorium-mediated insect infection. Together, the ASH1-PEX16 pathway plays a pivotal role in regulating peroxisome biogenesis to promote lipolysis for appressorium turgor generation, providing insights into the molecular mechanisms underlying fungal pathogenesis.

Sialic acids as attachment factors in mosquitoes mediating Japanese encephalitis virus infection.

The role of Culex mosquitoes in the transmission of Japanese encephalitis virus (JEV) is crucial, yet the mechanisms of JEV infection in these vectors remain unclear. Previous research has indicated that various host factors participate in JEV infection. Herein, we present evidence that mosquito sialic acids enhance JEV infection both in vivo and in vitro. By treating mosquitoes and C6/36 cells with neuraminidase or lectin, the function of sialic acids is effectively blocked, resulting in significant inhibition of JEV infection. Furthermore, knockdown of the sialic acid biosynthesis genes in Culex mosquitoes also leads to a reduction in JEV infection. Moreover, our research revealed that sialic acids play a role in the attachment of JEV to mosquito cells, but not in its internalization. To further explore the mechanisms underlying the promotion of JEV attachment by sialic acids, we conducted immunoprecipitation experiments to confirm the direct binding of sialic acids to the last α-helix in JEV envelope protein domain III. Overall, our study contributes to a molecular comprehension of the interaction between mosquitoes and JEV and offers potential strategies for preventing the dissemination of flavivirus in natural environments.IMPORTANCEIn this study, we aimed to investigate the impact of glycoconjugate sialic acids on mosquito infection with Japanese encephalitis virus (JEV). Our findings demonstrate that sialic acids play a crucial role in enhancing JEV infection by facilitating the attachment of the virus to the cell membrane. Furthermore, our investigation revealed that sialic acids directly bind to the final α-helix in the JEV envelope protein domain III, thereby accelerating virus adsorption. Collectively, our results highlight the significance of mosquito sialic acids in JEV infection within vectors, contributing to a better understanding of the interaction between mosquitoes and JEV.

Mosquito saliva enhances virus infection through sialokinin-dependent vascular leakage.

Viruses transmitted by Aedes mosquitoes are an increasingly important global cause of disease. Defining common determinants of host susceptibility to this large group of heterogenous pathogens is key for informing the rational design of panviral medicines. Infection of the vertebrate host with these viruses is enhanced by mosquito saliva, a complex mixture of salivary-gland-derived factors and microbiota. We show that the enhancement of infection by saliva was dependent on vascular function and was independent of most antisaliva immune responses, including salivary microbiota. Instead, the Aedes gene product sialokinin mediated the enhancement of virus infection through a rapid reduction in endothelial barrier integrity. Sialokinin is unique within the insect world as having a vertebrate-like tachykinin sequence and is absent from Anopheles mosquitoes, which are incompetent for most arthropod-borne viruses, whose saliva was not proviral and did not induce similar vascular permeability. Therapeutic strategies targeting sialokinin have the potential to limit disease severity following infection with Aedes-mosquito-borne viruses.

Neofunctionalization driven by positive selection led to the retention of the loqs2 gene encoding an Aedes specific dsRNA binding protein.

BACKGROUND: Mosquito borne viruses, such as dengue, Zika, yellow fever and Chikungunya, cause millions of infections every year. These viruses are mostly transmitted by two urban-adapted mosquito species, Aedes aegypti and Aedes albopictus. Although mechanistic understanding remains largely unknown, Aedes mosquitoes may have unique adaptations that lower the impact of viral infection. Recently, we reported the identification of an Aedes specific double-stranded RNA binding protein (dsRBP), named Loqs2, that is involved in the control of infection by dengue and Zika viruses in mosquitoes. Preliminary analyses suggested that the loqs2 gene is a paralog of loquacious (loqs) and r2d2, two co-factors of the RNA interference (RNAi) pathway, a major antiviral mechanism in insects. RESULTS: Here we analyzed the origin and evolution of loqs2. Our data suggest that loqs2 originated from two independent duplications of the first double-stranded RNA binding domain of loqs that occurred before the origin of the Aedes Stegomyia subgenus, around 31 million years ago. We show that the loqs2 gene is evolving under relaxed purifying selection at a faster pace than loqs, with evidence of neofunctionalization driven by positive selection. Accordingly, we observed that Loqs2 is localized mainly in the nucleus, different from R2D2 and both isoforms of Loqs that are cytoplasmic. In contrast to r2d2 and loqs, loqs2 expression is stage- and tissue-specific, restricted mostly to reproductive tissues in adult Ae. aegypti and Ae. albopictus. Transgenic mosquitoes engineered to express loqs2 ubiquitously undergo developmental arrest at larval stages that correlates with massive dysregulation of gene expression without major effects on microRNAs or other endogenous small RNAs, classically associated with RNA interference. CONCLUSIONS: Our results uncover the peculiar origin and neofunctionalization of loqs2 driven by positive selection. This study shows an example of unique adaptations in Aedes mosquitoes that could ultimately help explain their effectiveness as virus vectors.

