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Sustainable reductions in African malaria transmission require innovative tools for mosquito control. One proposal involves the use of low-threshold gene drive in Anopheles vector species, where a 'causal pathway' would be initiated by (i) the release of a gene drive system in target mosquito vector species, leading to (ii) its transmission to subsequent generations, (iii) its increase in frequency and spread in target mosquito populations, (iv) its simultaneous propagation of a linked genetic trait aimed at reducing vectorial capacity for Plasmodium, and (v) reduced vectorial capacity for parasites in target mosquito populations as the gene drive system reaches fixation in target mosquito populations, causing (vi) decreased malaria incidence and prevalence. Here the scope, objectives, trial design elements, and approaches to monitoring for initial field releases of such gene dive systems are considered, informed by the successful implementation of field trials of biological control agents, as well as other vector control tools, including insecticides, Wolbachia, larvicides, and attractive-toxic sugar bait systems. Specific research questions to be addressed in initial gene drive field trials are identified, and adaptive trial design is explored as a potentially constructive and flexible approach to facilitate testing of the causal pathway. A fundamental question for decision-makers for the first field trials will be whether there should be a selective focus on earlier points of the pathway, such as genetic efficacy via measurement of the increase in frequency and spread of the gene drive system in target populations, or on wider interrogation of the entire pathway including entomological and epidemiological efficacy. How and when epidemiological efficacy will eventually be assessed will be an essential consideration before decisions on any field trial protocols are finalized and implemented, regardless of whether initial field trials focus exclusively on the measurement of genetic efficacy, or on broader aspects of the causal pathway. Statistical and modelling tools are currently under active development and will inform such decisions on initial trial design, locations, and endpoints. Collectively, the considerations here advance the realization of developer ambitions for the first field trials of low-threshold gene drive for malaria vector control within the next 5 years.

The mosquito Aedes aegypti is an important vector of diseases including dengue, Zika, chikungunya, and yellow fever. Olfaction is a critical modality for mosquitoes enabling them to locate hosts, sources of nectar, and sites for oviposition. GABA is an essential neurotransmitter in olfactory processing in the insect brain, including the primary olfactory center, the antennal lobe. Previous work with Ae. aegypti has suggested that antennal lobe inhibition via GABA may be involved in the processing of odors. However, little is known about GABA receptor expression in the mosquito brain, or how they may be involved in odor attraction. In this context, generating mutants that target the mosquitos olfactory responses, and particularly the GABAergic system, is essential to achieve a better understanding of these diverse processes and olfactory coding in these disease vectors. Here we demonstrate the potential of a transgenic line using the QF2 transcription factor, GABA-B1QF2-ECFP, as a new neurogenetic tool to investigate the neural basis of olfaction in Ae. aegypti. Our results show that the gene insertion has a moderate impact on mosquito fitness. Moreover, the line presented here was crossed with a QUAS reporter line expressing the green fluorescent protein and used to determine the location of the metabotropic GABA-B1 receptor expression. We find high receptor expression in the antennal lobes, especially the cell bodies surrounding the antennal lobes. In the mushroom bodies, receptor expression was high in the Kenyon cells, but had low expression in the mushroom body lobes. Behavioral experiments testing the fruit odor attractants showed that the mutants lost their behavioral attraction. Together, these results show that the GABA-B1QF2-ECFP line provides a new tool to characterize GABAergic systems in the mosquito nervous system.

Mosquitoes transmit pathogens that pose a threat to millions of people globally. Unfortunately, widespread insecticide resistance makes it difficult to control these public health pests. General mechanisms of resistance, such as target site mutations or increased metabolic activity, are well established. However, many questions regarding the dynamics of these adaptations in the context of developmental and environmental conditions require additional exploration. One aspect of resistance that deserves further study is the role of heat shock proteins (HSPs) in insecticide tolerance. Studies show that mosquitoes experiencing heat stress before insecticide exposure demonstrate decreased mortality. This is similar to the observed reciprocal reduction in mortality in mosquitoes exposed to insecticide prior to heat stress. The environmental shifts associated with climate change will result in mosquitoes occupying environments with higher ambient temperatures, which could enhance existing insecticide resistance phenotypes. This physiological relationship adds a new dimension to the problem of insecticide resistance and further complicates the challenges that vector control and public health personnel face. This article reviews studies illustrating the relationship between insecticide resistance and HSPs or hsp genes as well as the intersection of thermotolerance and insecticide resistance. Further study of HSPs and insecticide resistance could lead to a deeper understanding of how environmental factors modulate the physiology of these important disease vectors to prepare for changing climatic conditions and the development of novel strategies to prevent vector-borne disease transmission.

Though the circumsporozoite protein (CSP) is the major surface protein of the Plasmodium sporozoite, and the basis for the currently used malaria vaccines, many questions regarding its function and structure remain open. CSP has a conserved domain structure among all Plasmodium species and is important for sporozoite formation, migration, and invasion, with no clear binding partners known. Proteolytic processing of CSP is essential for its function as are dynamic changes to its conformation, but only one domain has so far been crystallized and the enzymes processing CSP are unknown. The circumsporozoite protein (CSP) is one of the most studied proteins of the malaria parasite. It is the target of the only licensed malaria vaccines and is essential for sporozoite formation and infectivity. Yet, the mechanisms by which CSP functions and its interactions with other proteins are only beginning to be understood. Here we review the current state of knowledge of CSP structure and function, as sporozoites develop in the mosquito and establish infection in the mammalian host, and outline outstanding questions that need to be addressed. [ABSTRACT FROM AUTHOR]

Background: Gene drive-modified mosquitoes (GDMMs) have been promoted as one of the innovative technologies that may control and eliminate malaria and other mosquito-borne diseases. Several products are in early stages of development, targeting either population suppression or population modification of the mosquito vector. However, there is no direct experience of conducting risk assessment for environmental releases and subsequent policies regarding conditions for post-release. This study was carried out to gain additional insights on the possible post-release concerns that may arise, as they may inform future risk assessment and planning for deployment. Methods: This study involved desktop reviews on post release monitoring experiences with previously released biological control products. Stakeholder consultations involving online surveys, and face to face workshop with experts from selected African countries from Eastern, Western, and Southern African regions was then carried out to establish post-release monitoring concerns for GDMMs. Results: Review of genetic biocontrol technologies showed only limited lessons from post-release monitoring regimes with a focus largely limited to efficacy. For genetically modified organisms general surveillance and case-specific monitoring is expected in some of the regions. A number of post-release monitoring concerns in relation to the protection goals of human and animal health, biodiversity, and water quality were identified. Conclusion: Based on established- protection goals, several post-release monitoring concerns have been identified. Subject to a rigorous risk assessment process for future GDMMs products, the concerns may then be prioritized for post-release monitoring. [ABSTRACT FROM AUTHOR]

Background: Dengue is an emerging disease in Argentina due to the colonization of Aedes aegypti, the mosquito vector. Buenos Aires Province is the biggest and most populated district in Argentina, suffering dengue outbreaks of growing magnitude. During epidemic periods, pyrethroid insecticides are used in this country to control adult mosquitoes. Pyrethroid resistance in dengue vectors has been reported worldwide, making it necessary to implement resistance management strategies. The voltage-gated sodium channel is the target site of pyrethroids. Mutations in the gene encoding this protein, called kdr mutations, are usually the molecular cause of pyrethroid resistance in insects. In Ae. aegypti from the Americas, three kdr substitutions were described: V410L, V1016I, and F1534C. The diagnostic of kdr mutations is recommended for the early detection of pyrethroid resistance as well as the consequent planning of evidence-based control policies. Methods: We distributed ovitraps across 16 localities in Buenos Aires Province, collecting 22,123 eggs. A total of 522 mosquitoes were genotyped in positions 1016 and 1534 of voltage-gated channel using multiplex high-resolution melting and/or TaqMan probe methods. A subset of 449 samples was also genotyped by a singleplex high-resolution melting method developed ad hoc and/or Sanger sequencing. Results: We have documented, for the first time to our knowledge in the central region of Argentina, the presence of the 1016Ikdr + 1534Ckdr allele. Additionally, our study reports the first identification of the V410L mutation in central Argentina. These results underscore a growing trend of pyrethroid resistance in Ae. aegypti, fueled by the widespread use of these insecticides. Conclusions: We detected 1016Ikdr + 1534Ckdr and 410Lkdr mutations in central Argentina for the first time and improved the processivity and accuracy of kdr genotyping methods. The results are both a tool for resistance monitoring and a sign of alarm to direct efforts towards finding sustainable methods for vector control to complement or replace pyrethroids. Joint efforts between academia and authorities to develop and implement public policies for vector control are a productive way to transfer scientific results for their application in public health. [ABSTRACT FROM AUTHOR]

Background: The decision to imbibe a blood meal is predominantly dependent on the sensitivity and specificity of haematophagous arthropods to blood-derived adenine nucleotides, in particular adenosine triphosphate (ATP). Despite previous efforts to identify and characterise the specificity and sensitivity to ATP and other adenine nucleotides, as well as the role of other blood-derived phagostimulants across the Culicidae, comparisons across species remain difficult. Methods: The feeding response of the yellow fever mosquito Aedes aegypti and the African malaria vector Anopheles gambiae to adenine nucleotides in the presence of a carbonate buffer was assessed using a membrane feeding assay. The proportion of mosquitoes engorged and the volume imbibed by all mosquitoes was scored visually and spectrophotometrically. In addition, the proportion of prediuresing An. gambiae, as well as the volume engorged and prediuresed, was examined. Results: Aedes aegypti was more sensitive to adenine nucleotides than An. gambiae, but both species maintained specificity to these phagostimulants, demonstrating a dose-dependent bimodal feeding pattern, thereby expanding our understanding of the all-or-none blood-feeding hypothesis. Feeding on the bicarbonate buffer by An. gambiae—but not that of Ae. aegypti—demonstrated a species-specific variation in how blood phagostimulants are encoded. Adenine nucleotides, with and without bovine serum albumin, were observed to dose-dependently regulate the proportion of An. gambiae prediuresing and the volumes prediuresed but not volumes engorged. Conclusions: Taken together, the results of this study expand our understanding of how mosquitoes differentially assess and respond to blood meal constituents, and provide a basis for further physiological and molecular studies. [ABSTRACT FROM AUTHOR]

