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The Anopheles gambiae 1000 Genomes (Ag1000G) Consortium previously utilized deep sequencing methods to catalogue genetic diversity across African An. gambiae populations. We analyzed the complete datasets of 1142 individually sequenced mosquitoes through Microsoft Premonition's Bayesian mixture model based (BMM) metagenomics pipeline. All specimens were confirmed as either An. gambiae sensu stricto (s.s.) or An. coluzzii with a high degree of confidence ( > 98% identity to reference). Homo sapiens DNA was identified in all specimens indicating contamination may have occurred either at the time of specimen collection, preparation and/or sequencing. We found evidence of vertebrate hosts in 162 specimens. 59 specimens contained validated Plasmodium falciparum reads. Human hepatitis B and primate erythroparvovirus-1 viral sequences were identified in fifteen and three mosquito specimens, respectively. 478 of the 1,142 specimens were found to contain bacterial reads and bacteriophage-related contigs were detected in 27 specimens. This analysis demonstrates the capacity of metagenomic approaches to elucidate important vector-host-pathogen interactions of epidemiological significance., (© 2024. The Author(s).)

Mosquitoes belonging to the genus Anopheles are the only vectors of human malaria. Anopheles gibbinsi has been linked to malaria transmission in Kenya, with recent collections in Zambia reporting the mosquito species exhibiting zoophilic and exophilic behavioral patterns with occasional contact with humans. Given the paucity of genetic data, and challenges to identification and molecular taxonomy of the mosquitoes belonging to the Anopheles genus; we report the first complete mitochondrial genome of An. gibbinsi using a genome skimming approach. An Illumina Novaseq 6000 platform was used for sequencing, the length of the mitochondrial genome was 15401 bp, with 78.5% AT content comprised of 37 genes. Phylogenetic analysis by maximum likelihood using concatenation of the 13 protein coding genes demonstrated that An. marshallii was the closest relative based on existing sequence data. This study demonstrates that the skimming approach is an inexpensive and efficient approach for mosquito species identification and concurrent taxonomic rectification, which may be a useful alternative for generating reference sequence data for evolutionary studies among the Culicidae., Competing Interests: No competing interests were disclosed., (Copyright: © 2024 Ali R et al.)

Anopheles gambiae is a highly anthropophilic (human-preferring) malaria vector that prefers to blood feed frequently and selectively on humans. This mosquito species exhibits a strong innate olfactory preference to seek out human scent over other animals, and certain humans over others-key behavioral traits with the potential to drive heterogeneity in biting risk and malaria transmission. Here, we describe the application of a large-scale, semi-field system in Zambia for the quantification of An. gambiae olfactory preferences toward whole body odor sourced from individual humans. We detail steps for modifying one-person canvas tents to duct odor from sleeping humans into a central, semi-field flight cage arena that is securely screened. Using this system, we describe a protocol to perform two-choice olfactory preference assays with two human volunteers using laboratory-reared An. gambiae and odor-guided thermotaxis assays that leverage infrared video tracking to quantify mosquito landings on heated targets baited with each body odor sample. This multichoice olfactory preference assay has the potential to be applied with expanded cohorts of humans to define the chemosensory basis of An. gambiae host preference and interindividual differences in human attractiveness to mosquitoes and to be used to quantify the effects of protective measures such as personal and spatial repellents on mosquito landing behavior., (© 2024 Cold Spring Harbor Laboratory Press.)

The African malaria mosquito Anopheles gambiae is strongly attracted to human body odor and skin temperature. Quantitative behavioral assays suitable for use in semi-field environments with this nocturnal mosquito species are essential to gain improved insights into An. gambiae sensory biology, the mechanistic basis of mosquito attraction to humans, and host preference. In this protocol, we describe steps for engineering equipment for a novel behavioral assay for An. gambiae , which we have termed the odor-guided thermotaxis assay (OGTA). The OGTA uses infrared videography to quantify landings of female An. gambiae on an aluminum platform heated to human skin temperature that can be baited with volatile odorants such as carbon dioxide or human whole body odor. The OGTA facilitates high-content recordings of An. gambiae landing behavior during odor-guided thermotaxis under naturalistic semi-field conditions without the requirement for domestic power., (© 2024 Cold Spring Harbor Laboratory Press.)

The most dangerous mosquito species for human health are those that blood feed preferentially and frequently on humans (anthropophilic mosquitoes). These include prolific disease vectors such as the African malaria mosquito Anopheles gambiae and yellow fever mosquito Aedes aegypti The chemosensory basis for anthropophilic behavior exhibited by these disease vectors, as well as the factors that drive interindividual differences in human attractiveness to mosquitoes, remain largely uncharacterized. Here, we concisely review established methods to quantify mosquito interspecific and intraspecific host preference in the laboratory, as well as semi-field and field environments. Experimental variables for investigator consideration during assays of mosquito host preference across these settings are highlighted., (© 2024 Cold Spring Harbor Laboratory Press.)

Anopheles gambiae uses its sense of smell to hunt humans. We report a two-step method yielding cell-type-specific driver lines for enhanced neuroanatomical and functional studies of its olfactory system. We first integrated a driver-responder-marker (DRM) system cassette consisting of a linked T2A-QF2 driver, QUAS-GFP responder, and a gut-specific transgenesis marker into four chemoreceptor genes (Ir25a, Ir76b, Gr22, and orco) using CRISPR-Cas9-mediated homology-directed repair. The DRM system facilitated rapid selection of in-frame integrations via screening for GFP+ olfactory sensory neurons (OSNs) in G 1 larval progeny, even at genomic loci such as orco where we found the transgenesis marker was not visible. Next, we converted these DRM integrations into T2A-QF2 driver-marker lines by Cre-loxP excision of the GFP responder, making them suitable for binary use in transcuticular calcium imaging. These cell-type-specific driver lines tiling key OSN subsets will support systematic efforts to decode olfaction in this prolific malaria vector., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Long-acting injectable medications, such as atovaquone, offer the prospect of a "chemical vaccine" for malaria, combining drug efficacy with vaccine durability. However, selection and transmission of drug-resistant parasites is of concern. Laboratory studies have indicated that atovaquone resistance disadvantages parasites in mosquitoes, but lack of data on clinically relevant Plasmodium falciparum has hampered integration of these variable findings into drug development decisions. Here we generate atovaquone-resistant parasites that differ from wild type parent by only a Y268S mutation in cytochrome b, a modification associated with atovaquone treatment failure in humans. Relative to wild type, Y268S parasites evidence multiple defects, most marked in their development in mosquitoes, whether from Southeast Asia (Anopheles stephensi) or Africa (An. gambiae). Growth of asexual Y268S P. falciparum in human red cells is impaired, but parasite loss in the mosquito is progressive, from reduced gametocyte exflagellation, to smaller number and size of oocysts, and finally to absence of sporozoites. The Y268S mutant fails to transmit from mosquitoes to mice engrafted with human liver cells and erythrocytes. The severe-to-lethal fitness cost of clinically relevant atovaquone resistance to P. falciparum in the mosquito substantially lessens the likelihood of its transmission in the field., (© 2023. Springer Nature Limited.)