Effects of circadian clock disruption on gene expression and biological processes in Aedes aegypti.

BACKGROUND: This study explores the impact of disrupting the circadian clock through a Cycle gene knockout (KO) on the transcriptome of Aedes aegypti mosquitoes. The investigation aims to uncover the resulting alterations in gene expression patterns and physiological processes. RESULTS: Transcriptome analysis was conducted on Cyc knockout (AeCyc-/-) and wild-type mosquitoes at four time points in a light-dark cycle. The study identified system-driven genes that exhibit rhythmic expression independently of the core clock machinery. Cyc disruption led to altered expression of essential clock genes, affecting metabolic processes, signaling pathways, stimulus responses and immune responses. Notably, gene ontology enrichment of odorant binding proteins, indicating the clock's role in sensory perception. The absence of Cyc also impacted various regulation of metabolic and cell cycle processes was observed in all time points. CONCLUSIONS: The intricate circadian regulation in Ae. aegypti encompasses both core clock-driven and system-driven genes. The KO of Cyc gene instigated extensive gene expression changes, impacting various processes, thereby potentially affecting cellular and metabolic functions, immune responses, and sensory perception. The circadian clock's multifaceted involvement in diverse biological processes, along with its role in the mosquito's daily rhythms, forms a nexus that influences the vector's capacity to transmit diseases. These insights shed light on the circadian clock's role in shaping mosquito biology and behavior, opening new avenues for innovative disease control strategies.

CRISPR-Cas9 and Cas12a target site richness reflects genomic diversity in natural populations of Anopheles gambiae and Aedes aegypti mosquitoes.

Due to limitations in conventional disease vector control strategies including the rise of insecticide resistance in natural populations of mosquitoes, genetic control strategies using CRISPR gene drive systems have been under serious consideration. The identification of CRISPR target sites in mosquito populations is a key aspect for developing efficient genetic vector control strategies. While genome-wide Cas9 target sites have been explored in mosquitoes, a precise evaluation of target sites focused on coding sequence (CDS) is lacking. Additionally, target site polymorphisms have not been characterized for other nucleases such as Cas12a, which require a different DNA recognition site (PAM) and would expand the accessibility of mosquito genomes for genetic engineering. We undertook a comprehensive analysis of potential target sites for both Cas9 and Cas12a nucleases within the genomes of natural populations of Anopheles gambiae and Aedes aegypti from multiple continents. We demonstrate that using two nucleases increases the number of targets per gene. Also, we identified differences in nucleotide diversity between North American and African Aedes populations, impacting the abundance of good target sites with a minimal degree of polymorphisms that can affect the binding of gRNA. Lastly, we screened for gRNAs targeting sex-determination genes that could be widely applicable for developing field genetic control strategies. Overall, this work highlights the utility of employing both Cas9 and Cas12a nucleases and underscores the importance of designing universal genetic strategies adaptable to diverse mosquito populations.

Molecular Confirmation of Anopheles stephensi Mosquitoes in the Al Hudaydah Governorate, Yemen, 2021 and 2022.

We detected malaria vector Anopheles stephensi mosquitoes in the Al Hudaydah governorate in Yemen by using DNA sequencing. We report 2 cytochrome c oxidase subunit I haplotypes, 1 previously found in Ethiopia, Somalia, Djibouti, and Yemen. These findings provide insight into invasive An. stephensi mosquitoes in Yemen and their connection to East Africa.

Loop-Mediated Isothermal Amplification Assay to Detect Invasive Malaria Vector Anopheles stephensi Mosquitoes.