The diversity and abundance of vectors are essential parameters in the transmission dynamics of West Nile virus (WNV) between its avian reservoirs and clinically susceptible mammalian species. Knowing the determinants of vector abundance could be thus useful in preventing West Nile fever (WNF) cases and associated socio‐economic impact. We designed a survey at the wildlife–livestock interface to test the hypothesis that variations in environmental favourability between anthropized and wild scenarios modulate WNV vector abundance and transmission risk. In a continental Mediterranean region where WNF has recently emerged, we selected nine sampling sites and allocated three areas to every site with a decreasing gradient of wildlife–livestock interaction: A1—a horse farm where interaction is maximal; A2—a zone of intermediate interaction 500–1000 m from the farm; and A3—an entirely wild zone of low interaction 1–5 km from the farm. At a fortnightly frequency, we estimated mosquito abundance at each of the 27 study sites in May–December 2018 and April–July 2019. We estimated bird and mammal abundance, collected meteorological information and characterised mosquito habitat at the site scale. Thereafter, we studied the determinants of Culex spp., Culex pipiens sensu lato (s.l.) Linnaeus, 1758 (Diptera: Culicidae) and Culex theileri  Theobald, 1903 abundance by constructing negative binomial generalised linear mixed models. We identified 20 mosquito species, with a notable predominance of Culex spp. and, particularly, of Cx. pipiens s.l. We found differences in the spatiotemporal distribution of Culex spp. abundance and confirmed our hypothesis by finding important effects of local environmental variations in abundance. The accumulated rainfall in fortnights 4–14 and the mean temperature of the two fortnights before sampling were positively and statistically significantly associated with the abundance of Cx. pipiens s.l. (Z = 13.09, p < 0.001, and Z = 9.91, p < 0. 001, respectively) and Culex spp. (Z = 13.35, p < 0.001, and Z = 6.99, p < 0.001, respectively), while the mean temperature of the two previous fortnights was a positive statistically significant predictor (Z = 14.69, p < 0.001) of the abundance of Cx. theileri. The farm environment was the most conducive predictor to hosting Culex spp. compared with wild settings. Our results indicate that continental Mediterranean environments are favourable for WNV circulation and maintenance, especially the environment of anthropized rural settings such as farms. These results will have an impact on the spatiotemporal risk prediction of WNF emergence in continental Mediterranean environments. [ABSTRACT FROM AUTHOR]

Arboviruses, transmitted by medical arthropods, pose a serious health threat worldwide. During viral infection, Post Translational Modifications (PTMs) are present on both host and viral proteins, regulating multiple processes of the viral lifecycle. In this study, a mammalian E3 ubiquitin ligase WWP2 (WW domain containing E3 ubiquitin ligase 2) is identified, which interacts with the NS1 protein of Zika virus (ZIKV) and mediates K63 and K48 ubiquitination of Lys 265 and Lys 284, respectively. WWP2‐mediated NS1 ubiquitination leads to NS1 degradation via the ubiquitin‐proteasome pathway, thereby inhibiting ZIKV infection in mammalian hosts. Simultaneously, it is found Su(dx), a protein highly homologous to host WWP2 in mosquitoes, is capable of ubiquitinating NS1 in mosquito cells. Unexpectedly, ubiquitination of NS1 in mosquitoes does not lead to NS1 degradation; instead, it promotes viral infection in mosquitoes. Correspondingly, the NS1 K265R mutant virus is less infectious to mosquitoes than the wild‐type (WT) virus. The above results suggest that the ubiquitination of the NS1 protein confers different adaptations of ZIKV to hosts and vectors, and more importantly, this explains why NS1 K265‐type strains have become predominantly endemic in nature. This study highlights the potential application in antiviral drug and vaccine development by targeting viral proteins' PTMs. [ABSTRACT FROM AUTHOR]

AbstractMosquitoes, as major vectors of dangerous diseases, demand effective control measures for public health. Larvicides, while offering a potential solution, can exhibit varying effects on different mosquito species and developmental stages during efficacy studies. Standardizing mosquito developmental stages and ensuring optimal, synchronized development rates of various larval stages are crucial for larvicidal experiments and maximizing pupal production. Manual separation of different stages of larvae is labor intensive, time consuming and error prone. Therefore, this study aimed to investigate the use of a stereolithography 3D-printed device for the separation of mosquito larvae. This device is designed to have three modules to collect different larval stages based on their radial size differences. Each module of the device was equipped with small vibration motors to enable vibration-assisted filtration to prevent clogging. The accuracy of the larvae separation device was studied by repeated experiments and collected samples were analyzed under an optical microscope. The 3D-printed device was demonstrated to be orders of magnitude faster and more accurate compared to manual separation process. A larvicidal bioassay was also designed to investigate larvicidal assay impact on the survival of collected L3 and L4 specimens. Overall, it was shown that the presented device carries an important potential for rapid and accurate separation of different size larvae for larvicidal studies. [ABSTRACT FROM AUTHOR]

Background: Non-inferiority trials are recommended by the World Health Organization (WHO) to demonstrate that health products show comparable efficacy to that of existing standard of care. As part of the WHO Global Malaria Programme (GMP) process of assessment of malaria vector control products, a second-in-class insecticide-treated net (ITN) must be shown to be non-inferior to a first-in-class product based on mosquito mortality. The public health impact of the first-in-class pyrethroid-piperonyl butoxide (PBO) ITN, Olyset® Plus, has been demonstrated in epidemiological trials in areas with insecticide-resistant mosquitoes, but there is a need to determine the efficacy of other pyrethroid-PBO nets to ensure timely market availability of nets in order to increase access to ITNs. The non-inferiority of a deltamethrin-PBO ITN Yorkool® G3 was evaluated entomologically against Olyset® Plus in experimental huts in Tanzania, following WHO guidelines for non-inferiority trials. Methods: The trial of the two pyrethroid-PBO ITNs was conducted in experimental huts in Lupiro, Tanzania, using a randomized 7 × 7 Latin square block design. The study ran for 49 nights in 14 huts assessing the mosquito mortality and blood-feeding of wild, free-flying, pyrethroid-resistant Anopheles arabiensis. Using the non-inferiority approach, the comparative efficacy (primary endpoint was mosquito mortality at 24 h and secondary endpoint was blood-feeding) of unwashed and 20 times field-washed pyrethroid-PBO Yorkool® G3 ITNs, were compared with the first-in-class product Olyset® Plus and against a pyrethroid-only ITN, PermaNet® 2.0 ITNs, as a standard comparator. Results: The experimental hut trial demonstrated non-inferiority and superiority of Yorkool® G3 to Olyset® Plus based on mosquito mortality [51% vs. 39%, OR 1.68 (95% CI 1.50–1.88)], given that lower 95% CI exceeded 0.74 (delta of 39%) and the margin of no difference (1). Blood-feeding inhibition was high for all treated ITNs (> 90%) and Yorkool® G3 was non-inferior to Olyset® Plus [4% vs. 2%, OR 1.81 (95% CI 1.46–2.39)], given that upper 95% CI was less than 4.85 (delta of 4%). The pyrethroid-PBO ITNs were superior to the pyrethroid-only net, PermaNet® 2.0, as determined by both the proportion of mortality and blood-feeding of mosquitoes (p-value < 0.05). Conclusion: Yorkool® G3 ITNs demonstrated non-inferiority to the first-in-class Olyset® Plus and superiority over the standard pyrethroid-only ITN, PermaNet® 2.0 as measured by mortality and blood-feeding inhibition of wild pyrethroid-resistant An. arabiensis mosquitoes. Yorkool® G3 ITNs are potential tools for the control of metabolic insecticide-resistant malaria vectors, and their market availability will contribute to the cost-effective selection of ITNs by malaria control programmes to improve population access to ITNs. [ABSTRACT FROM AUTHOR]

Background: Vector-borne diseases cause morbidity and mortality globally. However, some areas are more impacted than others, especially with climate change. Controlling vectors remains the primary means to prevent these diseases, but new, more effective tools are needed. The World Health Organization (WHO) prioritized evaluating novel control methods, such as sterile insect technique (SIT) for control of Aedes-borne diseases. In response, a multiagency partnership between the U.S. Centers for Disease Control and Prevention (CDC), the Special Programme for Research and Training in Tropical Diseases (TDR), WHO, and the International Atomic Energy Agency (IAEA) supported the operational implementation and evaluation of SIT against Aedes aegypti and arboviral diseases in the Pacific through a consortium of regional partners (PAC-SIT Consortium). Main text: A workshop was held from 2 to 6 May 2023, during which PAC-SIT country participants, researchers, and stakeholders in SIT, scientific advisory committee members, and organizational partners came together to review the principles and components of SIT, share experiences, visit field sites and the SIT facility, and officially launch the PAC-SIT project. Working in groups focused on entomology, epidemiology, and community engagement, participants addressed challenges, priorities, and needs for SIT implementation. Conclusions: The PAC-SIT workshop brought together researchers and stakeholders engaged in evaluating SIT for arboviral diseases in the Pacific region and globally. This training workshop highlighted that many countries are actively engaged in building operational capacities and phased testing of SIT. The workshop identified a key need for robust larger-scale studies tied with epidemiological endpoints to provide evidence for the scalability and impact on mosquito-borne diseases. [ABSTRACT FROM AUTHOR]