The mosquito larval midgut is responsible for acquiring and storing most of the nutrients that will sustain the events of metamorphosis and the insect's adult life. Despite its importance, the basic biology of this larval organ is poorly understood. To help fill this gap, we carried out a comparative morphophysiological investigation of three larval midgut regions (gastric caeca, anterior midgut, and posterior midgut) of phylogenetically distant mosquitoes: Anopheles gambiae (Anopheles albimanus was occasionally used as an alternate), Aedes aegypti, and Toxorhynchites theobaldi. Larvae of Toxorhynchites mosquitoes are predacious, in contrast to the other two species, that are detritivorous. In this work, we show that the larval gut of the three species shares basic histological characteristics, but differ in other aspects. The lipid and carbohydrate metabolism of the An. gambiae larval midgut is different compared with that of Ae. aegypti and Tx. theobaldi. The gastric caecum is the most variable region, with differences probably related to the chemical composition of the diet. The peritrophic matrix is morphologically similar in the three species, and processes involved in the post-embryonic development of the organ, such as cell differentiation and proliferation, were also similar. FMRF-positive enteroendocrine cells are grouped in the posterior midgut of Tx. theobaldi, but individualized in An. gambiae and Ae. aegypti. We hypothesize that Tx. theobaldi larval predation is an ancestral condition in mosquito evolution., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The African malaria mosquito Anopheles gambiae exhibits a strong innate drive to seek out humans in its sensory environment, classically entering homes to land on human skin in the hours flanking midnight. To gain insight into the role that olfactory cues emanating from the human body play in generating this epidemiologically important behavior, we developed a large-scale multi-choice preference assay in Zambia with infrared motion vision under semi-field conditions. We determined that An. gambiae prefers to land on arrayed visual targets warmed to human skin temperature during the nighttime when they are baited with carbon dioxide (CO 2 ) emissions reflective of a large human over background air, body odor from one human over CO 2 , and the scent of one sleeping human over another. Applying integrative whole body volatilomics to multiple humans tested simultaneously in competition in a six-choice assay, we reveal high attractiveness is associated with whole body odor profiles from humans with increased relative abundances of the volatile carboxylic acids butyric acid, isobutryic acid, and isovaleric acid, and the skin microbe-generated methyl ketone acetoin. Conversely, those least preferred had whole body odor that was depleted of carboxylic acids among other compounds and enriched with the monoterpenoid eucalyptol. Across expansive spatial scales, heated targets without CO 2 or whole body odor were minimally or not attractive at all to An. gambiae. These results indicate that human scent acts critically to guide thermotaxis and host selection by this prolific malaria vector as it navigates towards humans, yielding intrinsic heterogeneity in human biting risk., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Despite efforts to minimize the impacts of malaria and reduce the number of primary vectors, malaria has yet to be eliminated in Zambia. Understudied vector species may perpetuate malaria transmission in pre-elimination settings. Anopheles squamosus is one of the most abundantly caught mosquito species in southern Zambia and has previously been found with Plasmodium falciparum sporozoites, a causal agent of human malaria. This species may be a critical vector of malaria transmission, however, there is a lack of genetic information available for An. squamosus. We report the first genome data and the first complete mitogenome (Mt) sequence of An. squamosus. The sequence was extracted from one individual mosquito from the Chidakwa area in Macha, Zambia. The raw reads were obtained using Illumina Novaseq 6000 and assembled through NOVOplasty alignment with related species. The length of the An. squamosus Mt was 15,351 bp, with 77.9 % AT content. The closest match to the whole mitochondrial genome in the phylogenetic tree is the African malaria mosquito, Anopheles gambiae. Its genome data is available through National Center for Biotechnology Information (NCBI) Sequencing Reads Archive (SRA) with accession number SRR22114392. The mitochondrial genome was deposited in NCBI GenBank with the accession number OP776919. The ITS2 containing contig sequence was deposited in GenBank with the accession number OQ241725. Mitogenome annotation and a phylogenetic tree with related Anopheles mosquito species are provided., Competing Interests: No competing interests were disclosed., (Copyright: © 2023 Nguyen VT et al.)

As the malaria parasite becomes resistant to every drug that we develop, the identification and development of novel drug candidates are essential. Many studies have screened compounds designed to target the clinically important blood stages. However, if we are to shrink the malaria map, new drugs that block the transmission of the parasite are needed. Sporozoites are the infective stage of the malaria parasite, transmitted to the mammalian host as mosquitoes probe for blood. Sporozoite motility is critical to their ability to exit the inoculation site and establish infection, and drug-like compounds targeting motility are effective at blocking infection in the rodent malaria model. In this study, we established a moderate-throughput motility assay for sporozoites of the human malaria parasite Plasmodium falciparum, enabling us to screen the 400 drug-like compounds from the pathogen box provided by the Medicines for Malaria Venture for their activity. Compounds exhibiting inhibitory effects on P. falciparum sporozoite motility were further assessed for transmission-blocking activity and asexual-stage growth. Five compounds had a significant inhibitory effect on P. falciparum sporozoite motility in the nanomolar range. Using membrane feeding assays, we demonstrate that four of these compounds had inhibitory activity against the transmission of P. falciparum to the mosquito. Interestingly, of the four compounds with inhibitory activity against both transmission stages, three are known kinase inhibitors. Together with a previous study that found that several of these compounds could inhibit asexual blood-stage parasite growth, our findings provide new antimalarial drug candidates that have multistage activity.

Background: Nchelenge District in northern Zambia suffers from holoendemic malaria transmission despite a decade of yearly indoor residual spraying (IRS) and insecticide-treated net (ITN) distributions. One hypothesis for this lack of impact is that some vectors in the area may forage in the early evening or outdoors. Anopheles gibbinsi specimens were identified in early evening mosquito collections performed in this study area, and further insight was gleaned into this taxon, including characterizing its genetic identity, feeding preferences, and potential role as a malaria vector., Methods: Mosquitoes were collected in July and August 2019 by CDC light traps in Nchelenge District in indoor sitting rooms, outdoor gathering spaces, and animal pens from 16:00-22:00. Host detection by PCR, COI and ITS2 PCR, and circumsporozoite (CSP) ELISA were performed on all samples morphologically identified as An. gibbinsi, and a subset of specimens were selected for COI and ITS2 sequencing. To determine risk factors for increased abundance of An. gibbinsi, a negative binomial generalized linear mixed-effects model was performed with household-level variables of interest., Results: Comparison of COI and ITS2 An. gibbinsi reference sequences to the NCBI database revealed > 99% identity to "Anopheles sp. 6" from Kenya. More than 97% of specimens were morphologically and molecularly consistent with An. gibbinsi. Specimens were primarily collected in animal pen traps (59.2%), followed by traps outdoors near where humans gather (24.3%), and traps set indoors (16.5%). Host DNA detection revealed a high propensity for goats, but 5% of specimens with detected host DNA had fed on humans. No specimens were positive for Plasmodium falciparum sporozoites. Animal pens and inland households > 3 km from Lake Mweru were both associated with increased An. gibbinsi abundance., Conclusions: This is the first report of An. gibbinsi in Nchelenge District, Zambia. This study provided a species identity for unknown "An. sp. 6" in the NCBI database, which has been implicated in malaria transmission in Kenya. Composite data suggest that this species is largely zoophilic and exophilic, but comes into contact with humans and the malaria parasites they carry. This species should continue to be monitored in Zambia and neighbouring countries as a potential malaria vector., (© 2022. The Author(s).)

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) administration has been accomplished primarily via injection, which requires significant time and is not suitable for field applications. To overcome these limitations, here we present a more efficient method for robust activation of RNAi by oral delivery of dsRNA to adult Anopheles gambiae. Long dsRNAs were produced in Escherichia coli strain HT115 (DE3), and a concentrated suspension of heat-killed dsRNA-containing bacteria in 10% sucrose was offered on cotton balls ad-libitum to adult mosquitoes. Cotton balls were replaced every 2 days for the duration of the treatment. Use of this method to target doublesex (a gene involved in sex differentiation) or fork head (which encodes a salivary gland transcription factor) resulted in reduced target gene expression and/or protein immunofluorescence signal, as measured by quantitative Real-Time PCR (qRT-PCR) or fluorescence confocal microscopy, respectively. Defects in salivary gland morphology were also observed. This highly flexible, user-friendly, low-cost, time-efficient method of dsRNA delivery could be broadly applicable to target genes important for insect vector physiology and beyond.