Spread of the Anopheles stephensi mosquito, an invasive malaria vector, threatens to put an additional 126 million persons per year in Africa at risk for malaria. To accelerate the early detection and rapid response to this mosquito species, confirming its presence and geographic extent is critical. However, existing molecular species assays require specialized laboratory equipment, interpretation, and sequencing confirmation. We developed and optimized a colorimetric rapid loop-mediated isothermal amplification assay for molecular An. stephensi species identification. The assay requires only a heat source and reagents and can be used with or without DNA extraction, resulting in positive color change in 30-35 minutes. We validated the assay against existing PCR techniques and found 100% specificity and analytical sensitivity down to 0.0003 ng of genomic DNA. The assay can successfully amplify single mosquito legs. Initial testing on samples from Marsabit, Kenya, illustrate its potential as an early vector detection and malaria mitigation tool.

Delayed Plasmodium falciparum Malaria in Pregnant Patient with Sickle Cell Trait 11 Years after Exposure, Oregon, USA.

Delayed Plasmodium falciparum malaria in immigrants from disease-endemic countries is rare. Such cases pose a challenge for public health because mosquitoborne transmission must be rigorously investigated. We report a case of delayed P. falciparum malaria in a pregnant woman with sickle cell trait 11 years after immigration to the United States.

Plasmodium vivax Infections among Immigrants from China Traveling to the United States.

Beginning in 2023, we observed increased Plasmodium vivax malaria cases at an institution in Los Angeles, California, USA. Most cases were among migrants from China who traveled to the United States through South and Central America. US clinicians should be aware of possible P. vivax malaria among immigrants from China.

Potential Performance of Two New RT-PCR and RT-qPCR Methods for Multiplex Detection of Dengue Virus Serotypes 1-4 and Chikungunya Virus in Mosquitoes.

Mosquitoes of the genus Aedes are the most important arthropod disease vector. Dengue virus (DENV) and Chikungunya virus (CHIKV) are the main arboviruses distributed throughout the world. Based on entomo-virological surveillance, appropriate public health strategies can be adopted to contain cases and control outbreaks. This study aims to show the potential performance of two new molecular methods for detecting DENV serotypes and CHIKV in mosquitoes. Mosquitoes were collected in urban and sylvatic areas of Bobo-Dioulasso, Burkina Faso, between July and August 2023. DENV and CHIKV were screened using new multiplex RT-PCR and RT-qPCR methods. A total of 2150 mosquitoes were trapped, consisting of 976 Aedes (959 Ae. aegypti, 6 Ae. furcifer, and 11 Ae. vittatus) and 1174 Culex sp. These were grouped into 39 pools, with each pool containing a maximum of 30 mosquitoes. Molecular screening revealed that 7.7% (3/39) of the pools were positive for DENV. Specifically, DENV-1 was detected in one pool (1/3), and DENV-3 was found in two pools (2/3). All pools tested negative for CHIKV. The overall minimum infection rate (MIR) of DENV in this study was 3.07 (95% CI: 2.24-19.86). This study shows the usefulness of our new molecular tools for the surveillance of DENV serotypes and CHIKV.

Supreme glutathione-dependent ketosteroid isomerase in the yellow-fever transmitting mosquito Aedes aegypti.

The steroid hormone ecdysone is essential for the reproduction and survival of insects. The hormone is synthesized from dietary sterols such as cholesterol, yielding ecdysone in a series of consecutive enzymatic reactions. In the insect orders Lepidoptera and Diptera a glutathione transferase called Noppera-bo (Nobo) plays an essential, but biochemically uncharacterized, role in ecdysteroid biosynthesis. The Nobo enzyme is consequently a possible target in harmful dipterans, such as disease-carrying mosquitoes. Flavonoid compounds inhibit Nobo and have larvicidal effects in the yellow-fever transmitting mosquito Aedes aegypti, but the enzyme is functionally incompletely characterized. We here report that within a set of glutathione transferase substrates the double-bond isomerase activity with 5-androsten-3,17-dione stands out with an extraordinary specific activity of 4000 µmol min-1 mg-1. We suggest that the authentic function of Nobo is catalysis of a chemically analogous ketosteroid isomerization in ecdysone biosynthesis.