Background: Study of malaria parasite cell biology is challenged by their small size, which can make visualisation of individual organelles difficult or impossible using conventional light microscopy. In recent years, the field has attempted to overcome this challenge through the application of ultrastructure expansion microscopy (U-ExM), which physically expands a biological sample approximately 4.5-fold. To date, U-ExM has mostly been used to visualise blood-stage parasites and used exclusively on parasites in vitro. Methods: Here we develop Mosquito Tissue U-ExM (MoTissU-ExM), a method for preparing dissected mosquito salivary glands and midguts by U-ExM. MoTissU-ExM preserves both host and parasite ultrastructure, enabling visualisation of oocysts and sporozoites in situ. We also provide a point-by-point protocol for how to perform MoTissU-ExM. Results: We validate that MoTissU-ExM samples expand as expected, provide a direct comparison of the same dissected tissues before and after MoTissU-ExM, and highlight some of the key host and parasite structures that can be visualised following MoTissU-ExM. Discussion: We discuss potential use cases for MoTissU-ExM for study of malaria parasite biology, and more broadly. We detail drawbacks or challenges MoTissU-ExM and imaging these expanded tissues, along with information troubleshooting this technique. Finally, we discuss how MoTissU-ExM could be applied and adapted in future to increase its utility. [ABSTRACT FROM AUTHOR]

Codon optimality refers to the effect that codon composition has on messenger RNA (mRNA) stability and translation level and implies that synonymous codons are not silent from a regulatory point of view. Here, we investigated the adaptation of virus genomes to the host optimality code using mosquito-borne dengue virus (DENV) as a model. We demonstrated that codon optimality exists in mosquito cells and showed that DENV preferentially uses nonoptimal (destabilizing) codons and avoids codons that are defined as optimal (stabilizing) in either human or mosquito cells. Human genes enriched in the codons preferentially and frequently used by DENV are upregulated during infection, and so is the tRNA decoding the nonoptimal and DENV preferentially used codon for arginine. We found that adaptation during single-host passaging in human or mosquito cells results in the selection of synonymous mutations towards DENV's preferred nonoptimal codons that increase virus fitness. Finally, our analyses revealed that hundreds of viruses preferentially use nonoptimal codons, with those infecting a single host displaying an even stronger bias, suggesting that host–pathogen interaction shapes virus-synonymous codon choice. Synopsis: Analyses of viral genome adaptation to the host optimality code, reveal that hundreds of human-infecting viruses preferentially use non-optimal codons and suggest that host-pathogen interactions can shape virus synonymous codon choice. Dengue virus (DENV) preferentially uses non-optimal codons and avoids codons that are defined as optimal in either human or mosquito cells. Codon optimality mechanism exists in mosquito cells. Human genes enriched in the codons preferentially and frequently used by DENV are up-regulated during DENV infection, as well as tRNAs decoding the non-optimal codon that DENV preferentially uses. Hundreds of human-infecting viruses preferentially use non-optimal codons suggesting that host-pathogen interaction shapes virus synonymous codon choice. Analyses of viral genome adaptation to the host optimality code, reveal that hundreds of human-infecting viruses preferentially use non-optimal codons and suggest that host-pathogen interactions can shape virus synonymous codon choice. [ABSTRACT FROM AUTHOR]

It is commonly recognized that predation and the risk of predation can have profound effects on behaviors and population ecology of prey. Comparatively, little attention has been paid to the trade-offs hosts make to manage the risk of parasitism, but there is evidence that hosts make behavioral trade-offs to avoid parasitism in ways that resemble those made by prey animals under the risk of predation. Mosquitoes are common hematophagous ectoparasites which parasitize a wide range of vertebrates, resulting in blood loss, distraction, and disease transmission. Many hosts engage in defensive behaviors to repel or kill mosquitoes and these behaviors likely come at the expense of activities such as foraging and vigilance for predators. We placed a mosquito attractant (BG-Sweetscent) and repellant (allethrin) near artificial foraging patches in areas where mosquitoes were common and measured the amount of grain left after a night of foraging (giving-up density) to investigate whether mosquitoes influenced Cotton Mouse (Peromyscus gossypinus) foraging. We predicted that in patches with mosquito attractants, mosquito harassment would cause mice to engage in defensive behaviors which would distract or deter them from foraging, resulting in less grain consumption relative to control patches. Conversely, we predicted that in mosquito-repellent patches, mice would experience less harassment, resulting in more grain consumption. Indoor arena trials found no evidence that the mosquito treatments influenced cotton mouse behavior in the absence of mosquitoes. However, we found no evidence that mosquito treatments affected giving-up densities. [ABSTRACT FROM AUTHOR]

Abstract Background Gene drive-modified mosquitoes (GDMMs) have been promoted as one of the innovative technologies that may control and eliminate malaria and other mosquito-borne diseases. Several products are in early stages of development, targeting either population suppression or population modification of the mosquito vector. However, there is no direct experience of conducting risk assessment for environmental releases and subsequent policies regarding conditions for post-release. This study was carried out to gain additional insights on the possible post-release concerns that may arise, as they may inform future risk assessment and planning for deployment. Methods This study involved desktop reviews on post release monitoring experiences with previously released biological control products. Stakeholder consultations involving online surveys, and face to face workshop with experts from selected African countries from Eastern, Western, and Southern African regions was then carried out to establish post-release monitoring concerns for GDMMs. Results Review of genetic biocontrol technologies showed only limited lessons from post-release monitoring regimes with a focus largely limited to efficacy. For genetically modified organisms general surveillance and case-specific monitoring is expected in some of the regions. A number of post-release monitoring concerns in relation to the protection goals of human and animal health, biodiversity, and water quality were identified. Conclusion Based on established- protection goals, several post-release monitoring concerns have been identified. Subject to a rigorous risk assessment process for future GDMMs products, the concerns may then be prioritized for post-release monitoring.

Abstract Background Dengue is an emerging disease in Argentina due to the colonization of Aedes aegypti, the mosquito vector. Buenos Aires Province is the biggest and most populated district in Argentina, suffering dengue outbreaks of growing magnitude. During epidemic periods, pyrethroid insecticides are used in this country to control adult mosquitoes. Pyrethroid resistance in dengue vectors has been reported worldwide, making it necessary to implement resistance management strategies. The voltage-gated sodium channel is the target site of pyrethroids. Mutations in the gene encoding this protein, called kdr mutations, are usually the molecular cause of pyrethroid resistance in insects. In Ae. aegypti from the Americas, three kdr substitutions were described: V410L, V1016I, and F1534C. The diagnostic of kdr mutations is recommended for the early detection of pyrethroid resistance as well as the consequent planning of evidence-based control policies. Methods We distributed ovitraps across 16 localities in Buenos Aires Province, collecting 22,123 eggs. A total of 522 mosquitoes were genotyped in positions 1016 and 1534 of voltage-gated channel using multiplex high-resolution melting and/or TaqMan probe methods. A subset of 449 samples was also genotyped by a singleplex high-resolution melting method developed ad hoc and/or Sanger sequencing. Results We have documented, for the first time to our knowledge in the central region of Argentina, the presence of the 1016Ikdr + 1534Ckdr allele. Additionally, our study reports the first identification of the V410L mutation in central Argentina. These results underscore a growing trend of pyrethroid resistance in Ae. aegypti, fueled by the widespread use of these insecticides. Conclusions We detected 1016Ikdr + 1534Ckdr and 410Lkdr mutations in central Argentina for the first time and improved the processivity and accuracy of kdr genotyping methods. The results are both a tool for resistance monitoring and a sign of alarm to direct efforts towards finding sustainable methods for vector control to complement or replace pyrethroids. Joint efforts between academia and authorities to develop and implement public policies for vector control are a productive way to transfer scientific results for their application in public health. Graphical Abstract

Abstract Background The decision to imbibe a blood meal is predominantly dependent on the sensitivity and specificity of haematophagous arthropods to blood-derived adenine nucleotides, in particular adenosine triphosphate (ATP). Despite previous efforts to identify and characterise the specificity and sensitivity to ATP and other adenine nucleotides, as well as the role of other blood-derived phagostimulants across the Culicidae, comparisons across species remain difficult. Methods The feeding response of the yellow fever mosquito Aedes aegypti and the African malaria vector Anopheles gambiae to adenine nucleotides in the presence of a carbonate buffer was assessed using a membrane feeding assay. The proportion of mosquitoes engorged and the volume imbibed by all mosquitoes was scored visually and spectrophotometrically. In addition, the proportion of prediuresing An. gambiae, as well as the volume engorged and prediuresed, was examined. Results Aedes aegypti was more sensitive to adenine nucleotides than An. gambiae, but both species maintained specificity to these phagostimulants, demonstrating a dose-dependent bimodal feeding pattern, thereby expanding our understanding of the all-or-none blood-feeding hypothesis. Feeding on the bicarbonate buffer by An. gambiae—but not that of Ae. aegypti—demonstrated a species-specific variation in how blood phagostimulants are encoded. Adenine nucleotides, with and without bovine serum albumin, were observed to dose-dependently regulate the proportion of An. gambiae prediuresing and the volumes prediuresed but not volumes engorged. Conclusions Taken together, the results of this study expand our understanding of how mosquitoes differentially assess and respond to blood meal constituents, and provide a basis for further physiological and molecular studies. Graphical Abstract