The stalling global progress in the fight against malaria prompts the urgent need to develop new intervention strategies. Whilst engineered symbiotic bacteria have been shown to confer mosquito resistance to parasite infection, a major challenge for field implementation is to address regulatory concerns. Here, we report the identification of a Plasmodium-blocking symbiotic bacterium, Serratia ureilytica Su_YN1, isolated from the midgut of wild Anopheles sinensis in China that inhibits malaria parasites via secretion of an antimalarial lipase. Analysis of Plasmodium vivax epidemic data indicates that local malaria cases in Tengchong (Yunnan province, China) are significantly lower than imported cases and importantly, that the local vector A. sinensis is more resistant to infection by P. vivax than A. sinensis from other regions. Analysis of the gut symbiotic bacteria of mosquitoes from Yunnan province led to the identification of S. ureilytica Su_YN1. This bacterium renders mosquitoes resistant to infection by the human parasite Plasmodium falciparum or the rodent parasite Plasmodium berghei via secretion of a lipase that selectively kills parasites at various stages. Importantly, Su_YN1 rapidly disseminates through mosquito populations by vertical and horizontal transmission, providing a potential tool for blocking malaria transmission in the field.

Mosquitoes are the greatest animal threat to human health, causing hundreds of millions of infections and around 1 million deaths each year. All mosquito-borne pathogens must traverse the salivary glands (SGs) to be transmitted to the next host, making this organ an ideal target for interventions. The adult SG develops from precursor cells located in the larval SG duct bud. Characterization of the larval SG has been limited. We sought to better understand larval SG architecture, secretion and gene expression. We developed an optimized method for larval SG staining and surveyed hundreds of larval stage 4 (L4) SGs using fluorescence confocal microscopy. Remarkable variation in SG cell and chromatin organization differed among individuals and across the L4 stage. Lumen formation occurred during L4 stage through secretion likely involving a coincident cellular apical lipid enrichment and extracellular vesicle-like structures. Meta-analysis of microarray data showed that larval SG gene expression is divergent from adult SGs, more similar to larval gastric cecae, but different from other larval gut compartments. This work highlights the variable cell architecture of larval Anopheles gambiae SGs and provides candidate targets for genetic strategies aiming to disrupt SGs and transmission of mosquito-borne pathogens., (© 2020 The Royal Entomological Society.)

The global malaria burden has decreased substantially, but gains have been uneven both within and between countries. In Zambia, the malaria burden remains high in northern and eastern regions of the country. To effectively reduce malaria transmission in these areas, evidence-based intervention strategies are needed. Zambia's National Malaria Control Centre conducted targeted indoor residual spraying (IRS) in 40 high-burden districts from 2014 to 2016 using the novel organophosphate insecticide pirimiphos-methyl. The Southern and Central Africa International Centers of Excellence for Malaria Research conducted an evaluation of the impact of the IRS campaign on household vector abundance in Nchelenge District, Luapula Province. From April 2012 to July 2017, field teams conducted indoor overnight vector collections from 25 to 30 households per month using Centers for Disease Control light traps. Changes in indoor anopheline counts before versus after IRS were assessed by species using negative binomial regression models with robust standard errors, controlling for geographic and climatological covariates. Counts of Anopheles funestus declined by approximately 50% in the study area and within areas targeted for IRS, and counts of Anopheles gambiae declined by approximately 40%. Within targeted areas, An. funestus counts declined more in sprayed households than in unsprayed households; however, this relationship was not observed for An. gambiae. The moderate decrease in indoor vector abundance indicates that IRS with pirimiphos-methyl is an effective vector control measure, but a more comprehensive package of interventions is needed with sufficient coverage to effectively reduce the malaria burden in this setting.

Despite ongoing malaria control efforts implemented throughout sub-Saharan Africa, malaria remains an enormous public health concern. Current interventions such as indoor residual spraying with insecticides and use of insecticide-treated bed nets are aimed at targeting the key malaria vectors that are primarily endophagic and endophilic. Anopheles coustani s.l., an understudied vector of malaria, is a species previously thought to exhibit mostly zoophilic behavior. Like many of these understudied species, An. coustani has greater anthropophilic tendencies than previously appreciated, is often both endophagic and exophagic, and carries Plasmodium falciparum sporozoites. The aim of this study was to explore genetic variation of An. coustani mosquitoes and the potential of this species to contribute to malaria parasite transmission in high transmission settings in Zambia and the Democratic Republic of the Congo (DRC). Morphologically identified An. coustani specimens that were trapped outdoors in these study sites were analyzed by PCR and sequencing for species identification and bloodmeal sources, and malaria parasite infection was determined by ELISA and qPCR. Fifty An. coustani s.s. specimens were confirmed by analysis of mitochondrial DNA cytochrome c oxidase subunit I (COI) and ribosomal internal transcribed spacer region 2 (ITS2). Maximum likelihood phylogenetic analysis of COI and ITS2 sequences revealed two distinct phylogenetic groups within this relatively small regional collection. Our findings indicate that both An. coustani groups have anthropophilic and exophagic habits and come into frequent contact with P. falciparum, suggesting that this potential alternative malaria vector might elude current vector control measures in northern Zambia and southern DRC., (© The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Malaria remains one of the most important public health problems, causing significant morbidity and mortality. Malaria is a mosquito borne disease transmitted through an infectious bite from the female Anopheles mosquito. Malaria control will eventually rely on a multitude of approaches, which includes ways to block transmission to, through and from mosquitoes. To study mosquito stages of malaria parasites in the laboratory, we have optimized a protocol to culture highly infectious Plasmodium falciparum gametocytes, a parasite stage required for transmission from the human host to the mosquito vector. P. falciparum gametocytes mature through five morphologically distinct steps, which takes approximately 1-2 weeks. Gametocyte culture described in this protocol is completed in 15 days and are infectious to mosquitoes from days 15-18. These protocols were developed to maintain a continuous cycle of infection competent gametocytes and to maintain uninterrupted supply of mosquito stages of the parasite. Here, we describe the methodology of gametocyte culture and how to infect mosquitoes with these parasites using glass membrane feeders.

Although transmission of malaria and other mosquito-borne diseases is geographically heterogeneous, in sub-Saharan Africa risk maps are rarely used to determine which communities receive vector control interventions. We compared outcomes in areas receiving different indoor residual spray (IRS) strategies in Eastern Province, Zambia: (1) concentrating IRS interventions within a geographical area, (2) prioritizing communities to receive IRS based on predicted probabilities of Anopheles funestus, and (3) prioritizing communities to receive IRS based on observed malaria incidence at nearby health centers. Here we show that the use of predicted probabilities of An. funestus to guide IRS implementation saw the largest decrease in malaria incidence at health centers, a 13% reduction (95% confidence interval = 5-21%) compared to concentrating IRS geographically and a 37% reduction (95% confidence interval = 30-44%) compared to targeting IRS based on health facility incidence. These results suggest that vector control programs could produce better outcomes by prioritizing IRS according to malaria-vector risk maps.