Nested patterns of commensals and endosymbionts in microbial communities of mosquito vectors.

BACKGROUND: Mosquitoes serve as vectors for numerous pathogens, posing significant health risks to humans and animals. Understanding the complex interactions within mosquito microbiota is crucial for deciphering vector-pathogen dynamics and developing effective disease management strategies. Here, we investigated the nested patterns of Wolbachia endosymbionts and Escherichia-Shigella within the microbiota of laboratory-reared Culex pipiens f. molestus and Culex quinquefasciatus mosquitoes. We hypothesized that Wolbachia would exhibit a structured pattern reflective of its co-evolved relationship with both mosquito species, while Escherichia-Shigella would display a more dynamic pattern influenced by environmental factors. RESULTS: Our analysis revealed different microbial compositions between the two mosquito species, although some microorganisms were common to both. Network analysis revealed distinct community structures and interaction patterns for these bacteria in the microbiota of each mosquito species. Escherichia-Shigella appeared prominently within major network modules in both mosquito species, particularly in module P4 of Cx. pipiens f. molestus, interacting with 93 nodes, and in module Q3 of Cx. quinquefasciatus, interacting with 161 nodes, sharing 55 nodes across both species. On the other hand, Wolbachia appeared in disparate modules: module P3 in Cx. pipiens f. molestus and a distinct module with a single additional taxon in Cx. quinquefasciatus, showing species-specific interactions and no shared taxa. Through computer simulations, we evaluated how the removal of Wolbachia or Escherichia-Shigella affects network robustness. In Cx. pipiens f. molestus, removal of Wolbachia led to a decrease in network connectivity, while Escherichia-Shigella removal had a minimal impact. Conversely, in Cx. quinquefasciatus, removal of Escherichia-Shigella resulted in decreased network stability, whereas Wolbachia removal had minimal effect. CONCLUSIONS: Contrary to our hypothesis, the findings indicate that Wolbachia displays a more dynamic pattern of associations within the microbiota of Culex pipiens f. molestus and Culex quinquefasciatus mosquitoes, than Escherichia-Shigella. The differential effects on network robustness upon Wolbachia or Escherichia-Shigella removal suggest that these bacteria play distinct roles in maintaining community stability within the microbiota of the two mosquito species.

Obesity's Unexpected Influence: Reduced Alphavirus Transmission and Altered Immune Activation in the Vector.

Chikungunya virus (CHIKV) and Mayaro virus (MAYV) are emerging/re-emerging alphaviruses transmitted by Aedes spp. mosquitoes and responsible for recent disease outbreaks in the Americas. The capacity of these viruses to cause epidemics is frequently associated with increased mosquito transmission, which in turn is governed by virus-host-vector interactions. Although many studies have explored virus-vector interactions, significant gaps remain in understanding how vertebrate host factors influence alphavirus transmission by mosquitoes. We previously showed that obesity, a ubiquitous vertebrate host biological factor, reduces alphavirus transmission potential in mosquitoes. We hypothesized that alphavirus-infected obese bloodmeals altered immune genes and/or pathways in mosquitoes, thereby inhibiting virus transmission. To test this, we conducted RNA sequencing (RNA-seq) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) on midgut RNA from mosquitoes fed on alphavirus-infected lean and obese mice. This approach aimed to identify potential antiviral or proviral genes and pathways altered in mosquitoes after consuming infected obese bloodmeals. We found upregulation of the Toll pathway and downregulation of several metabolic and other genes in mosquitoes fed on alphavirus-infected obese bloodmeals. Through gene knockdown studies, we demonstrated the antiviral role of Toll pathway and proviral roles of AAEL009965 and fatty acid synthase (FASN) in the transmission of alphaviruses by mosquitoes. Therefore, this study utilized obesity to identify factors influencing alphavirus transmission by mosquitoes and this research approach may pave the way for designing broadly effective antiviral measures to combat mosquito-borne viruses, such as releasing transgenic mosquitoes deficient in the identified genes.

Tyrosine transfer RNA levels and modifications during blood-feeding and vitellogenesis in the mosquito, Aedes aegypti.