Objective: To determine the current insecticide resistance status of Aedes (Ae.) aegypti and Ae. albopictus to four insecticides, namely 0.05% deltamethrin, 0.75% permethrin, 5% malathion and 0.25% pirimiphos–methyl using the World Health Organisation (WHO) susceptibility test kit. Methods: Adult bioassays were carried out using the standard protocol of the World Health Organisation. All F1 generation urban and suburban field strains of Ae. aegypti and Ae. albopictus were tested against pyrethroid and organophosphate insecticides, including the presence of piperonyl butoxide (PBO) in four replicates of 25 non-blood-fed female mosquitoes ranging from 3 to 5 days old. The Vector Control Research Unit (VCRU) laboratory strain served as a reference strain. Results: In this study, 0.05% deltamethrin demonstrated a lower value of knockdown time when 50% of the mosquito population died (KT50) and knockdown time when 95% of the mosquito population died (KT95), which is significantly more effective compared to 0.75% permethrin against adult female Ae. aegypti (urban and suburban) and Ae. albopictus (urban and suburban) (ANOVA, P

Abstract Background Vector-borne diseases cause morbidity and mortality globally. However, some areas are more impacted than others, especially with climate change. Controlling vectors remains the primary means to prevent these diseases, but new, more effective tools are needed. The World Health Organization (WHO) prioritized evaluating novel control methods, such as sterile insect technique (SIT) for control of Aedes-borne diseases. In response, a multiagency partnership between the U.S. Centers for Disease Control and Prevention (CDC), the Special Programme for Research and Training in Tropical Diseases (TDR), WHO, and the International Atomic Energy Agency (IAEA) supported the operational implementation and evaluation of SIT against Aedes aegypti and arboviral diseases in the Pacific through a consortium of regional partners (PAC-SIT Consortium). Main text A workshop was held from 2 to 6 May 2023, during which PAC-SIT country participants, researchers, and stakeholders in SIT, scientific advisory committee members, and organizational partners came together to review the principles and components of SIT, share experiences, visit field sites and the SIT facility, and officially launch the PAC-SIT project. Working in groups focused on entomology, epidemiology, and community engagement, participants addressed challenges, priorities, and needs for SIT implementation. Conclusions The PAC-SIT workshop brought together researchers and stakeholders engaged in evaluating SIT for arboviral diseases in the Pacific region and globally. This training workshop highlighted that many countries are actively engaged in building operational capacities and phased testing of SIT. The workshop identified a key need for robust larger-scale studies tied with epidemiological endpoints to provide evidence for the scalability and impact on mosquito-borne diseases.

A 51-year-old man presented with decreased vision, fever, confusion, headaches, agitation, nausea, vomiting and diarrhea. Magnetic resonance imaging of the brain demonstrated bilateral T2 hyperintense lesions in the region of the mesial temporal lobe and optic radiations. There was a predominantly polymorphonuclear leukocyte pleocytosis in the cerebrospinal fluid (CSF) with hyperproteinorachia. A meningoencephalitis was diagnosed. Intravenous fluorescein angiography (IVFA) demonstrated a multifocal chorioretinitis that was in a linear pattern in the left eye. CSF enzyme-linked immunosorbent assay was positive for West Nile virus (WNV) IgM. We review the clinical manifestations of WNV disease and highlight the value of IVFA in determining the diagnosis. [ABSTRACT FROM AUTHOR]

Abstract Background Malaria is a mosquito-transmitted disease that kills more than half a million people annually. The lack of effective malaria vaccines and recently increasing malaria cases urge innovative approaches to prevent malaria. Previously, we reported that the extract from the soil-dwelling fungus Purpureocillium lilacinum, a common fungus from the soil, reduced Plasmodium falciparum oocysts in Anopheles gambiae midguts after mosquitoes contacted the treated surface before feeding. Methods We used liquid chromatography to fraction fungal crude extract and tract the active fraction using a contact-wise approach and standard membrane feeding assays. The purified small molecules were analyzed using precise mass spectrometry and tandem mass spectrometry. Results We isolated four active small molecules from P. lilacinum and determined them as leucinostatin A, B, A2, and B2. Pre-exposure of mosquitoes via contact with very low-concentration leucinostatin A significantly reduced the number of oocysts. The half-maximal response or inhibition concentration (EC50) via pre-exposure was 0.7 mg/m2, similar to atovaquone but lower than other known antimalarials. The inhibitory effect of leucinostatin A against P. falciparum during intraerythrocytic development, gametogenesis, sporogonic development, and ookinete formation, with the exception of oocyst development, suggests that leucinostatins play a part during parasite invasion of new cells. Conclusions Leucinostatins, secondary metabolites from P. lilacinum disrupt malaria development, particular transmission to mosquitoes by contact. The contact-wise malaria control as a nonconventional approach is highly needed in malaria-endemic areas. Graphical Abstract

Background & objectives: Vector-borne diseases pose substantial global health challenges, with a notable impact on India. Effective vector control strategies are crucial to mitigating these diseases as there are no effective vaccines available. Synthetic chemicals are widely used but concerns about safety and environmental impact have prompted exploration of alternative natural solutions. Unani medicine offers a potential source of natural products. This study aims to systematically review classical Unani texts and contemporary scientific literature to evaluate the vector control potential of Unani medicines. Methods: A comprehensive review of classical Unani texts was carried out to compile data on herbal and mineral substances suggested in Unani medicine for control of insects. Furthermore, electronic databases like PubMed, Google Scholar, and Science Direct were explored for research studies evaluating the insect repellent, larvicidal, and insecticidal activities of the Unani medicines. Time restriction was not imposed to ensure comprehensive retrieval of pertinent and up-to-date information. Results: From classical Unani texts, 29 drugs were identified for control of insects, primarily of plant origin from diverse botanical families. Recent scientific investigations have confirmed the insect-repellent, insecticidal, and larvicidal activities of 19 of these Unani medicines against various insect species, including mosquitoes. Interpretation & conclusion: These findings underscore the potential of Unani medicines as a natural source for vector control. However, further research is essential to explore mechanism, delivery methods, and optimal concentrations for developing effective vector-control formulations.

Abstract Background Mosquitoes (Culicidae), as disease vectors, represent a risk for human health worldwide. Repeated introductions of alien mosquito species and the spread of invasive species have been recorded in different countries. Traditionally, identification of mosquitoes relies on morphological observation. However, morphology-based identification is associated with a number of potential disadvantages, such as the high level of specialisation of the operator and its limited applicability to damaged samples. In these cases, species identification is achieved through molecular methods based on DNA amplification. Molecular-based taxonomy has also enabled the development of techniques for the study of environmental DNA (eDNA). Previous studies indicated the 16S mitochondrial ribosomal RNA (rRNA) gene as a promising target for this application; however, 16S rRNA sequences are available for only a limited number of mosquito species. In addition, although primers for the 16S rRNA gene were designed years ago, they are based on limited numbers of mosquito sequences. Thus, the aims of this study were to: (i) design pan-mosquito 16S rRNA gene primers; (ii) using these primers, generate a 16S rRNA gene mosquito reference library (with a focus on mosquitoes present in Italy); and (iii) compare the discriminatory power of the 16S rRNA gene with two widely used molecular markers, cytochrome c oxidase subunit 1 mitochondrial gene (COI) and internal transcribed spacer 2 (ITS2). Methods A total of six mosquito genera (28 mosquito species) were included in this study: Aedes (n = 16 species), Anopheles (5 species), Coquillettidia (1 species), Culex (3 species), Culiseta (2 species) and Uranotaenia (1 species). DNA was extracted from the whole mosquito body, and more than one specimen for each species was included in the analysis. Sanger sequencing was used to generate DNA sequences that were then analysed through the Barcode of Life Data Systems (BOLD). Phylogenetic analyses were also performed. Results Novel 16S rDNA gene, COI and ITS2 sequences were generated. The 16S rRNA gene was shown to possess sufficient informativeness for the identification of mosquito species, with a discriminatory power equivalent to that of COI. Conclusions This study contributes to the generation of DNA barcode libraries, focussed on Italian mosquitoes, with a significant increase in the number of 16S rRNA gene sequences. We hope that these novel sequences will provide a resource for studies on the biodiversity, monitoring and metabarcoding of mosquitoes, including eDNA-based approaches. Graphical abstract

Abstract Background Most insects are poikilotherms and ectotherms, so their body temperature is predicated by environmental temperature. With climate change, insect body temperature is rising, which affects how insects develop, survive, and respond to infection. Aging also affects insect physiology by deteriorating body condition and weakening immune proficiency via senescence. Aging is usually considered in terms of time, or chronological age, but it can also be conceptualized in terms of body function, or physiological age. We hypothesized that warmer temperature decouples chronological and physiological age in insects by accelerating senescence. To investigate this, we reared the African malaria mosquito, Anopheles gambiae, at 27 °C, 30 °C and 32 °C, and measured survival starting at 1-, 5-, 10- and 15-days of adulthood after no manipulation, injury, or a hemocoelic infection with Escherichia coli or Micrococcus luteus. Then, we measured the intensity of an E. coli infection to determine how the interaction between environmental temperature and aging shapes a mosquito’s response to infection. Results We demonstrate that longevity declines when a mosquito is infected with bacteria, mosquitoes have shorter lifespans when the temperature is warmer, older mosquitoes are more likely to die, and warmer temperature marginally accelerates the aging-dependent decline in survival. Furthermore, we discovered that E. coli infection intensity increases when the temperature is warmer and with aging, and that warmer temperature accelerates the aging-dependent increase in infection intensity. Finally, we uncovered that warmer temperature affects both bacterial and mosquito physiology. Conclusions Warmer environmental temperature accelerates aging in mosquitoes, negatively affecting both longevity and infection outcomes. These findings have implications for how insects will serve as pollinators, agricultural pests, and disease vectors in our warming world.