Despite the annual implementation of a robust and extensive indoor residual spraying programme against malaria vectors in Limpopo Province (South Africa), significant transmission continues and is a serious impediment to South Africa's malaria elimination objectives. In order to gain a better understanding regarding possible causes of this residual malaria, we conducted a literature review of the historical species composition and abundance of malaria vector mosquitoes in the Limpopo River Valley region of the Vhembe District, northern Limpopo Province, the region with the highest remaining annual malaria cases in South Africa. In addition, mosquito surveys were carried out in the same region between October 2017 and October 2018. A total of 2225 adult mosquitoes were collected using CO 2 -baited tent and light traps, human landing catches and cow-baited traps. Of the 1443 Anopheles collected, 516 were members of the An. gambiae complex and 511 An. funestus group. In the malaria endemic rural areas outside the Kruger National Park, one specimen each of An. gambiae s.s. and An. funestus and only three of An. arabiensis were collected. The latter species was abundant at a remote hot spring in the neighboring Kruger National Park. Eighteen other species of Anopheles were collected. Our survey results support the historical findings that An. arabiensis , the species widely held to be the prime malaria vector in South Africa, is a rare species in the malaria endemic Limpopo River Valley. The implications of the mosquito surveys for malaria transmission, elimination and vector control in northern Limpopo Province and neighboring regions are discussed.

Competing Interests: The authors declare no competing interest.

The mosquito Anopheles gambiae s.s . is distributed across most of sub-Saharan Africa and is of major scientific and public health interest for being an African malaria vector. Here we present population genomic analyses of 111 specimens sampled from west to east Africa, including the first whole genome sequences from oceanic islands, the Comoros. Genetic distances between populations of A.   gambiae are discordant with geographic distances but are consistent with a stepwise migration scenario in which the species increases its range from west to east Africa through consecutive founder events over the last ~200,000 years. Geological barriers like the Congo River basin and the East African rift seem to play an important role in shaping this process. Moreover, we find a high degree of genetic isolation of populations on the Comoros, confirming the potential of these islands as candidate sites for potential field trials of genetically engineered mosquitoes for malaria control., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2019.)

Plasmodium sporozoites (SPZs) must traverse the mosquito salivary glands (SGs) to reach a new vertebrate host and continue the malaria disease cycle. Although SGs can harbor thousands of sporozoites, only 10 to 100 are deposited into a host during probing. To determine how the SGs might function as a bottleneck in SPZ transmission, we have characterized Anopheles stephensi SGs infected with the rodent malaria parasite Plasmodium berghei using immunofluorescence confocal microscopy. Our analyses corroborate findings from previous electron microscopy studies and provide new insights into the invasion process. We identified sites of SPZ accumulation within SGs across a range of infection intensities. Although SPZs were most often seen in the distal lateral SG lobes, they were also observed in the medial and proximal lateral lobes. Most parasites were associated with either the basement membrane or secretory cavities. SPZs accumulated at physical barriers, including fused salivary ducts and extensions of the chitinous salivary duct wall into the distal lumen. SPZs were observed only rarely within salivary ducts. SPZs appeared to contact each other in many different quantities, not just in the previously described large bundles. Within parasite bundles, all of the SPZs were oriented in the same direction. We found that moderate levels of infection did not necessarily correlate with major SG disruptions or abundant SG cell death. Altogether, our findings suggest that SG architecture largely acts as a barrier to SPZ transmission. IMPORTANCE Malaria continues to have a devastating impact on human health. With growing resistance to insecticides and antimalarial drugs, as well as climate change predictions indicating expansion of vector territories, the impact of malaria is likely to increase. Additional insights regarding pathogen migration through vector mosquitoes are needed to develop novel methods to prevent transmission to new hosts. Pathogens, including the microbes that cause malaria, must invade the salivary glands (SGs) for transmission. Since SG traversal is required for parasite transmission, SGs are ideal targets for transmission-blocking strategies. The work presented here highlights the role that mosquito SG architecture plays in limiting parasite traversal, revealing how the SG transmission bottleneck is imposed. Further, our data provide unprecedented detail about SG-sporozoite interactions and gland-to-gland variation not provided in previous studies., (Copyright © 2019 Wells and Andrew.)

Anopheles jeyporiensis and Anopheles nivipes appear to play an important role in contemporary malaria transmission in Bangladesh. However, very little is known about the natural host selection of these vectors. Therefore, host selection of these two species was investigated in Bandarban, the most malarious region of Bangladesh. A total of 480 engorged mosquitoes were analyzed. The human blood index (HBI) of An. jeyporiensis varied from 4.17% in outdoor to 19.17% in indoor collections. Similarly, HBI of An. nivipes ranged between 0.83% and 22.50% from outdoor to indoor. For both species, cow blood indices were significantly higher than HBI in both indoor and outdoor collections. These data demonstrate the opportunistic and zoophilic nature of An. jeyporiensis and An. nivipes , which suggests that approaches such as zooprophylasis may prove beneficial as a control strategy.

Background: The emergence of mosquitoes that can avoid indoor-deployed interventions, such as treated bed nets and indoor residual spraying, threatens the mainstay of malaria control in Zambia. Furthermore, the requirement for high coverage of these tools poses operational challenges. Spatial repellents are being assessed to supplement these vector control tools, but limitations exist in the residual effect of the repellent and the need for external power or heat for diffusion of the volatiles., Methods: A semi-field evaluation of a novel controlled release spatial repellent device (CRD) was conducted in Macha, Zambia. These devices emanate metofluthrin with no need for external power. Devices were deployed in huts within the semi-field system (SFS). Female Anopheles gambiae sensu stricto released within the SFS were trapped overnight by light traps and collected by aspiration the next morning inside and outside of huts to determine the extent of mosquito repellency and the impact on host-seeking and survival. Experiments studied the impact of number of devices as well as the presence of hut occupants. The study was complemented with numerical methods based on computational fluid dynamics to simulate spatial distribution of metofluthrin., Results: Presence of CRDs was associated with significant reductions in indoor counts of mosquitoes, regardless of whether huts were occupied or not. Repellency ranged from 15 to 60% compared to huts with no devices. Reducing the number of devices from 16 to 4 had little impact on repellency. When huts were occupied, indoor mosquito host-seeking was higher in the presence of CRDs, whilst survival was significantly reduced., Conclusions: This study demonstrated that deployment of as few as four CRDs within a hut was associated with reduced indoor mosquito densities. As would be expected, presence of occupants within huts, resulted in greater indoor catches (both with and without devices). The increased indoor mosquito host-seeking and mortality in huts when devices were present may be explained by the excito-repellency activity of metofluthrin. These semi-field experiments provide preliminary data on the utility of CRD spatial repellents to reduce indoor densities of An. gambiae mosquitoes. Studies will further investigate the impact of CRDs on mosquito behaviour as well as epidemiological protective efficacy.

Anopheles funestus s.s. is a primary vector of malaria in sub-Saharan Africa. Despite its important role in human Plasmodium transmission, evolutionary history, genetic diversity, and population structure of An. funestus in southern and central Africa remains understudied. We deep sequenced, assembled, and annotated the complete mitochondrial genome of An. funestus s.s. for the first time, providing a foundation for further genetic research of this important malaria vector species. We further analyzed the complete mitochondrial genomes of 43 An. funestus s.s. from three sites in Zambia, Democratic Republic of the Congo, and Tanzania. From these 43 mitogenomes we identified 41 unique haplotypes that comprised 567 polymorphic sites. Bayesian phylogenetic reconstruction confirmed the co-existence of two highly divergent An. funestus maternal lineages, herein defined as lineages I and II, in Zambia and Tanzania. The estimated coalescence time of these two mitochondrial lineages is ~500,000 years ago (95% HPD 426,000-594,000 years ago) with subsequent independent diversification. Haplotype network and phylogenetic analysis revealed two major clusters within lineage I, and genetic relatedness of samples with deep branching in lineage II. At this time, data suggest that the lineages are partially sympatric. This study illustrates that accurate retrieval of full mitogenomes of Anopheles vectors enables fine-resolution studies of intraspecies genetic relationships, population differentiation, and demographic history. Further investigations on whether An. funestus mitochondrial lineages represent biologically meaningful populations and their potential implications for malaria vector control are warranted.