Mosquitoes such as Aedes aegypti must consume a blood meal for the nutrients necessary for egg production. Several transcriptome and proteome changes occur post-blood meal that likely corresponds with codon usage alterations. Transfer RNA (tRNA) is the adapter molecule that reads messenger RNA codons to add the appropriate amino acid during protein synthesis. Chemical modifications to tRNA enhance codon decoding, improving the accuracy and efficiency of protein synthesis. Here, we examined tRNA modifications and transcripts associated with the blood meal and subsequent periods of vitellogenesis in A. aegypti. More specifically, we assessed tRNA transcript abundance and modification levels in the fat body at critical times post blood-feeding. Based on a combination of alternative codon usage and identification of particular modifications, we discovered that increased transcription of tyrosine tRNAs is likely critical during the synthesis of egg yolk proteins in the fat body following a blood meal. Altogether, changes in both the abundance and modification of tRNA are essential factors in the process of vitellogenin production after blood-feeding in mosquitoes.

Mosquito pollination of plants: an overview of their role and an assessment of the possible contribution of disease vectors.

Mosquitoes visit flowers to obtain sugar or other nutrients and therefore possibly serve as major or minor pollinators of some plant species. They also often derive plant nutrients from other sources, such as extrafloral nectaries and honeydew. In a few cases, the plant-mosquito relationship is close, and mosquito pollination has been confirmed. Most plant species visited by mosquitoes, however, appear to depend on multiple means of pollination, particularly other flower-feeding insects. In addition, most mosquito species visit the flowers of many kinds of plants, possibly dispersing pollen in both biologically meaningful and irrelevant ways. This apparent lack of selectivity by both plants and mosquitoes liberates each of them from dependence on an unreliable pollen vehicle or nutrient source. A hypothetical pollinating role for the two top vectors of devastating human-disease pathogens, Anopheles gambiae or Aedes aegypti, relies on indirect evidence. So far, this evidence suggests that their participation in pollen transfer of native, introduced, or beneficial plants is negligible. The few plant species likely to be pollinated by these vectors are mostly invasive, harmful weeds associated with humans. That conclusion draws support from four characteristics of these vectors: (1) the numerous alternative potential pollinators of the flowers they visit; (2) their common use of diverse non-floral sources of nutrients; (3) the females' infrequent sugar feeding and heavy reliance on human blood for energy; and (4) their relatively low population densities. From these traits it follows that focused suppression or elimination of these two vectors, by whatever means, is highly unlikely to have adverse effects on pollination in endemic biotic communities or on ornamental plants or food crops.

Detecting the impacts of humidity, rainfall, temperature, and season on chikungunya, dengue and Zika viruses in Aedes albopictus mosquitoes from selected sites in Cebu city, Philippines.

BACKGROUND: Aedes albopictus is the secondary vector for dengue virus (DENV) in the Philippines, and also harbors chikungunya (CHIKV) and Zika (ZIKV) viruses. This study aimed to determine the minimum infection rates (MIRs) of CHIKV, DENV serotypes, and ZIKV in Ae. albopictus collected from selected two-site categories by altitude (highland [H] and lowland [L] sites) in Cebu city, Philippines during the wet (WS) and dry seasons (DS) of 2021-2022, and to explore the relationships between these arboviral MIRs and the local weather. METHODS: The viral RNA extracts in pooled and reared adult Ae. albopictus collected during the DS and WS from two-site categories were subjected to RT-PCR to amplify and detect gene loci specific for CHIKV, DENV-1 to DENV-4, and ZIKV and analyzed with the weather data. RESULTS: The range of CHIKV MIRs was higher in the WS (13.61-107.38 infected individuals per 1,000 mosquitoes) than in the DS (13.22-44.12), but was similar between the two-site categories. Rainfall (RF) influenced the CHIKV MIR. The MIR ranges of both DENV-2 (WS: H = 0, L = 0; DS: H = 0-5.92; L = 0-2.6) and DENV-4 (WS: H = 0, L = 0-2.90; DS: H = 2.96-6.13, L = 0-15.63) differed by season but not between the two-site categories. Relative humidity (RH), RF, and temperature did not influence DENVs' MIRs. The MIR range of ZIKV was similar in both seasons (WS: 11.36-40.27; DS: 0-46.15) and two-site categories (H = 0-90.91, L = 0-55.56). RH and temperature influenced ZIKV MIR. CONCLUSIONS: RF influenced CHIKV MIR in Ae. albopictus, whereas RH and temperature influenced that of ZIKV. Season influenced the MIRs of CHIKV and DENVs but not in ZIKV. Ae. albopictus were co-infected with CHIKV, DENVs, and ZIKV in both highland and lowland sites in Cebu city. Recommendations include all-year-round implementation of the Philippine Department of Health's  4S enhanced strategy and installation of water pipelines in rural highlands for vector and disease control. Our findings are relevant to protect public health in the tropics in this climate change.

