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

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

Potential key genes involved in metabolic resistance to malathion in the southern house mosquito, Culex quinquefasciatus, and functional validation of CYP325BC1 and CYP9M12 as candidate genes using RNA interference.

BACKGROUND: Metabolic detoxification is one of the major mechanisms contributing to the development of resistance in mosquitoes, including the southern house mosquito, Culex quinquefasciatus. The three major detoxification supergene families, cytochrome P450s, glutathione S-transferases and general esterases, have been demonstrated to play an important role in metabolic resistance. In this study, we performed differential gene expression analysis based on high-throughput transcriptome sequencing on samples from four experimental groups to give insight into key genes involved in metabolic resistance to malathion in Cx. quinquefasciatus. We conducted a whole transcriptome analysis of field captured wild Cx. quinquefasciatus from Harris County (WI), Texas and a malathion susceptible laboratory-maintained Sebring colony (CO) to investigate metabolic insecticide resistance. Field captured mosquitoes were also phenotypically classified into the malathion resistant and malathion susceptible groups following a mortality response measure conducted using a Centers for Disease Control and Prevention (CDC) bottle assay. The live (MR) and dead (MS) specimens from the bottle assay, along with an unselected WI sample and a CO sample were processed for total RNA extraction and subjected to whole-transcriptome sequencing. RESULTS: We demonstrated that the genes coding for detoxification enzymes, particularly cytochrome P450s, were highly up-regulated in the MR group compared to the MS group with similar up-regulation observed in the WI group compared to the CO group. A total of 1,438 genes were differentially expressed in comparison between MR and MS group, including 614 up-regulated genes and 824 down-regulated genes. Additionally, 1,871 genes were differentially expressed in comparison between WI and CO group, including 1,083 up-regulated genes and 788 down-regulated genes. Further analysis on differentially expressed genes from three major detoxification supergene families in both comparisons resulted in 16 detoxification genes as candidates potentially associated with metabolic resistance to malathion. Knockdown of CYP325BC1 and CYP9M12 using RNA interference on the laboratory-maintained Sebring strain significantly increased the mortality of Cx. quinquefasciatus after exposure to malathion. CONCLUSION: We generated substantial transcriptomic evidence on metabolic detoxification of malathion in Cx. quinquefasciatus. We also validated the functional roles of two candidate P450 genes identified through DGE analysis. Our results are the first to demonstrate that knockdown of CYP325BC1 and CYP9M12 both significantly increased malathion susceptibility in Cx. quinquefasciatus, indicating involvement of these two genes in metabolic resistance to malathion.

Plasmodium relictum infection in Culex quinquefasciatus (Culicidae) decreases diel flight activity but increases peak dusk flight activity.

BACKGROUND: Parasites are recognized for their ability to modify host physiology and behaviours in ways that increase parasite fitness. Protozoan parasites of the genus Plasmodium are a group of widespread vector-borne parasites of vertebrates, causing disease to a wide range of hosts, but most notably to human and avian hosts. METHODS: The hypothesis that infection with the avian malaria, Plasmodium relictum (GRW4 lineage) impacts flight activity in one of their natural vectors, Culex quinquefasciatus, was tested using both parasites and mosquitoes colonized from local populations in East-Central Texas, USA. Groups of Cx. quinquefasciatus were allowed to feed directly on canaries with active P. relictum infections and control canaries with no P. relictum exposure history. Additionally, how P. relictum sporozoite invasion of mosquito salivary glands impacts mosquito flight activity behaviour was tested using a Locomotor Activity Monitor for both control and infected females. Generalized linear mixed models were used to evaluate the influence of infection status on the response variables of flight activity (continuous) and probability of flight occurring (binomial). RESULTS: Infection status was a significant predictor of flight activity and flight probability and interactions between infection status and experimental period of infection as well as infection status and dusk were statistically significant predictors of flight activity. Plasmodium relictum infected mosquitoes had a mean flight activity of 3.10 and control mosquitoes had an overall mean flight activity of 3.13. DISCUSSION: Based on these results, avian malaria parasites increase the flight activity of these mosquitoes at hours known for peak host-seeking behaviour but decrease overall diel activity. CONCLUSION: Although the ramifications of this behavioural change for P. relictum transmission are unclear, these results provide additional empirical evidence suggesting that avian malaria can influence mosquito behaviour and modulate transmission potential.