Abstract Background Mosquito host feeding patterns are an important factor of the species-specific vector capacity determining pathogen transmission routes. Culex pipiens s.s./Cx. torrentium are competent vectors of several arboviruses, such as West Nile virus and Usutu virus. However, studies on host feeding patterns rarely differentiate the morphologically indistinguishable females. Methods We analyzed the host feeding attraction of Cx. pipiens and Cx. torrentium in host-choice studies for bird, mouse, and a human lure. In addition, we summarized published and unpublished data on host feeding patterns of field-collected specimens from Germany, Iran, and Moldova from 2012 to 2022, genetically identified as Cx. pipiens biotype pipiens, Cx. pipiens biotype molestus, Cx. pipiens hybrid biotype pipiens × molestus, and Cx. torrentium, and finally put the data in context with similar data found in a systematic literature search. Results In the host-choice experiments, we did not find a significant attraction to bird, mouse, and human lure for Cx. pipiens pipiens and Cx. torrentium. Hosts of 992 field-collected specimens were identified for Germany, Iran, and Moldova, with the majority determined as Cx. pipiens pipiens, increasing the data available from studies known from the literature by two-thirds. All four Culex pipiens s.s./Cx. torrentium taxa had fed with significant proportions on birds, humans, and nonhuman mammals. Merged with the data from the literature from 23 different studies showing a high prevalence of blood meals from birds, more than 50% of the blood meals of Cx. pipiens s.s. were identified as birds, while up to 39% were human and nonhuman mammalian hosts. Culex torrentium fed half on birds and half on mammals. However, there were considerable geographical differences in the host feeding patterns. Conclusions In the light of these results, the clear characterization of the Cx. pipiens s.s./Cx. torrentium taxa as ornithophilic/-phagic or mammalophilic/-phagic needs to be reconsidered. Given their broad host ranges, all four Culex taxa could potentially serve as enzootic and bridge vectors. Graphical Abstract

Abstract Background Aedes aegypti, the primary vector of dengue, chikungunya, and Zika viruses, poses a significant public health threat worldwide. Traditional control methods using insecticides are increasingly challenged by resistance and environmental concerns. The sterile insect technique (SIT) offers an eco-friendly alternative that has been successfully applied to other insect pests. This article aims to briefly review Ae. aegypti management in Cuba, highlighting the accomplishments, challenges, and future directions of the SIT. Main body Here we provide a brief summary of the extensive history of Ae. aegypti control efforts in Cuba. After a successful eradication campaign in the 1980s, a resurgence of dengue cases has been observed in recent years, suggesting that traditional control methods may have limited effectiveness under current conditions. In response, Cuba initiated a phased approach to develop and evaluate the feasibility of SIT for Ae. aegypti control, starting in 2008. Initial research focused on Ae. aegypti mating behavior and sterilization methods, followed by successful laboratory and semi-field trials that demonstrated population suppression. The first open-field trial in 2020 confirmed the efficacy of the SIT in reducing Ae. aegypti populations under real-world conditions. Currently, the research is in a phase involving a cluster-randomized superiority-controlled trial. This planned trial will compare the standard vector control program with the same program augmented by the SIT, aiming to assess the impact of the SIT on dengue incidence as the primary outcome. Implementing robust epidemiological trials to evaluate the effectiveness of the SIT is complex due to potential spillover effects from mosquito and human movement across study areas. Additionally, conducting the SIT requires significant development and operational investments. Despite these challenges, the ongoing Cuban trial holds promise for establishing the SIT as an effective and sustainable tool for Ae. aegypti control and for reducing the burden of mosquito-borne diseases. Conclusions The phased evaluation conducted in Cuba confirms the efficacy of the SIT against Ae. aegypti, highlighting its potential for sustainable mosquito-borne disease management. The effective implementation of multi-site trials will be crucial in providing evidence of the potential of the sterile insect technique as part of a strategy to reduce the incidence of arboviral diseases. Graphical Abstract

Abstract Background Rift Valley fever virus (RVFV) is a zoonotic mosquito-borne virus with serious implications for livestock health, human health, and the economy in Africa, and is suspected to be endemic in north-eastern KwaZulu-Natal (KZN), South Africa. The vectors of RVFV in this area are poorly known, although several species, such as Aedes (Neomelaniconion) mcintoshi, Aedes (Neomelaniconion) circumluteolus, Aedes (Aedimorphus) durbanensis, and Culex (Lasioconops) poicilipes may be involved. The aim of the study was to determine the vertebrate blood meal sources of potential RVFV mosquito vectors in north-eastern KZN and to characterize the host-biting network. Methods Blood-fed mosquitoes were collected monthly from November 2019 to February 2023 using a backpack aspirator, CO2-baited Centers for Disease Control and Prevention (CDC) miniature light traps and tent traps, in the vicinity of water bodies and livestock farming households. The mosquitoes were morphologically identified. DNA was extracted from individual mosquitoes and used as templates to amplify the vertebrate cytochrome c oxidase I (COI) and cytochrome b (cytb) genes using conventional polymerase chain reaction (PCR). Amplicons were sequenced and queried in GenBank and the Barcode of Life Data systems to identify the vertebrate blood meal sources and confirm mosquito identifications. All mosquitoes were screened for RVFV using real time reverse transcription (RT)-PCR. Results We identified the mammalian (88.8%) and avian (11.3%) blood meal sources from 409 blood-fed mosquitoes. Aedes circumluteolus (n = 128) made up the largest proportion of collected mosquitoes. Cattle (n = 195) and nyala (n = 61) were the most frequent domestic and wild hosts, respectively. Bipartite network analysis showed that the rural network consisted of more host-biting interactions than the reserve network. All mosquitoes tested negative for RVFV. Conclusions Several mosquito species, including Ae. circumluteolus, and vertebrate host species, including cattle and nyala, could play a central role in RVFV transmission. Future research in this region should focus on these species to better understand RVFV amplification. Graphical Abstract

Abstract Codon optimality refers to the effect that codon composition has on messenger RNA (mRNA) stability and translation level and implies that synonymous codons are not silent from a regulatory point of view. Here, we investigated the adaptation of virus genomes to the host optimality code using mosquito-borne dengue virus (DENV) as a model. We demonstrated that codon optimality exists in mosquito cells and showed that DENV preferentially uses nonoptimal (destabilizing) codons and avoids codons that are defined as optimal (stabilizing) in either human or mosquito cells. Human genes enriched in the codons preferentially and frequently used by DENV are upregulated during infection, and so is the tRNA decoding the nonoptimal and DENV preferentially used codon for arginine. We found that adaptation during single-host passaging in human or mosquito cells results in the selection of synonymous mutations towards DENV’s preferred nonoptimal codons that increase virus fitness. Finally, our analyses revealed that hundreds of viruses preferentially use nonoptimal codons, with those infecting a single host displaying an even stronger bias, suggesting that host–pathogen interaction shapes virus-synonymous codon choice.

ObjectiveTo evaluate the field mosquito surveillance efficacy between the black box method and the light trap method.MethodsEight light traps and eight black boxes were placed in each of the eight field sites in Baoshan District of Shanghai, with a minimum distance of 10 meters between each traps. Surveillance was conducted twice a month from May to September 2023.ResultsThe total number of mosquitoes (273) and female mosquitoes (228) captured by the black box method were 0.795 and 0.774 times higher than those caught by the light trap method, respectively. There was no statistically significant difference (P>0.05) between the mosquito density (t=1.723 5, P=0.100 2) and the female mosquito density (t=1.805 9, P=0.101 1), and there was a correlation (P0.05) and female mosquitoes(χ2=0.394 4, P>0.05) captured by black box and light trap methods. In four different habitats, the composition ratios of mosquito species captured by the black box and light trap methods were: hospitals (38.10% and 38.77%) > rural housing areas (36.26% and 37.61%) > parks (14.65% and 12.54%)> urban housing areas (10.99% and 11.08%), and the composition ratios of female mosquitoes captured by the black box and light trap methods were: rural housing areas (40.35% and 42.52%) > hospitals (37.72% and 36.05%) > parks (13.16% and 13.27%) > urban housing areas (8.77% and 8.16%). In different habitats, there was no significant difference in the composition ratio of mosquitoes captured by two monitoring methods(χ2=0.5987, P>0.05), and there was no significant difference in the composition ratio of female mosquitoes(χ2=0.2993, P>0.05).ConclusionWith widen application and fewer requirements for equipment, operation skills, and use cost the black box method can be interchanged with the light trap method for monitoring mosquito density, especially in disaster areas or remote areas with difficult in having access to electricity.