Malaria is a major health burden in the border-belt areas of Bangladesh. There are recent data from adult mosquito collections that a number of vectors are involved in the transmission cycle. However, little information regarding the larval habitats of Anopheles mosquitoes are available in Bangladesh. To start filling this gap, a cross-sectional larval survey was conducted in Bandarban, Bangladesh from October 2011 to September 2012. Descriptive analysis, Poisson regression, spearman correlations and zero-inflated Poisson regression were used to calculate the degree of association between the abundance of larval Anopheles species and environmental factors. From the 300 larval habitats sampled, 5,568 Anopheles larvae were collected and of these, 2,263 (40.6%) were identified to species. Collections represented 16 Anopheles species with Anopheles vagus (26.4%, n = 598) as the dominant species. A total of 16 Anopheles larval habitat types were identified. Larval abundance was significantly different (P < 0.05) among habitats with pond (40%, n = 914) and rice field (34%, n = 779) implicated to be the most utilized. Larval abundance varied significantly (P < 0.05) with habitat characteristics. Most of the larvae were collected from sites with a range of pH from 7.0 to 8.0. Data obtained from this study revealed both natural and human-created larval habitats were favorable for anopheline larval survival and development. Such information elucidates plausible drivers of high anopheline diversity, high vector abundance, changes in relative species abundance from historic data, and sustained transmission of malaria in endemic areas of Bangladesh.

K + channels are integral membrane proteins, which contribute to maintain vital parameters such as the cellular membrane potential and cell volume. Malaria parasites encode two K + channel homologues, Kch1 and Kch2, which are well-conserved among members of the Plasmodium genus. In the rodent malaria parasite P. berghei, the functional significance of K + channel homologue PbKch2 was studied using targeted gene knock-out. The knockout parasites were characterized in a mouse model in terms of growth-kinetics and infectivity in the mosquito vector. Furthermore, using a tracer-uptake technique with 86 Rb + as a K + congener, the K + transporting properties of the knockout parasites were assessed., Results: Genetic disruption of Kch2 did not grossly affect the phenotype in terms of asexual replication and pathogenicity in a mouse model. In contrast to Kch1-null parasites, Kch2-null parasites were fully capable of forming oocysts in female Anopheles stephensi mosquitoes. 86 Rb + uptake in Kch2-deficient blood-stage P. berghei parasites (Kch2-null) did not differ from that of wild-type (WT) parasites. About two-thirds of the 86 Rb + uptake in WT and in Kch2-null parasites could be inhibited by K + channel blockers and could be inferred to the presence of functional Kch1 in Kch2 knockout parasites. Kch2 is therefore not required for transport of K + in P. berghei and is not essential to mosquito-stage sporogonic development of the parasite., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Mosquito-borne diseases cause one million deaths and hundreds of millions of human infections yearly. With all such diseases, the pathogen must traverse the mosquito salivary gland (SG) for transmission to a new host, making the SGs ideal targets for genetic strategies to block transmission. Prior studies have elucidated details of SG structure by light and electron microscopy and have deeply explored the salivary transcriptome and proteome. Very little is known, however, about how the unique functional architecture of mosquito SGs is achieved. Using immunohistochemistry and confocal microscopy, we address two questions regarding SGs of the malaria vector Anopheles gambiae. How does the distinct cup-shaped morphology of SG secretory cells arise? And, how does the salivary duct, the structure through which saliva and parasites exit the glands, form? We demonstrate that SG cells begin as cuboidal-shaped cells surrounding a matrix-filled lumen that mature into cup-shaped cells through the formation and fusion of a large pre-apical compartment (PAC) to the apical surface. The secretory duct begins as buds of chitin at the apical surface of individual secretory cells. Further chitin deposition connects these chitin buds to form a contiguous duct that largely separates from the apical surface during PAC fusion.

Background: A commonly used measure of malaria transmission intensity is the entomological inoculation rate (EIR), defined as the product of the human biting rate (HBR) and sporozoite infection rate (SIR). The EIR excludes molecular parameters that may influence vector control and surveillance strategies. The purpose of this study was to investigate Anopheles multiple blood feeding behavior (MBF) and Plasmodium falciparum multiplicity of infection (MOI) within the mosquito host in Nchelenge District, northern Zambia. Mosquitoes were collected from light traps and pyrethroid spray catch in Nchelenge in the 2013 wet season. All anophelines were tested for blood meal host, P. falciparum, and MOI using PCR. Circumsporozoite (CSP) ELISA and microsatellite analysis were performed to detect parasites in the mosquito and MBF, respectively. Statistical analyses used regression models to assess MBF and MOI and exact binomial test for human sex bias. Both MBF and MOI can enhance our understanding of malaria transmission dynamics beyond what is currently understood through conventional EIR estimates alone., Results: The dominant malaria vectors collected in Nchelenge were Anopheles funestus (sensu stricto) and An. gambiae (s.s.) The EIRs of An. funestus (s.s.) and An. gambiae (s.s.) were 39.6 infectious bites/person/6 months (ib/p/6mo) and 5.9 ib/p/6mo, respectively, and took multiple human blood meals at high rates, 23.2 and 25.7% respectively. There was no bias in human host sex preference in the blood meals. The SIR was further characterized for parasite genetic diversity. The overall P. falciparum MOI was 6.4 in infected vectors, exceeding previously reported average MOIs in humans in Africa., Conclusions: Both Anopheles MBF rates and P. falciparum MOI in Nchelenge were among some of the highest reported in sub-Saharan Africa. The results suggest an underestimation of the EIR and large numbers of circulating parasite clones. Together, the results describe important molecular aspects of transmission excluded from the traditional EIR measurement. These elements may provide more sensitive measures with which to assess changes in transmission intensity and risk in vector and parasite surveillance programs.

Southern Zambia is the focus of strategies to create malaria-free zones. Interventions being rolled out include test and treat strategies and distribution of insecticide-treated bed nets that target vectors that host-seek indoors and late at night. In Macha, Choma District, collections of mosquitoes were made outdoors using barrier screens within homesteads or UV bulb light traps set next to goats, cattle, or chickens during the rainy season of 2015. Anopheline mosquitoes were identified to species using molecular methods and Plasmodium falciparum infectivity was determined by ELISA and real-time qPCR methods. More than 40% of specimens caught were identified as Anopheles squamosus Theobald, 1901 of which six were found harboring malaria parasites. A single sample, morphologically identified as Anopheles coustani Laveran, 1900, was also found to be infectious. All seven specimens were caught outdoors next to goat pens. Parasite-positive specimens as well as a subset of An. squamosus specimens from either the same study or archive collections from the same area underwent sequencing of the mitochondrial cytochrome oxidase subunit I gene. Maximum parsimony trees constructed from the aligned sequences indicated presence of at least two clades of An. squamosus with infectious specimens falling in each clade. The single infectious specimen identified morphologically as An. coustani could not be matched to reference sequences. This is the first report from Zambia of infections in An. squamosus, a species which is described in literature to display exophagic traits. The bionomic characteristics of this species needs to be studied further to fully evaluate the implications for indoor-targeted vector control., (© The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background Information: Anopheles gambiae is the major mosquito vector for Plasmodium falciparum malaria in sub-Saharan Africa, where it survives in stressful climates. Aquaporin water channels are expressed in all life forms, where they provide environmental adaptation by conferring rapid trans-cellular movement of water (classical aquaporins) or water plus glycerol (aquaglyceroporins). Here, we report an aquaglyceroporin homolog in A. gambiae, AgAQP3 (A. gambiae aquaglyceroporin 3)., Results: Despite atypical pore-lining amino acids, AgAQP3 is permeated by water, glycerol and urea, and is not significantly inhibited by 1 mM HgCl 2 . AgAQP3 is expressed more heavily in male mosquitoes, yet adult female A. gambiae abundantly express AgAQP3 in Malpighian tubules and gut where large amounts of fluid exchange occur during blood meal digestion, water and nutrient absorption and waste secretion. Reducing expression of AgAQP3 by RNA interference reduces median mosquito survival at 39°C. After an infectious blood meal, mosquitoes with depleted AgAQP3 expression exhibit fewer P. falciparum oocysts in the midgut compared to control mosquitoes., Conclusions: Our studies reveal critical contributions of AgAQP3 to A. gambiae heat tolerance and P. falciparum development in vivo., Significance: This study indicates that AgAQP3 may be a major factor explaining why A. gambiae is an important malaria vector mosquito in sub-Saharan Africa, and may be a potential target for novel malaria control strategies., (© 2016 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.)