Rapid selection of a pyrethroid metabolic enzyme CYP9K1 by operational malaria control activities.

Since 2004, indoor residual spraying (IRS) and long-lasting insecticide-impregnated bednets (LLINs) have reduced the malaria parasite prevalence in children on Bioko Island, Equatorial Guinea, from 45% to 12%. After target site-based (knockdown resistance; kdr) pyrethroid resistance was detected in 2004 in Anopheles coluzzii (formerly known as the M form of the Anopheles gambiae complex), the carbamate bendiocarb was introduced. Subsequent analysis showed that kdr alone was not operationally significant, so pyrethroid-based IRS was successfully reintroduced in 2012. In 2007 and 2014-2015, mass distribution of new pyrethroid LLINs was undertaken to increase the net coverage levels. The combined selection pressure of IRS and LLINs resulted in an increase in the frequency of pyrethroid resistance in 2015. In addition to a significant increase in kdr frequency, an additional metabolic pyrethroid resistance mechanism had been selected. Increased metabolism of the pyrethroid deltamethrin was linked with up-regulation of the cytochrome P450 CYP9K1. The increase in resistance prompted a reversion to bendiocarb IRS in 2016 to avoid a resurgence of malaria, in line with the national Malaria Control Program plan.

Culex quinquefasciatus (Diptera: Culicidae) survivorship following the ingestion of bird blood infected with Haemoproteus sp. parasites.

Arthropod vectors are frequently exposed to a diverse assemblage of parasites, but the consequence of these infections on their biology and behavior are poorly understood. We experimentally evaluated whether the ingestion of a common protozoan parasite of avian hosts (Haemoproteus spp.; Haemosporida: Haemoproteidae) impacted the survivorship of Culex quinquefasciatus (Say) (Diptera: Culicidae). Blood was collected from wild northern cardinals (Cardinalis cardinalis) in College Station, Texas, and screened for the presence of Haemoproteus spp. parasites using microscopic and molecular methods. Experimental groups of Cx. quinquefasciatus mosquitoes were offered Haemoproteus-positive cardinal blood through an artificial feeding apparatus, while control groups received Haemoproteus-negative cardinal blood or domestic canary (Serinus canaria domestica) blood. Culex quinquefasciatus mosquitoes exposed to Haemoproteus infected cardinal blood survived significantly fewer days than mosquitoes that ingested Haemoproteus-negative cardinal blood. The survival of mosquitoes fed on positive cardinal blood had a median survival time of 18 days post-exposure and the survival of mosquitoes fed on negative cardinal blood exceeded 50% across the 30 day observation period. Additionally, mosquitoes that fed on canary controls survived significantly fewer days than cardinal negative controls, with canary control mosquitoes having a median survival time of 17 days. This study further supports prior observations that Haemoproteus parasites can be pathogenic to bird-biting mosquitoes, and suggests that Haemoproteus parasites may indirectly suppress the transmission of co-circulating vector-borne pathogens by modulating vector survivorship. Our results also suggest that even in the absence of parasite infection, bloodmeals from different bird species can influence mosquito survivorship.

Malaria outbreak in Riaba district, Bioko Island: lessons learned.

At the beginning of 2019, a sudden surge of malaria cases was observed in the district of Riaba, Bioko Island. Between January and April, confirmed malaria cases increased 3.8-fold compared to the same period in 2018. Concurrently, anopheline human biting rate (HBR) increased 2.1-fold. During the outbreak, 82.2% of the district population was tested for malaria with a rapid diagnostic test; 37.2% of those tested had a detectable infection and were treated according to national guidelines. Vector control interventions, including indoor residual spraying and larval source management were scaled-up. After the interventions, the number of confirmed cases decreased by 70% and the overall parasite prevalence in the communities by 43.8%. Observed prevalence in a follow up malaria indicator survey, however, was significantly higher than elsewhere on the island, and higher than in previous years. There was no significant reduction in HBR, which remained high for the rest of the year. The surge was attributed to various factors, chiefly increased rainfall and a large number of anthropogenic anopheline breeding sites created by construction works. This case study highlights the need for sustained vector control interventions and multi-sector participation, particularly in malaria control and elimination settings with persistently high local malaria receptivity.