Abstract Background Analyses of the temporal distribution of mosquitoes are presented in statistical charts, but it is difficult to prove in statistics whether differences in peak periods exist among different years or habitats. This study aimed to investigate the application of circular statistics in determining the peak period and a comparison of differences. Methods Surveillance of adult mosquitoes was conducted twice a month by light traps in five different habitats from March to November for 3 years (2021–2023) in Qingdao, Shandong Province, China. The Kruskal–Wallis test was performed to determine the differences in mosquito density among different years and habitats. Circular statistics and line charts were employed to determine the peak period and a comparison of differences. Results Among a total of 14,834 adult mosquitoes comprising five mosquito species from four genera, Culex pipiens pallens was dominant and accounted for 89.6% of the specimens identified. Aedes albopictus, Armigeres subalbatus, and Anopheles sinensis made up 5.7%, 4.2%, and 0.5%, respectively. Culex tritaeniorhynchus accounted for less than 0.1%. The mean mosquito density (females/trap night) for the trapping period was 10.3 in 2021, 5.6 in 2022, and 3.6 in 2023. Among five habitats, the highest mosquito density was 8.9 in livestock sheds, followed by 6.8 in parks, 5.9 in rural dwellings, 5.5 in urban dwellings, and 5.4 in hospitals. No statistically significant differences were found among different years (H = 1.96, d.f. 2, P = 0.376) and habitats (H = 0.45, d.f. 4, P = 0.978). Overall, the peak period of mosquito activity fell in the months from June to September. The peak period among 3 years differed significantly (F (2,7022) = 119.17, P 0.05). Conclusion Circular statistics could be effectively combined with statistical charts to elucidate the peak period of mosquitoes and determine the differences in statistics among different years and habitats. These findings will provide valuable information for mosquito control and public health management. Graphical Abstract

Background: Mosquitoes (Culicidae), as disease vectors, represent a risk for human health worldwide. Repeated introductions of alien mosquito species and the spread of invasive species have been recorded in different countries. Traditionally, identification of mosquitoes relies on morphological observation. However, morphology-based identification is associated with a number of potential disadvantages, such as the high level of specialisation of the operator and its limited applicability to damaged samples. In these cases, species identification is achieved through molecular methods based on DNA amplification. Molecular-based taxonomy has also enabled the development of techniques for the study of environmental DNA (eDNA). Previous studies indicated the 16S mitochondrial ribosomal RNA (rRNA) gene as a promising target for this application; however, 16S rRNA sequences are available for only a limited number of mosquito species. In addition, although primers for the 16S rRNA gene were designed years ago, they are based on limited numbers of mosquito sequences. Thus, the aims of this study were to: (i) design pan-mosquito 16S rRNA gene primers; (ii) using these primers, generate a 16S rRNA gene mosquito reference library (with a focus on mosquitoes present in Italy); and (iii) compare the discriminatory power of the 16S rRNA gene with two widely used molecular markers, cytochrome c oxidase subunit 1 mitochondrial gene (COI) and internal transcribed spacer 2 (ITS2). Methods: A total of six mosquito genera (28 mosquito species) were included in this study: Aedes (n = 16 species), Anopheles (5 species), Coquillettidia (1 species), Culex (3 species), Culiseta (2 species) and Uranotaenia (1 species). DNA was extracted from the whole mosquito body, and more than one specimen for each species was included in the analysis. Sanger sequencing was used to generate DNA sequences that were then analysed through the Barcode of Life Data Systems (BOLD). Phylogenetic analyses were also performed. Results: Novel 16S rDNA gene, COI and ITS2 sequences were generated. The 16S rRNA gene was shown to possess sufficient informativeness for the identification of mosquito species, with a discriminatory power equivalent to that of COI. Conclusions: This study contributes to the generation of DNA barcode libraries, focussed on Italian mosquitoes, with a significant increase in the number of 16S rRNA gene sequences. We hope that these novel sequences will provide a resource for studies on the biodiversity, monitoring and metabarcoding of mosquitoes, including eDNA-based approaches. [ABSTRACT FROM AUTHOR]

Background: Malaria is a mosquito-transmitted disease that kills more than half a million people annually. The lack of effective malaria vaccines and recently increasing malaria cases urge innovative approaches to prevent malaria. Previously, we reported that the extract from the soil-dwelling fungus Purpureocillium lilacinum, a common fungus from the soil, reduced Plasmodium falciparum oocysts in Anopheles gambiae midguts after mosquitoes contacted the treated surface before feeding. Methods: We used liquid chromatography to fraction fungal crude extract and tract the active fraction using a contact-wise approach and standard membrane feeding assays. The purified small molecules were analyzed using precise mass spectrometry and tandem mass spectrometry. Results: We isolated four active small molecules from P. lilacinum and determined them as leucinostatin A, B, A2, and B2. Pre-exposure of mosquitoes via contact with very low-concentration leucinostatin A significantly reduced the number of oocysts. The half-maximal response or inhibition concentration (EC50) via pre-exposure was 0.7 mg/m2, similar to atovaquone but lower than other known antimalarials. The inhibitory effect of leucinostatin A against P. falciparum during intraerythrocytic development, gametogenesis, sporogonic development, and ookinete formation, with the exception of oocyst development, suggests that leucinostatins play a part during parasite invasion of new cells. Conclusions: Leucinostatins, secondary metabolites from P. lilacinum disrupt malaria development, particular transmission to mosquitoes by contact. The contact-wise malaria control as a nonconventional approach is highly needed in malaria-endemic areas. [ABSTRACT FROM AUTHOR]

Background: Most insects are poikilotherms and ectotherms, so their body temperature is predicated by environmental temperature. With climate change, insect body temperature is rising, which affects how insects develop, survive, and respond to infection. Aging also affects insect physiology by deteriorating body condition and weakening immune proficiency via senescence. Aging is usually considered in terms of time, or chronological age, but it can also be conceptualized in terms of body function, or physiological age. We hypothesized that warmer temperature decouples chronological and physiological age in insects by accelerating senescence. To investigate this, we reared the African malaria mosquito, Anopheles gambiae, at 27 °C, 30 °C and 32 °C, and measured survival starting at 1-, 5-, 10- and 15-days of adulthood after no manipulation, injury, or a hemocoelic infection with Escherichia coli or Micrococcus luteus. Then, we measured the intensity of an E. coli infection to determine how the interaction between environmental temperature and aging shapes a mosquito's response to infection. Results: We demonstrate that longevity declines when a mosquito is infected with bacteria, mosquitoes have shorter lifespans when the temperature is warmer, older mosquitoes are more likely to die, and warmer temperature marginally accelerates the aging-dependent decline in survival. Furthermore, we discovered that E. coli infection intensity increases when the temperature is warmer and with aging, and that warmer temperature accelerates the aging-dependent increase in infection intensity. Finally, we uncovered that warmer temperature affects both bacterial and mosquito physiology. Conclusions: Warmer environmental temperature accelerates aging in mosquitoes, negatively affecting both longevity and infection outcomes. These findings have implications for how insects will serve as pollinators, agricultural pests, and disease vectors in our warming world. [ABSTRACT FROM AUTHOR]

Background: Fibrinogen-related protein 1 (frep1) is a member of the pattern-recognizing receptor family (PRR) which generates an innate immune response after recognizing the pattern associated molecular pattern (PAMP) that occurs on the surface of microorganisms. The main objective of this study is to characterize frep1 and its in-silico analysis in Anopheles stephensi. Methods and result: The DNA was extracted from female Anopheles stephensi. PCR was performed for complete analysis of frep1 using specific primers. The gene sequence of frep1 was identified by Sanger sequencing. The bioinformatics structure analysis approach revealed the presence of 3 exons and 4 introns in the frep1. The sequence of frep1 was submitted to NCBI GeneBank with accession number ON817187.1. Quantitative real-time PCR was performed to analyze frep1 expression. At the developmental stage, frep1 is highly expressed in the L1 stage, egg, and adult female mosquito. In addition, frep1 is highly expressed in the tissue fat body, midgut, and salivary gland. After blood-fed, an upregulation of frep1 at 48 h in the midgut, and downregulation in fat body were observed at different time intervals. Conclusion: The genomic data of frep1 is encoded by 12,443 bp. The frep1 has a significant role in the early metamorphosis. Its expression in fat body and midgut suggests it could be important for fat metabolism and post-blood digestion. The conserved domain could be targeted for vector control. Further study is required to elucidate its function against malaria parasites to confirm its agonist role in malaria transmission. [ABSTRACT FROM AUTHOR]

Simple Summary: The mosquito species Culex pipiens, has two ecotypes, pipiens and molestus. The two forms differ in mating behavior and how they manage overwintering, with the molestus form thriving in closed indoor surroundings but without an ability to manage cold winter conditions, and the pipiens form that requiring open spaces for mating and going into diapause during winter. Both ecotypes are important vectors for mosquito-borne viruses. Cx. pipiens mosquitoes often carry endosymbiotic Wolbachia bacteria that are transmitted from the mother to the egg, but this varies frequency of occurrence. The Wolbachia endosymbiont affects the reproductive success of mosquitoes so that only eggs infected with a compatible Wolbachia can be fertilized by an infected male. In artificially infected Aedes mosquitoes, Wolbachia affects whether the mosquito can transmit a virus though bites. We studied how Cx. pipiens mosquitoes from Sweden carried Wolbachia and compared them to mosquitoes from other European countries, and found that all tested Cx. pipiens mosquitoes carried Wolbachia. We characterized the Wolbachia from these mosquitoes and found that Cx. pipiens of the two ecotypes carry different strains of Wolbachia in Sweden and Norway but not in sampled mosquitoes from Italy or England. This highlights the differences between the two ecotypes in northern Europe. The mosquito Culex pipiens occurs in two ecotypes differing in their mating and overwintering behavior: pipiens mate in open environments and diapause, and molestus also mate in small spaces and is active throughout the year. Cx. pipiens carry Wolbachia endosymbionts of the wPip strain, but the frequency of infection differs between studied populations. Wolbachia infection affects the host reproductive success through cytoplasmic incompatibility. wPip Wolbachia is divided into five types, wPip I–V. The type of wPip carried varies among Cx. pipiens populations. In northern European locations different wPip types are found in the two ecotypes, whereas in southern locations, they often carry the same type, indicating differences in hybridization between ecotypes. In this study, Cx. pipiens specimens of both ecotypes were collected from Sweden and compared to specimens from Norway, England, Italy, and the Netherlands, as well as Cx. quinquefasciatus from Mali and Thailand. The abundance varied, but all specimens were infected by Wolbachia, while the tested specimens of other mosquito species were often uninfected. The wPip strains were determined through the sequence analysis of Wolbachia genes ank2 and pk1, showing that Cx. pipiens ecotypes in Scandinavia carry different wPip strains. The observed differences in wPip strains indicate that hybridization is not frequent and may contribute to barriers against hybridization of the ecotypes in Sweden and Norway. [ABSTRACT FROM AUTHOR]