Nchelenge District in Luapula Province, northern Zambia, experiences holoendemic malaria despite implementation of vector control programs. The major Anopheles vectors that contribute to Plasmodium falciparum transmission in this area had not previously been well defined. Three collections performed during the 2012 wet and dry seasons and the 2013 wet season revealed Anopheles funestus sensu stricto and Anopheles gambiae sensu stricto as the main vectors, where 80-85% of each collection was composed of An. funestus Both vectors were found to be highly anthropophilic, and An. funestus has higher sporozoite infection rates (SIRs) and entomological inoculation rates (EIRs) year-round compared with An. gambiae: SIRs of 1.8-3.0% and 0-2.5%, respectively, and EIRs of 3.7-41.5 infectious bites per 6-month period (ib/p/6mo) and 0-5.9 ib/p/6mo, respectively. Spatial and temporal changes in each vector's dynamics and bionomics were also observed. Anopheles funestus was the predominant vector in the villages near Kenani Stream in both wet and dry seasons, whereas An. gambiae was found to be the main vector in areas near Lake Mweru during the wet season. The vector data illustrate the need for broader temporal and spatial sampling in Nchelenge and present unique opportunities to further our understanding of malarial transmission and implications for malarial control in high-risk areas., (© The American Society of Tropical Medicine and Hygiene.)

The success of mosquito-based malaria control is dependent upon susceptible bionomic traits in local malaria vectors. It is crucial to have accurate and reliable methods to determine mosquito species composition in areas subject to malaria. An unexpectedly diverse set of Anopheles species was collected in the western Kenyan highlands, including unidentified and potentially new species carrying the malaria parasite Plasmodium falciparum. This study identified 2,340 anopheline specimens using both ribosomal DNA internal transcribed spacer region 2 and mitochondrial DNA cytochrome oxidase subunit 1 loci. Seventeen distinct sequence groups were identified. Of these, only eight could be molecularly identified through comparison to published and voucher sequences. Of the unidentified species, four were found to carry P. falciparum by circumsporozoite enzyme-linked immunosorbent assay and polymerase chain reaction, the most abundant of which had infection rates comparable to a primary vector in the area, Anopheles funestus. High-quality adult specimens of these unidentified species could not be matched to museum voucher specimens or conclusively identified using multiple keys, suggesting that they may have not been previously described. These unidentified vectors were captured outdoors. Diverse and unknown species have been incriminated in malaria transmission in the western Kenya highlands using molecular identification of unusual morphological variants of field specimens. This study demonstrates the value of using molecular methods to compliment vector identifications and highlights the need for accurate characterization of mosquito species and their associated behaviors for effective malaria control., (© The American Society of Tropical Medicine and Hygiene.)

The understanding of malaria vector species in association with their bionomic traits is vital for targeting malaria interventions and measuring effectiveness. Many entomological studies rely on morphological identification of mosquitoes, limiting recognition to visually distinct species/species groups. Anopheles species assignments based on ribosomal DNA ITS2 and mitochondrial DNA COI were compared to morphological identifications from Luangwa and Nyimba districts in Zambia. The comparison of morphological and molecular identifications determined that interpretations of species compositions, insecticide resistance assays, host preference studies, trap efficacy, and Plasmodium infections were incorrect when using morphological identification alone. Morphological identifications recognized eight Anopheles species while 18 distinct sequence groups or species were identified from molecular analyses. Of these 18, seven could not be identified through comparison to published sequences. Twelve of 18 molecularly identified species (including unidentifiable species and species not thought to be vectors) were found by PCR to carry Plasmodium sporozoites - compared to four of eight morphological species. Up to 15% of morphologically identified Anopheles funestus mosquitoes in insecticide resistance tests were found to be other species molecularly. The comprehension of primary and secondary malaria vectors and bionomic characteristics that impact malaria transmission and intervention effectiveness are fundamental in achieving malaria elimination.

Background: The human population in the highlands of Nyanza Province, western Kenya, is subject to sporadic epidemics of Plasmodium falciparum. Indoor residual spraying (IRS) and long-lasting insecticide treated nets (LLINs) are used widely in this area. These interventions are most effective when Anopheles rest and feed indoors and when biting occurs at times when individuals use LLINs. It is therefore important to test the current assumption of vector feeding preferences, and late night feeding times, in order to estimate the extent to which LLINs protect the inhabitants from vector bites., Methods: Mosquito collections were made for six consecutive nights each month between June 2011 and May 2012. CDC light-traps were set next to occupied LLINs inside and outside randomly selected houses and emptied hourly. The net usage of residents, their hours of house entry and exit and times of sleeping were recorded and the individual hourly exposure to vectors indoors and outdoors was calculated. Using these data, the true protective efficacy of nets (P*), for this population was estimated, and compared between genders, age groups and from month to month., Results: Primary vector species (Anopheles funestus s.l. and Anopheles arabiensis) were more likely to feed indoors but the secondary vector Anopheles coustani demonstrated exophagic behaviour (p < 0.05). A rise in vector biting activity was recorded at 19:30 outdoors and 18:30 indoors. Individuals using LLINs experienced a moderate reduction in their overall exposure to malaria vectors from 1.3 to 0.47 bites per night. The P* for the population over the study period was calculated as 51% and varied significantly with age and season (p < 0.01)., Conclusions: In the present study, LLINs offered the local population partial protection against malaria vector bites. It is likely that P* would be estimated to be greater if the overall suppression of the local vector population due to widespread community net use could be taken into account. However, the overlap of early biting habit of vectors and human activity in this region indicates that additional methods of vector control are required to limit transmission. Regular surveillance of both vector behaviour and domestic human-behaviour patterns would assist the planning of future control interventions in this region.

Background: Despite the efforts of the National Malaria Control Programme, malaria remains as an important public health problem in Bangladesh, particularly in the south-eastern region bordering India. Successful malaria control strategies rely on a detailed understanding of the underlying causes of malaria transmission. Here, an entomological survey was conducted in a malaria endemic area of Bangladesh bordering India to investigate the Anopheles mosquito community and assess their Plasmodium infection status., Methods: Monthly entomological collections were undertaken from October 2010 to September 2011 in five villages in the Matiranga sub-district, Khagrachari district in Bangladesh, bordering the Indian State of Tripura. CDC miniature light traps were placed inside houses to collect adult Anopheles mosquitoes. Following morphological and molecular identification of the female Anopheles mosquitoes collected, they were screened for circumsporozoite proteins (CSP) of Plasmodium falciparum (Pf), Plasmodium vivax-210 (Pv-210) and Plasmodium vivax-247 (Pv-247), by ELISA to determine natural infection rates. Variation in Anopheles species composition, relative abundance and Plasmodium infection rates were analysed between sampled villages., Results: A total of 2,027 female Anopheles were collected, belonging to 20 species. Anopheles nivipes was the most abundant species in our test villages during the peak malaria transmission season, and was observed sympatrically with An. philippinensis in the studied area. However, in the dry off-peak season, An. jeyporiensis was the most abundant species. Shannon's diversity index was highest in October (2.12) and evenness was highest in May (0.91). The CSP ELISA positive rate overall was 0.44%. Anopheles karwari (n=2), An. barbirostris s.l. (n=1) and An. vagus (n=1) were recorded positive for Pf. Anopheles kochi (n=1) was positive for Pv-210 while An. umbrosus (n=1), An. nivipes (n=1) and An. kochi (n=1) were positive for Pv-247. A mixed infection of Pf and Pv-247 was detected in An. barbirostris s.l.., Conclusion: High diversity of Anopheles species was observed in areas close to the international border where species that were underestimated for malaria transmission significantly outnumbered principal vector species and these may play a significantly heightened role in malaria transmission.