Immunomodulation in dengue: towards deciphering dengue severity markers.

BACKGROUND: Dengue is a vector-borne debilitating disease that is manifested as mild dengue fever, dengue with warning signs, and severe dengue. Dengue infection provokes a collective immune response; in particular, the innate immune response plays a key role in primary infection and adaptive immunity during secondary infection. In this review, we comprehensively walk through the various markers of immune response against dengue pathogenesis and outcome. MAIN BODY: Innate immune response against dengue involves a collective response through the expression of proinflammatory cytokines, such as tumor necrosis factors (TNFs), interferons (IFNs), and interleukins (ILs), in addition to anti-inflammatory cytokines and toll-like receptors (TLRs) in modulating viral pathogenesis. Monocytes, dendritic cells (DCs), and mast cells are the primary innate immune cells initially infected by DENV. Such immune cells modulate the expression of various markers, which can influence disease severity by aiding virus entry and proinflammatory responses. Adaptive immune response is mainly aided by B and T lymphocytes, which stimulate the formation of germinal centers for plasmablast development and antibody production. Such antibodies are serotype-dependent and can aid in virus entry during secondary infection, mediated through a different serotype, such as in antibody-dependent enhancement (ADE), leading to DENV severity. The entire immunological repertoire is exhibited differently depending on the immune status of the individual. SHORT CONCLUSION: Dengue fever through severe dengue proceeds along with the modulated expression of several immune markers. In particular, TLR2, TNF-α, IFN-I, IL-6, IL-8, IL-17 and IL-10, in addition to intermediate monocytes (CD14+CD16+) and Th17 (CD4+IL-17+) cells are highly expressed during severe dengue. Such markers could assist greatly in severity assessment, prompt diagnosis, and treatment.

Tips and tools to obtain and assess mosquito viromes.

Due to their vectorial capacity, mosquitoes (Diptera: Culicidae) receive special attention from health authorities and entomologists. These cosmopolitan insects are responsible for the transmission of many viral diseases, such as dengue and yellow fever, causing huge impacts on human health and justifying the intensification of research focused on mosquito-borne diseases. In this context, the study of the virome of mosquitoes can contribute to anticipate the emergence and/or the reemergence of infectious diseases. The assessment of mosquito viromes also contributes to the surveillance of a wide variety of viruses found in these insects, allowing the early detection of pathogens with public health importance. However, the study of mosquito viromes can be challenging due to the number and complexities of steps involved in this type of research. Therefore, this article aims to describe, in a straightforward and simplified way, the steps necessary for obtention and assessment of mosquito viromes. In brief, this article explores: the capture and preservation of specimens; sampling strategies; treatment of samples before DNA/RNA extraction; extraction methodologies; enrichment and purification processes; sequencing choices; and bioinformatics analysis.

Senescence of humoral antimicrobial immunity occurs in infected mosquitoes when the temperature is higher.

Mosquitoes cannot use metabolism to regulate their body temperature, and therefore, climate warming is altering their physiology. Mosquitoes also experience a physiological decline with aging, a phenomenon called senescence. Because both high temperature and aging are detrimental to mosquitoes, we hypothesized that high temperatures accelerate senescence. Here, we investigated how temperature and aging, independently and interactively, shape the antimicrobial immune response of the mosquito, Anopheles gambiae. Using a zone-of-inhibition assay that measures the antimicrobial activity of hemolymph, we found that antimicrobial activity increases following infection. Moreover, in infected mosquitoes, antimicrobial activity weakens as the temperature rises to 32C, and antimicrobial activity increases from 1 to 5 days of age and stabilizes with further aging. Importantly, in E. coli-infected mosquitoes, higher temperature causes an aging-dependent decline in antimicrobial activity. Altogether, this study demonstrates that higher temperature can accelerate immune senescence in infected mosquitoes, thereby interactively shaping their ability to fight an infection.