Oral infection of mosquitoes by arboviruses often results in a large degree of variation in the amount of infectious virus between individual mosquitoes, even when the mosquitoes are from inbred laboratory strains. This variability in arbovirus load has been shown to affect virus transmissibility. Previously, our group described population genetic and specific infectivity differences between the virus populations found in high and low titer Aedes aegypti mosquitoes that had been orally infected with Sindbis virus (SINV). In this study, we sought to investigate whether there were also differences in transcriptomic response between these high and low titer mosquitoes. Results from the transcriptomic data analysis showed that more genes involved in antiviral activity, endopeptidase activity, and methyltransferase activity were upregulated in low titer mosquitoes than in high titer mosquitoes, relative to blood-fed controls. Meanwhile, genes involved in ion transport, energy metabolism, acetylation, glycosylation, lipid metabolism, and transport tended to be upregulated in high titer mosquitoes more than in low titer mosquitoes, relative to blood-fed mosquitoes. Overall, genes involved in antiviral activities tended to be upregulated in low titer mosquitoes while genes involved in proviral activities were mostly upregulated in high titer mosquitoes. This study has identified a number of candidate mosquito genes that are putatively associated with SINV titer variability after oral infection of Ae. aegypti, and these can now be investigated in order to ascertain their roles in virus replication and their contributions to determining vector competence. [ABSTRACT FROM AUTHOR]

Viral diseases like yellow fever, dengue, and Zika have an alarming impact on public health. These diseases can be transmitted by Aedes mosquito species, such as Ae. albopictus, which is now found in many countries outside its original range. Xenorhabdus and Photorhabdus spp. are enteric bacterial symbionts of insect-preying nematodes and are known to produce an array of natural products with various activities including larvicidal activity. In this study, the effects of natural products produced by four Xenorhabdus and one Photorhabdus bacteria on the ovipositional behavior of Ae. albopictus mosquitoes were assessed. Utilizing a binary choice assay in insect cages, gravid female mosquitoes were presented with two oviposition cups containing water supplemented with varying concentrations of bacterial supernatants (50–1% concentrations) versus control medium. After 72 h, the eggs deposited on filter papers were counted. The oviposition attractant index (OAI) feature of the bacterial supernatant was evaluated using the number of eggs laid in the cups. Notably, all tested supernatants exhibited concentration-dependent deterrence of oviposition. Xenorhabdus cabanillasii displayed the strongest deterrent effect, inhibiting egg-laying at 50–5% concentrations (OAI: − 0.87 to − 0.35), followed by X. nematophila (50–10%, OAI: − 0.82 to − 0.52). Xenorhabdus szentirmaii, X. doucetiae, and P. kayaii showed significant deterrence at ≥ 20% concentrations. Using promoter exchange mutants generated by the easyPACId approach, fabclavine from X. szentirmaii was identified as the bioactive compound with evident deterrent effects. Such deterrents targeting egg-laying could be valuable for controlling populations by disrupting their breeding in suitable habitats. [ABSTRACT FROM AUTHOR]

Larval habitat availability, Typha phytotelmata use and the relation of environmental factors to annual dynamics of the mosquito Wyeomyia leucostigma Lutz, 1904 (larvae and females) were studied in Costanera Sur Ecological Reserve at Buenos Aires, Argentina. Bimonthly, 1520 plants were examined, detecting W. leucostigma in 80.8% of surveys. Larval habitat availability was associated with temperature, but not with rainfall. In addition, the high temperature was associated with greater biting rate. Dynamics of host-seeking were recorded over 10 months of the year and were associated with temperature as well. Furthermore, an accidental fire led to an increase of W. leucostigma abundance likely based on an increase of the Thypha marshes productivity that occurred after the fire. [ABSTRACT FROM AUTHOR]

The present study was carried out for the first time in Morocco to valorise plant species of the Artemisia genus. Its aim was to evaluate the larvicidal activity of essential oils from six Artemisia plants (A. herba alba, A. absinthium, A. campestris L., A. negrei, A. pontica, and A. arborescens) against Culex pipiens mosquitoes, which are recognised as potential vectors of West Nile virus. Bioinsecticides derived from EOs have shown promise as effective mosquito control agents. The EOs were analysed using GC–MS and GC‐FID and were obtained by hydrodistillation from the aerial parts of the plants. Besides, the experiment was carried out according to the standard WHO protocol. Thujone and camphor were identified as the predominant compounds in all six Artemisia plants. A. herba alba demonstrated the highest larvicidal activity with LC50 and LC90 values of 4.85 and 15.39 μg/mL, respectively. Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) were employed to explore the correlation between major EO compounds and larvicidal efficacy. The results suggest that these findings could pave the way for the development of tailored biocides that specifically target important disease vectors. [ABSTRACT FROM AUTHOR]

Our knowledge of alphavirus genetic diversity is mainly based on viruses isolated from anthropophilic mosquito species, humans, and livestock during outbreaks. Studies on alphaviruses from sylvatic amplification cycles in sub-Saharan Africa have been conducted less often than from epizootic environments. To gain insight into alphavirus diversity in enzootic transmission cycles, we collected over 23,000 mosquitoes in lowland rainforest and savannah gallery forest in southwestern Uganda and tested them for alphavirus infections. We detected Sindbis virus (SINV) in a Culex Culex sp. mosquito and Middelburg virus (MIDV) in Eretmapodites intermedius and Mansonia africana. MIDV is a mosquito-borne alphavirus that causes febrile illness in sheep, goats, and horses and was previously not known to occur in Uganda. SINV, also a mosquito-borne alphavirus, causes mild infections in humans. Full genomes of SINV and MIDV were sequenced, showing a nucleotide identity of 99% to related strains. Both isolates replicated to high titres in a wide variety of vertebrate cells. Our data suggest endemic circulation of SINV and MIDV in Uganda. [ABSTRACT FROM AUTHOR]

Mosquitoes are known to choose oviposition habitats actively based on their suitability for survival of their offspring. They thus avoid laying eggs in dangerous habitats with aquatic predators through detection of their cues. Visual, tactile, and chemical cues emitted by certain predators have been shown to reduce oviposition in some mosquitoes, which in turn may affect their adult population sizes. However, those tests tend to be limited to species from temperate regions. Moreover, mosquito oviposition response to predator cues have been shown to be predator specific, but this has not been investigated for many potential predators worldwide. In the African subtropics, temporary ponds can be important mosquito breeding habitats and the same habitats are also used by a group of specialized killifish that can survive dry periods using drought resistant eggs. Although they are reported as mosquito predators, it is unknown whether mosquitoes actively avoid laying eggs in ponds with annual killifish. We investigated this using the mosquito Culex quinquefasciatus and the free and caged killifish Nothobranchius neumanni in the outdoor mesocosm experiments in Tanzania. We found that Cx. quinquefasciatus had 71% lower oviposition in mesocosms with free swimming killifish and 68% lower oviposition in mesocosms with caged killifish. We conclude that predator-released chemical cues alone are enough to explain the observed responses. If the cues can be isolated and chemically identified can be developed into a biological mosquito control strategy and used to safeguard public health in areas where mosquito borne diseases are endemic. [ABSTRACT FROM AUTHOR]

In an age of emerging zoonoses, it is important to understand the intricate system of vectors and reservoirs, or hosts, and their relation to humans. West Nile Virus (WNV) has been detected in a myriad of nonhuman hosts. Transmission of the virus to humans is reliant on amplified seroprevalence within the host, which occurs primarily in birds. However, recent studies have found that other animal groups, including crocodilians, can obtain seroprevalence amplification to levels that make them competent hosts able to transmit WNV to mosquitoes, which can then transmit to humans. Climate change could exacerbate this transmission risk by shifting the distributions of mosquito vectors towards novel geographic ranges. Here, we use maximum entropy models to map the current and future distributions of three mosquito vector species and four crocodilian species in North America to determine the emerging risk of WNV outbreaks associated with changing climates and WNV associated with crocodilians in North America. From our models, we determined that one mosquito species in particular, Culex quinquefasciatus, will increase its distribution across the ranges of all crocodilian species in all tested climate change scenarios. This poses a potential risk to public health for people visiting and living near crocodilian farms and high-density natural crocodilian populations. [ABSTRACT FROM AUTHOR]

Background: Mosquito host feeding patterns are an important factor of the species-specific vector capacity determining pathogen transmission routes. Culex pipiens s.s./Cx. torrentium are competent vectors of several arboviruses, such as West Nile virus and Usutu virus. However, studies on host feeding patterns rarely differentiate the morphologically indistinguishable females. Methods: We analyzed the host feeding attraction of Cx. pipiens and Cx. torrentium in host-choice studies for bird, mouse, and a human lure. In addition, we summarized published and unpublished data on host feeding patterns of field-collected specimens from Germany, Iran, and Moldova from 2012 to 2022, genetically identified as Cx. pipiens biotype pipiens, Cx. pipiens biotype molestus, Cx. pipiens hybrid biotype pipiens × molestus, and Cx. torrentium, and finally put the data in context with similar data found in a systematic literature search. Results: In the host-choice experiments, we did not find a significant attraction to bird, mouse, and human lure for Cx. pipiens pipiens and Cx. torrentium. Hosts of 992 field-collected specimens were identified for Germany, Iran, and Moldova, with the majority determined as Cx. pipiens pipiens, increasing the data available from studies known from the literature by two-thirds. All four Culex pipiens s.s./Cx. torrentium taxa had fed with significant proportions on birds, humans, and nonhuman mammals. Merged with the data from the literature from 23 different studies showing a high prevalence of blood meals from birds, more than 50% of the blood meals of Cx. pipiens s.s. were identified as birds, while up to 39% were human and nonhuman mammalian hosts. Culex torrentium fed half on birds and half on mammals. However, there were considerable geographical differences in the host feeding patterns. Conclusions: In the light of these results, the clear characterization of the Cx. pipiens s.s./Cx. torrentium taxa as ornithophilic/-phagic or mammalophilic/-phagic needs to be reconsidered. Given their broad host ranges, all four Culex taxa could potentially serve as enzootic and bridge vectors. [ABSTRACT FROM AUTHOR]