Background: Defining the anopheline mosquito vectors and their foraging behaviour in malaria endemic areas is crucial for disease control and surveillance. The standard protocol for molecular identification of host blood meals in mosquitoes is to morphologically identify fed mosquitoes and then perform polymerase chain reaction (PCR), precipitin tests, or ELISA assays. The purpose of this study was to determine the extent to which the feeding rate and human blood indices (HBIs) of malaria vectors were underestimated when molecular confirmation by PCR was performed on both visually fed and unfed mosquitoes., Methods: In association with the Southern Africa International Centers of Excellence in Malaria Research (ICEMR), mosquito collections were performed at three sites: Choma district in southern Zambia, Nchelenge district in northern Zambia, and Mutasa district in eastern Zimbabwe. All anophelines were classified visually as fed or unfed, and tested for blood meal species using PCR methods. The HBIs of visually fed mosquitoes were compared to the HBIs of overall PCR confirmed fed mosquitoes by Pearson's Chi-Square Test of Independence., Results: The mosquito collections consisted of Anopheles arabiensis from Choma, Anopheles funestus s.s., Anopheles gambiae s.s. and Anopheles leesoni from Nchelenge, and An. funestus s.s. and An. leesoni from Mutasa. The malaria vectors at all three sites had large human blood indices (HBI) suggesting high anthropophily. When only visually fed mosquitoes tested by PCR for blood meal species were compared to testing those classified as both visually fed and unfed mosquitoes, it was found that the proportion blooded was underestimated by up to 18.7%. For most Anopheles species at each site, there was a statistically significant relationship (P < 0.05) between the HBIs of visually fed mosquitoes and that of the overall PCR confirmed fed mosquitoes., Conclusion: The impact on HBI of analysing both visually fed and unfed mosquitoes varied from site to site. This discrepancy may be due to partial blood feeding behaviour by mosquitoes, digestion of blood meals, sample condition, and/or expertise of entomology field staff. It is important to perform molecular testing on all mosquitoes to accurately characterize vector feeding behaviour and develop interventions in malaria endemic areas.

Efforts to develop a successful malaria vaccine are hampered due to lack of assays that are predictive of protective immunity without conducting large clinical studies. The effect of experimental vaccines and drugs on malaria transmission is yet more difficult to measure. Knowledge on the Plasmodium infection rate in mosquito populations will aid the measurement of effects from intervention measures for malaria control. Here, we report the development of a chemiluminescent sandwich ELISA (ECL-ELISA) that can detect Plasmodium falciparum circumsporozoite protein (Pf CSP) produced in recombinant form at concentrations of 4.4pg and in P. falciparum sporozoites (Pf SPZ) derived from mosquito salivary glands at levels corresponding to 5 Pf SPZ. Most importantly, we demonstrate reliable Pf CSP-based detection of 0.056day 8 P. falciparum oocysts developing inside mosquito midguts in whole mosquito lysates. Cumulatively, the ECL-ELISA is 47× more sensitive for the detection of Pf CSP than a colorimetric ELISA while greatly simplifying sample preparation, obviating the need for cumbersome midgut dissections and allowing high throughput screening of Plasmodium infection in mosquito populations. The ECL-ELISA may also have broader application in diagnosis of infectious diseases and the prognostic value in cancer and other diseases such as auto-immunity and genetic disorders based on antigen detection, or quality validation of biological vaccine components., (Published by Elsevier B.V.)

Mosquitoes possess an innate immune system that is capable of limiting infection by a variety of pathogens, including the Plasmodium spp. parasites responsible for human malaria. The Anopheles immune deficiency (IMD) innate immune signaling pathway confers resistance to Plasmodium falciparum. While some previously identified Anopheles anti-Plasmodium effectors are regulated through signaling by Rel2, the transcription factor of the IMD pathway, many components of this defense system remain uncharacterized. To begin to better understand the regulation of immune effector proteins by the IMD pathway, we used oligonucleotide microarrays and iTRAQ to analyze differences in mRNA and protein expression, respectively, between transgenic Anopheles stephensi mosquitoes exhibiting blood meal-inducible overexpression of an active recombinant Rel2 and their wild-type conspecifics. Numerous genes were differentially regulated at both the mRNA and protein levels following induction of Rel2. While multiple immune genes were up-regulated, a majority of the differentially expressed genes have no known immune function in mosquitoes. Selected up-regulated genes from multiple functional categories were tested for both anti-Plasmodium and anti-bacterial action using RNA interference (RNAi). Based on our experimental findings, we conclude that increased expression of the IMD immune pathway-controlled transcription factor Rel2 affects the expression of numerous genes with diverse functions, suggesting a broader physiological impact of immune activation and possible functional versatility of Rel2. Our study has also identified multiple novel genes implicated in anti-Plasmodium defense., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Anopheles gambiae is a major vector mosquito for Plasmodium falciparum, the deadly pathogen causing most human malaria in sub-Saharan Africa. Synthesized in the fat body, trehalose is the predominant sugar in mosquito hemolymph. It not only provides energy but also protects the mosquito against desiccation and heat stresses. Trehalose enters the mosquito hemolymph by the trehalose transporter AgTreT1. In adult female A. gambiae, AgTreT1 is predominantly expressed in the fat body. We found that AgTreT1 expression is induced by environmental stresses such as low humidity or elevated temperature. AgTreT1 RNA silencing reduces the hemolymph trehalose concentration by 40%, and the mosquitoes succumb sooner after exposure to desiccation or heat. After an infectious blood meal, AgTreT1 RNA silencing reduces the number of P. falciparum oocysts in the mosquito midgut by over 70% compared with mock-injected mosquitoes. These data reveal important roles for AgTreT1 in stress adaptation and malaria pathogen development in a major vector mosquito. Thus, AgTreT1 may be a potential target for malaria vector control.

Anopheles mosquitoes transmit malaria, a major public health problem among many African countries. One of the most effective methods to control malaria is by controlling the Anopheles mosquito vectors that transmit the parasites. Mathematical models have both predictive and explorative utility to investigate the pros and cons of different malaria control strategies. We have developed a C++ based, stochastic spatially explicit model (ANOSPEX; Ano pheles Spatially-Explicit) to simulate Anopheles metapopulation dynamics. The model is biologically rich, parameterized by field data, and driven by field-collected weather data from Macha, Zambia. To preliminarily validate ANOSPEX, simulation results were compared to field mosquito collection data from Macha; simulated and observed dynamics were similar. The ANOSPEX model will be useful in a predictive and exploratory manner to develop, evaluate and implement traditional and novel strategies to control malaria, and for understanding the environmental forces driving Anopheles population dynamics.