A cluster randomized trial comparing deltamethrin and bendiocarb as insecticides for indoor residual spraying to control malaria on Bioko Island, Equatorial Guinea.

BACKGROUND: Indoor residual spraying (IRS) has been used on Bioko for malaria control since 2004. In 2013 the insecticide was changed from bendiocarb to deltamethrin. Shortly after this change, there was a marked increase in malaria prevalence on the island. This trial was carried out to compare the effectiveness of bendiocarb and deltamethrin for use in IRS on Bioko. METHODS: Twenty-four clusters of houses were randomized to receive IRS with either bendiocarb or deltamethrin. Approximately 3 months after the intervention, the prevalence of malaria and levels of haemoglobin were measured in children aged 2-14 years in each cluster. RESULTS: Prevalence of malaria in 2-14 year olds was lower in the bendiocarb arm (16.8, 95 % CI 11.1-24.7, N = 1374) than in the deltamethrin arm (23.2, 95 % CI 16.0-32.3, N = 1330) but this difference was not significant (p = 0.390), even after adjusting for covariates (p = 0.119). Mean haemoglobin in children was marginally higher in the bendiocarb clusters (11.6 g/dl, 95 % CI 11.5-11.8, N = 1326) than in the deltamethrin clusters (11.5 g/dl, 95 % CI 11.3-11.7, N = 1329). This difference was borderline significant after adjusting for covariates (p = 0.049). CONCLUSIONS: The results are suggestive of bendiocarb being more effective at preventing malaria on Bioko although evidence for this was weak. The results are likely due to the fact that local vectors remain fully susceptible to bendiocarb whereas subsequent tests have shown resistance to deltamethrin.

Increasing outdoor host-seeking in Anopheles gambiae over 6 years of vector control on Bioko Island.

BACKGROUND: Vector control through indoor residual spraying (IRS) has been employed on Bioko Island, Equatorial Guinea, under the Bioko Island Malaria Control Project (BIMCP) since 2004. This study analyses the change in mosquito abundance, species composition and outdoor host-seeking proportions from 2009 to 2014, after 11 years of vector control on Bioko Island. METHODS: All-night indoor and outdoor human landing catches were performed monthly in the Bioko Island villages of Mongola, Arena Blanca, Biabia and Balboa from 2009 to 2014. Collected mosquitoes were morphologically identified and a subset of Anopheles gambiae sensu lato (s.l.) were later identified molecularly to their sibling species. Mosquito collection rates, species composition and indoor/outdoor host-seeking sites were analysed using generalized linear mixed models to assess changes in mosquito abundance and behaviour. RESULTS: The overall mosquito collection rate declined in each of the four Bioko Island villages. Anopheles coluzzii and Anopheles melas comprised the An. gambiae s.l. mosquito vector population, with a range of species proportions across the four villages. The proportion of outdoor host-seeking An. gambiae s.l. mosquitoes increased significantly in all four villages with an average increase of 58.8 % [57.9, 59.64 %] in 2009 to 70.0 % [67.8, 72.0 %] in 2014. Outdoor host-seeking rates did not increase in the month after an IRS spray round compared to the month before, suggesting that insecticide repellency has little impact on host-seeking behaviour. CONCLUSION: While vector control on Bioko Island has succeeded in substantial reduction in overall vector biting rates, populations of An. coluzzii and An. melas persist. Host-seeking behaviour has changed in these An. gambiae s.l. populations, with a shift towards outdoor host-seeking. During this study period, the proportion of host-seeking An. gambiae s.l. caught outdoors observed on Bioko Island increased to high levels, exceeding 80 % in some locations. It is possible that there may be a genetic basis underlying this large shift in host-seeking behaviour, in which case outdoor feeding could pose a serious threat to current vector control programmes. Currently, the BIMCP is preparing for this potential challenge by testing source reduction as a complementary control effort that also targets outdoor transmission.

The effective population size of malaria mosquitoes: large impact of vector control.