Background: Aedes aegypti, the primary vector of dengue, chikungunya, and Zika viruses, poses a significant public health threat worldwide. Traditional control methods using insecticides are increasingly challenged by resistance and environmental concerns. The sterile insect technique (SIT) offers an eco-friendly alternative that has been successfully applied to other insect pests. This article aims to briefly review Ae. aegypti management in Cuba, highlighting the accomplishments, challenges, and future directions of the SIT. Main body: Here we provide a brief summary of the extensive history of Ae. aegypti control efforts in Cuba. After a successful eradication campaign in the 1980s, a resurgence of dengue cases has been observed in recent years, suggesting that traditional control methods may have limited effectiveness under current conditions. In response, Cuba initiated a phased approach to develop and evaluate the feasibility of SIT for Ae. aegypti control, starting in 2008. Initial research focused on Ae. aegypti mating behavior and sterilization methods, followed by successful laboratory and semi-field trials that demonstrated population suppression. The first open-field trial in 2020 confirmed the efficacy of the SIT in reducing Ae. aegypti populations under real-world conditions. Currently, the research is in a phase involving a cluster-randomized superiority-controlled trial. This planned trial will compare the standard vector control program with the same program augmented by the SIT, aiming to assess the impact of the SIT on dengue incidence as the primary outcome. Implementing robust epidemiological trials to evaluate the effectiveness of the SIT is complex due to potential spillover effects from mosquito and human movement across study areas. Additionally, conducting the SIT requires significant development and operational investments. Despite these challenges, the ongoing Cuban trial holds promise for establishing the SIT as an effective and sustainable tool for Ae. aegypti control and for reducing the burden of mosquito-borne diseases. Conclusions: The phased evaluation conducted in Cuba confirms the efficacy of the SIT against Ae. aegypti, highlighting its potential for sustainable mosquito-borne disease management. The effective implementation of multi-site trials will be crucial in providing evidence of the potential of the sterile insect technique as part of a strategy to reduce the incidence of arboviral diseases. [ABSTRACT FROM AUTHOR]

Background: Rift Valley fever virus (RVFV) is a zoonotic mosquito-borne virus with serious implications for livestock health, human health, and the economy in Africa, and is suspected to be endemic in north-eastern KwaZulu-Natal (KZN), South Africa. The vectors of RVFV in this area are poorly known, although several species, such as Aedes (Neomelaniconion) mcintoshi, Aedes (Neomelaniconion) circumluteolus, Aedes (Aedimorphus) durbanensis, and Culex (Lasioconops) poicilipes may be involved. The aim of the study was to determine the vertebrate blood meal sources of potential RVFV mosquito vectors in north-eastern KZN and to characterize the host-biting network. Methods: Blood-fed mosquitoes were collected monthly from November 2019 to February 2023 using a backpack aspirator, CO2-baited Centers for Disease Control and Prevention (CDC) miniature light traps and tent traps, in the vicinity of water bodies and livestock farming households. The mosquitoes were morphologically identified. DNA was extracted from individual mosquitoes and used as templates to amplify the vertebrate cytochrome c oxidase I (COI) and cytochrome b (cytb) genes using conventional polymerase chain reaction (PCR). Amplicons were sequenced and queried in GenBank and the Barcode of Life Data systems to identify the vertebrate blood meal sources and confirm mosquito identifications. All mosquitoes were screened for RVFV using real time reverse transcription (RT)-PCR. Results: We identified the mammalian (88.8%) and avian (11.3%) blood meal sources from 409 blood-fed mosquitoes. Aedes circumluteolus (n = 128) made up the largest proportion of collected mosquitoes. Cattle (n = 195) and nyala (n = 61) were the most frequent domestic and wild hosts, respectively. Bipartite network analysis showed that the rural network consisted of more host-biting interactions than the reserve network. All mosquitoes tested negative for RVFV. Conclusions: Several mosquito species, including Ae. circumluteolus, and vertebrate host species, including cattle and nyala, could play a central role in RVFV transmission. Future research in this region should focus on these species to better understand RVFV amplification. [ABSTRACT FROM AUTHOR]

Introduction: This research aimed to assess housing design consideration features and architects' perceptions of their effectiveness in malaria prevention. Previous studies have highlighted various architectural design elements that can reduce mosquito entry into homes, thus contributing to malaria prevention. This study aims to build on these insights by evaluating specific design features and gathering professional opinions on their effectiveness. Methods: The methodological approach adopted was quantitative, with data collected using a structured questionnaire containing closed-ended questions and a checklist. The observation checklist was used to ascertain mosquito entry reduction features incorporated in 208 systematically selected houses in Garneton East, Kitwe, Zambia. Additionally, a structured questionnaire containing fourteen key design features identified from the literature was administered to 76 architects purposively sampled to establish their perceptions of the effectiveness of these design factors in malaria prevention. Data collected was analyzed using descriptive statistics and exploratory factor analysis. Results: The findings revealed that most houses needed to incorporate design features to reduce mosquito entry. The common features incorporated were ceilings (86.80%), screened air ventilation (59.80%), and window screens (54.40%). The use of window and door screens, as well as mosquito-repellent paint, were perceived as the most effective measures by the architects. Discussion: The results suggest a keen awareness among architects regarding the efficacy of certain design features in malaria prevention. Despite this awareness, the implementation of these features in actual housing designs needs to be improved. This indicates a need for greater effort and adaptation in building regulations to promote the use of mosquito entry reduction features. Enhancing housing designs with these features can significantly reduce malaria morbidity and mortality. Therefore, it is recommended that building regulations be updated to support and encourage the incorporation of mosquito entry reduction features in housing designs. [ABSTRACT FROM AUTHOR]

In the absence of an effective vaccine, integrated vector control (IVC) is the key method for controlling dengue cases. The COVID-19 pandemic lockdown significantly weakened the surveillance, preventive insecticide application, and vector control effectiveness. Therefore, the Department of Health issued a memorandum urging barangays to keep implementing interventions to prevent dengue cases from growing in number. This study aimed to describe the status of the implementation of the IVC component of the National Dengue Prevention and Control Program by the Barangay Health Stations (BHSs) of District 5, Manila City, the Philippines, before the COVID-19 pandemic in 2019 and during the pandemic in 2020. The pandemic affected numerous government programs, so the IVC status before and amid the pandemic is worth describing. The study used a cross-sectional descriptive design with a remote survey being conducted with 144 BHS from 08-23 Feb 2021 using a self-administered questionnaire. Results showed that the BHS conducted larviciding (84.02%), adulticiding either through indoor residual spraying [IRS (86.81%)] or space spraying (77.08%) in 2019. Distributions of insecticide-treated nets [ITN (27.78%)], insecticide-treated curtains [ITC (17.36%)], smoke repellents (54.17%), and skin repellents (50.00%) were also implemented. In 2020 amid the pandemic, 81.94% of BHS implemented larviciding, whereas 83.33% and 77.08% conducted IRS and space spraying, respectively. In addition, ITNs (24.31%), ITCs (17.36%), smoke repellents (52.08%), and skin repellents (44.44%) were also distributed in 2020. Despite the pandemic, most barangays in District 5 continued to implement IVC strategies, which contributed to vector density and possibly prevented an increase in mosquito density that could have led to dengue outbreaks. [ABSTRACT FROM AUTHOR]

Temperature is commonly acknowledged as one of the primary forces driving ectotherm vector populations, most notably by influencing metabolic rates and survival. Although numerous experiments have shown this for a wide variety of organisms, the vast majority has been conducted at constant temperatures and changes therein, while temperature is far from constant in nature, and includes seasonal and diurnal cycles. As fluctuating temperatures have been described to affect metabolic processes at (sub)cellular level, this calls for studies evaluating the relative importance of temperature fluctuations and the changes therein. To gain insight in the effects of temperature fluctuations on ectotherm development, survival, and sex ratio, we developed an inexpensive, easily reproducible, and open‐source, Arduino‐based temperature control system, which emulates natural sinusoidal fluctuations around the average temperature. We used this novel setup to compare the effects of constant (mean) temperatures, most commonly used in experiments, block schemes, and natural sinusoidal fluctuations as well as an extreme variant with twice its amplitude using the cosmopolitan mosquito species Culex pipiens s.l. as a study organism. Our system accurately replicated the preprogrammed temperature treatments under outdoor conditions, even more accurately than traditional methods. While no effects were detected on survival and sex ratio within the ranges of variation evaluated, development was sped up considerably by including temperature fluctuations, especially during pupation, where development under constant temperatures took almost a week (30%) longer than under natural fluctuations. Doubling the amplitude further decreased development time by 1.5 days. These results highlight the importance of including (natural) oscillations in experiments on ectotherm organisms – both aquatic and terrestrial – that use temperature as a variable. Ultimately, these results have major repercussions for downstream effects at larger scales that may be studied with applications such as ecological niche models, disease risk models, and assessing ecosystem services that rely on ectotherm organisms. [ABSTRACT FROM AUTHOR]

Dengue is an arbovirus transmitted by the Aedes spp. mosquito. Approximately 390 million infections occur annually per World Health Organization estimates, with significant increases in infections throughout the last decade. The disease is endemic in warmer climates throughout the world, though cases may also be imported to non-endemic regions by returning travelers. Patients experience a wide variety of symptoms ranging from asymptomatic infection to severe disease requiring critical care. Emergency clinicians should consider the diagnosis of dengue in patients from endemic areas presenting with a flu-like illness, rash, and evidence of bleeding. [ABSTRACT FROM AUTHOR]