In 2007, the first free mass distribution of insecticide-treated bed nets (ITNs) occurred in southern Zambia. To determine the effect of ITNs on heterogeneity in biting rates, human DNA from Anopheles arabiensis blood meals was genotyped to determine the number of hosts that had contributed to the blood meals. The multiple feeding rate decreased from 18.9% pre-ITN to 9.1% post-ITN, suggesting that mosquito biting had focused onto a smaller fraction of the population. Pre-ITN, 20% of persons in a household provided 40% of blood meals, which increased to 59% post-ITN. To measure heterogeneity over a larger scale, mosquitoes were collected in 90 households in two village areas. Of these households, 25% contributed 78.1% of An. arabiensis, and households with high frequencies of An. arabiensis were significantly spatially clustered. The results indicate that substantial heterogeneity in malaria risk exists at local and household levels, and household-level heterogeneity may be influenced by interventions, such as ITNs.

EVALUATION OF: Fang W, Vega-Rodríguez J, Ghosh AK et al. Development of transgenic fungi that kill human malaria parasites in mosquitoes. Science 331(6020), 1074-1077 (2011). Paratransgenesis is the genetic manipulation of insect endosymbiotic microorganisms such as bacteria, viruses or fungi. Paratransgenesis has been proposed as a potential method to control vector-borne diseases such as malaria. In this article, Fang and colleagues have used genetic manipulation to insert multiple antimalaria effector genes into the entomopathogenic fungus Metarhizium anisopliae. When the modified fungus was used to infect Anopheles mosquitoes, it expressed the antimalaria effector molecules in the mosquito hemolymph. When several different effector molecules were coexpressed, malaria levels in the mosquito salivary glands were inhibited by up to 98% compared with controls. Significant inhibition could be initiated by as little as seven fungal spores and was very rapid and long lasting. These data suggest that recombinant entomopathogenic fungi could be deployed as part of a strategy to control malaria.

Anopheles coustani s.l. and Anopheles squamosus are sub-Saharan mosquito species that have been implicated in malaria transmission. Although generally believed to be of negligible importance due to their overwhelmingly zoophilic behavior, An. coustani s.l. and An. squamosus made up a large proportion of the anophelines collected by human landing catches during the 2007-2008 and 2008-2009 rainy seasons in Macha, Zambia. Further, polymerase chain reaction-based blood meal identification showed that the majority of blood meals from these mosquito species caught in human-baited Centers for Disease Control light traps were from human hosts. Although no An. coustani s.l. or An. squamosus were found to be positive for Plasmodium, the demonstrated anthropophilic tendencies of these mosquitoes in southern Zambia suggest their potential as secondary malaria vectors.

Altered patterns of malaria endemicity reflect, in part, changes in feeding behavior and climate adaptation of mosquito vectors. Aquaporin (AQP) water channels are found throughout nature and confer high-capacity water flow through cell membranes. The genome of the major malaria vector mosquito Anopheles gambiae contains at least seven putative AQP sequences. Anticipating that transmembrane water movements are important during the life cycle of A. gambiae, we identified and characterized the A. gambiae aquaporin 1 (AgAQP1) protein that is homologous to AQPs known in humans, Drosophila, and sap-sucking insects. When expressed in Xenopus laevis oocytes, AgAQP1 transports water but not glycerol. Similar to mammalian AQPs, water permeation of AgAQP1 is inhibited by HgCl(2) and tetraethylammonium, with Tyr185 conferring tetraethylammonium sensitivity. AgAQP1 is more highly expressed in adult female A. gambiae mosquitoes than in males. Expression is high in gut, ovaries, and Malpighian tubules where immunofluorescence microscopy reveals that AgAQP1 resides in stellate cells but not principal cells. AgAQP1 expression is up-regulated in fat body and ovary by blood feeding but not by sugar feeding, and it is reduced by exposure to a dehydrating environment (42% relative humidity). RNA interference reduces AgAQP1 mRNA and protein levels. In a desiccating environment (<20% relative humidity), mosquitoes with reduced AgAQP1 protein survive significantly longer than controls. These studies support a role for AgAQP1 in water homeostasis during blood feeding and humidity adaptation of A. gambiae, a major mosquito vector of human malaria in sub-Saharan Africa.

Human landing catches (HLCs) are currently the preferred method to determine vector human biting rates (HBRs), which are key determinants of entomologic inoculation rates and important measures for assessing the impact of vector control efforts. Although HLCs are the most direct means of establishing HBRs, they are labor-intensive, and their use is facing increasing ethical concerns. The relationship between Centers for Disease Control (CDC) light traps and HLC collections was evaluated in Macha, Zambia during the 2007-2008 and 2008-2009 rainy seasons. A CDC light trap captured on average 1.91 (95% confidence interval = 1.16-2.28) times as many An. arabiensis per night as an indoor HLC. Additionally, nets treated with deltamethrin did not affect the numbers of An. arabiensis collected. Our results suggest that in regions where use of vector control interventions is high and vector densities are low, CDC light traps can be used to monitor An. arabiensis HBRs.

Anopheles arabiensis mosquitoes are the primary vector responsible for Plasmodium falciparum transmission in Macha, Zambia. Because insecticide-treated bed nets (ITNs) have the potential to alter host feeding behavior, the extent of the zoophilic and exophagic tendencies of the vector was evaluated during the two rainy seasons after ITN introduction. Centers for Disease Control light traps, paired indoor/outdoor human landing catches, and outdoor cattle-baited collections were used to assess potential changes in host preference. Results support the hypothesis that An. arabiensis mosquitoes in Macha remain highly anthropophilic despite high ITN use. Anopheles arabiensis mosquitoes in Macha appear to be relatively exophagic and have been caught biting outdoors immediately after sunset and before sunrise, potentially circumventing some of the protective effects of ITNs.

An increase in Anopheles arabiensis showing endophilic behavior was observed in Macha, Zambia during March 2007. To determine whether this shift in resting behavior was accompanied by a change in feeding preference, an attempt was made to calculate the human blood index. However, only 46.2% of blood meals were successfully identified with existing polymerase chain reaction (PCR) diagnostics. This failure was hypothesized to be caused by the limitations of existing methods that are not capable of identifying host source from anophelines resting for extended time periods. Using an assay we developed that allows for the identification of mammalian host DNA out to 60 hours post-feeding, we were able to successfully determine the host source of 94.3% of recovered blood meals. The data show that, although An. arabiensis in Macha experienced a period of higher endophily, the degree of anthropophily and the sporozoite rate in the population remained comparable to the previous malaria season.

Paratransgenesis, the genetic manipulation of insect symbiotic microorganisms, is being considered as a potential method to control vector-borne diseases such as malaria. The feasibility of paratransgenic malaria control has been hampered by the lack of candidate symbiotic microorganisms for the major vector Anopheles gambiae. In other systems, densonucleosis viruses (DNVs) are attractive agents for viral paratransgenesis because they infect important vector insects, can be genetically manipulated and are transmitted to subsequent generations. However, An. gambiae has been shown to be refractory to DNV dissemination. We discovered, cloned and characterized the first known DNV (AgDNV) capable of infection and dissemination in An. gambiae. We developed a flexible AgDNV-based expression vector to express any gene of interest in An. gambiae using a two-plasmid helper-transducer system. To demonstrate proof-of-concept of the viral paratransgenesis strategy, we used this system to transduce expression of an exogenous gene (enhanced green fluorescent protein; EGFP) in An. gambiae mosquitoes. Wild-type and EGFP-transducing AgDNV virions were highly infectious to An. gambiae larvae, disseminated to and expressed EGFP in epidemiologically relevant adult tissues such as midgut, fat body and ovaries and were transmitted to subsequent mosquito generations. These proof-of-principle data suggest that AgDNV could be used as part of a paratransgenic malaria control strategy by transduction of anti-Plasmodium peptides or insect-specific toxins in Anopheles mosquitoes. AgDNV will also be extremely valuable as an effective and easy-to-use laboratory tool for transient gene expression or RNAi in An. gambiae.