Malaria vectors in sub-Saharan Africa have proven themselves very difficult adversaries in the global struggle against malaria. Decades of anti-vector interventions have yielded mixed results--with successful reductions in transmission in some areas and limited impacts in others. These varying successes can be ascribed to a lack of universally effective vector control tools, as well as the development of insecticide resistance in mosquito populations. Understanding the impact of vector control on mosquito populations is crucial for planning new interventions and evaluating existing ones. However, estimates of population size changes in response to control efforts are often inaccurate because of limitations and biases in collection methods. Attempts to evaluate the impact of vector control on mosquito effective population size (N(e)) have produced inconclusive results thus far. Therefore, we obtained data for 13-15 microsatellite markers for more than 1,500 mosquitoes representing multiple time points for seven populations of three important vector species--Anopheles gambiae, An. melas, and An. moucheti--in Equatorial Guinea. These populations were exposed to indoor residual spraying or long-lasting insecticidal nets in recent years. For comparison, we also analyzed data from two populations that have no history of organized vector control. We used Approximate Bayesian Computation to reconstruct their demographic history, allowing us to evaluate the impact of these interventions on the effective population size. In six of the seven study populations, vector control had a dramatic impact on the effective population size, reducing N(e) between 55%-87%, the exception being a single An. melas population. In contrast, the two negative control populations did not experience a reduction in effective population size. This study is the first to conclusively link anti-vector intervention programs in Africa to sharply reduced effective population sizes of malaria vectors.

Species-specific chemosensory gene expression in the olfactory organs of the malaria vector Anopheles gambiae.

BACKGROUND: The malaria mosquito Anopheles gambiae has a high preference for human hosts, a characteristic that contributes greatly to its capacity for transmitting human malaria. A sibling species, An. quadriannulatus, has a quite different host preference and feeds mostly on bovids. For this reason it does not contribute to human malaria transmission. Host seeking in mosquitoes is modulated by the olfactory system, which is primarily housed in the antennae and maxillary palps. Therefore, the detection of differing host odors by sibling species may be reflected in the expression level of the olfactory genes involved. Accordingly, we compared the transcriptomes of the antennae and maxillary palps of An. gambiae and An. quadriannulatus. RESULTS: We identified seven relatively abundant olfactory receptors, nine ionotropic receptors and three odorant binding proteins that are substantially up-regulated in An. gambiae antennae. Interestingly, we find that the maxillary palps of An. gambiae contain a species-specific olfactory receptor, Or52, and five An. gambiae-specific gustatory receptors (AgGr48-52) that are relatively abundant. These five gustatory receptors are also expressed in An. gambiae antennae, although at lower level, indicating a likely role in olfaction, rather than gustation. We also document an approximately three-fold higher overall expression of olfaction genes in the maxillary palps of An. quadriannulatus, indicating an important role of this organ in the olfaction system of this species. Finally, the expression of the CO2 receptor genes is five to six-fold higher in the zoophilic An. quadriannulatus, implying a much higher sensitivity for detecting CO2. CONCLUSIONS: These results identify potential human host preference genes in the malaria vector An. gambiae. Interestingly, species-specific expression of several gustatory receptors in the olfactory organs indicate a role in olfaction rather than gustation. Additionally, a more expansive role for maxillary palps in olfaction is implicated than previously thought, albeit more so in the zoophilic An. quadriannulatus.

The role of the major chemosensory organs in the host-seeking activity of Anopheles coluzzii (Diptera: Culicidae).

Anopheles coluzzii (Coetzee & Wilkerson) and its sibling species Anopheles gambiae s.s. (Giles) are highly anthropophilic and among the major malaria vectors in sub-Saharan Africa. Mosquitoes use various senses to find hosts, but rely primarily on olfaction. Therefore, the mosquito olfactory system has been studied extensively, including a variety of studies comparing chemosensory gene expression between An. coluzzii and its zoophilic sibling species Anopheles quadriannulatus (Theobald). These studies revealed species-specific chemosensory gene expression in the antennae and maxillary palps, which raised the question of a potential role for the palps in determining species-specific host preferences. To answer this question, we mechanically ablated the antennae, maxillary palps, and labella, and ran both control and ablated mosquitoes through a dual-port olfactometer. While we aimed to identify the organs responsible for vertebrate host choice, the ablated mosquitoes exclusively responded to human odor, so we were unable to do so. However, we were able to refine our understanding of the roles of these organs in host-seeking activation (leaving the release cage) as well as odor response (entering an odor port). As expected, the antennae are the most important organs to both behaviors: activation was roughly halved and vertebrate odor response was abolished in antennae-ablated mosquitoes. Maxillary palp ablation had little impact on activation, but reduced odor response to a similar degree as the exclusion of CO2. Finally, while labellar ablation dramatically reduced activation (probably associated with the inability to feed), it had little impact on odor response, suggesting that any labellar role in host choice is likely not olfactory.

Unveiling candidate genes for metabolic resistance to malathion in Aedes albopictus through RNA sequencing-based transcriptome profiling.

Aedes albopictus, also known as the Asian tiger mosquito, is indigenous to the tropical forests of Southeast Asia. Ae. albopictus is expanding across the globe at alarming rates, raising concern over the transmission of mosquito-borne diseases, such as dengue, West Nile fever, yellow fever, and chikungunya fever. Since Ae. albopictus was reported in Houston (Harris County, Texas) in 1985, this species has rapidly expanded to at least 32 states across the United States. Public health efforts aimed at controlling Ae. albopictus, including surveillance and adulticide spraying operations, occur regularly in Harris County. Despite rotation of insecticides to mitigate the development of resistance, multiple mosquito species including Culex quinquefasciatus and Aedes aegypti in Harris County show organophosphate and pyrethroid resistance. Aedes albopictus shows relatively low resistance levels as compared to Ae. aegypti, but kdr-mutation and the expression of detoxification genes have been reported in Ae. albopictus populations elsewhere. To identify potential candidate detoxification genes contributing to metabolic resistance, we used RNA sequencing of field-collected malathion-resistant and malathion-susceptible, and laboratory-maintained susceptible colonies of Ae. albopictus by comparing the relative expression of transcripts from three major detoxification superfamilies involved in malathion resistance due to metabolic detoxification. Between these groups, we identified 12 candidate malathion resistance genes and among these, most genes correlated with metabolic detoxification of malathion, including four P450 and one alpha esterase. Our results reveal the metabolic detoxification and potential cuticular-based resistance mechanisms associated with malathion resistance in Ae. albopictus in Harris County, Texas.

Five years of malaria control in the continental region, Equatorial Guinea.

BACKGROUND: A successful malaria control programme began in 2004 on Bioko Island, Equatorial Guinea. From 2007, the same multiple malaria interventions, though reduced in scope for funding reasons, were introduced to the four mainland provinces of Equatorial Guinea (the continental region) aiming to recreate Bioko's success. Two provinces received long-lasting insecticidal nets (LLINs) and two provinces received biannual indoor residual spraying (IRS). Enhanced case management and communications were introduced throughout. METHODS: Estimates of intervention coverage and indicators of malaria transmission for 2007 to 2011 were derived from annual malaria indicator surveys (MIS). Results were complemented by health information system (HIS) and entomological data. The personal protection offered by LLINs and IRS against Plasmodium falciparum infection was estimated with logistic regression. RESULTS: The estimated proportion of children aged 1-4 using either an LLIN the previous night or living in a house sprayed in the last six months was 23% in 2007 and 42% in 2011. The estimated prevalence of P. falciparum in children aged 1-4 was 68% (N=1,770; 95% confidence interval [CI]: 58-76%) in 2007 and 52% (N=1,602; 95% CI: 44-61%) in 2011. Children 1-4 years had lower prevalence if they used an LLIN the previous night (N=1,124, 56%; adjusted odds ratio [aOR] 0.64, 95% CI: 0.55-0.74) or if they lived in a sprayed house (N=1,150, 57%; aOR 0.80, 95% CI: 0.62-1.03) compared to children with neither intervention (N=4,131, 66%, reference group). The minority of children who both used an LLIN and lived in a sprayed house had the lowest prevalence of infection (N=171, 45%; aOR 0.52, 95% CI: 0.35-0.78). High site-level intervention coverage did not always correlate with lower site-level P. falciparum prevalence. The malaria season peaked in either June or July, not necessarily coinciding with MIS data collection. CONCLUSIONS: Though moderate impact was achieved after five years of vector control, case management, and communications, prevalence remained high due to an inability to sufficiently scale-up coverage with either IRS or LLINs. Both LLINs and IRS provided individual protection, but greater protection was afforded to children who benefitted from both.

Effective population size of Culex quinquefasciatus under insecticide-based vector management and following Hurricane Harvey in Harris County, Texas.

Introduction: Culex quinquefasciatus is a mosquito species of significant public health importance due to its ability to transmit multiple pathogens that can cause mosquito-borne diseases, such as West Nile fever and St. Louis encephalitis. In Harris County, Texas, Cx. quinquefasciatus is a common vector species and is subjected to insecticide-based management by the Harris County Public Health Department. However, insecticide resistance in mosquitoes has increased rapidly worldwide and raises concerns about maintaining the effectiveness of vector control approaches. This concern is highly relevant in Texas, with its humid subtropical climate along the Gulf Coast that provides suitable habitat for Cx. quinquefasciatus and other mosquito species that are known disease vectors. Therefore, there is an urgent and ongoing need to monitor the effectiveness of current vector control programs. Methods: In this study, we evaluated the impact of vector control approaches by estimating the effective population size of Cx. quinquefasciatus in Harris County. We applied Approximate Bayesian Computation to microsatellite data to estimate effective population size. We collected Cx. quinquefasciatus samples from two mosquito control operation areas; 415 and 802, during routine vector monitoring in 2016 and 2017. No county mosquito control operations were applied at area 415 in 2016 and 2017, whereas extensive adulticide spraying operations were in effect at area 802 during the summer of 2016. We collected data for eighteen microsatellite markers for 713 and 723 mosquitoes at eight timepoints from 2016 to 2017 in areas 415 and 802, respectively. We also investigated the impact of Hurricane Harvey's landfall in the Houston area in August of 2017 on Cx. quinquefasciatus population fluctuation. Results: We found that the bottleneck scenario was the most probable historical scenario describing the impact of the winter season at area 415 and area 802, with the highest posterior probability of 0.9167 and 0.4966, respectively. We also detected an expansion event following Hurricane Harvey at area 802, showing a 3.03-fold increase in 2017. Discussion: Although we did not detect significant effects of vector control interventions, we found considerable influences of the winter season and a major hurricane on the effective population size of Cx. quinquefasciatus. The fluctuations in effective population size in both areas showed a significant seasonal pattern. Additionally, the significant population expansion following Hurricane Harvey in 2017 supports the necessity for post-hurricane vector-control interventions.

Genetic isolation within the malaria mosquito Anopheles melas.

Anopheles melas is a brackish water-breeding member of the Anopheles gambiae complex that is distributed along the coast of West Africa and is a major malaria vector within its range. Because little is known about the population structure of this species, we analysed 15 microsatellite markers and 1161 bp of mtDNA in 11 A. melas populations collected throughout its range. Compared with its sibling species A. gambiae, A. melas populations have a high level of genetic differentiation between them, representing its patchy distribution due to its fragmented larval habitat that is associated with mangroves and salt marsh grass. Populations clustered into three distinct groups representing Western Africa, Southern Africa and Bioko Island populations that appear to be mostly isolated. Fixed differences in the mtDNA are present between all three clusters, and a Bayesian clustering analysis of the microsatellite data found no evidence for migration from mainland to Bioko Island populations, and little migration was evident between the Southern to the Western cluster. Surprisingly, mtDNA divergence between the three A. melas clusters is on par with levels of divergence between other species of the A. gambiae complex, and no support for monophyly was observed in a maximum-likelihood phylogenetic analysis. Finally, an approximate Bayesian analysis of microsatellite data indicates that Bioko Island A. melas populations were connected to the mainland populations in the past, but became isolated, presumably when sea levels rose after the last glaciation period (≥10 000-11 000 bp). This study has exposed species-level genetic divergence within A. melas and also has implications for control of this malaria vector.

Light traps fail to estimate reliable malaria mosquito biting rates on Bioko Island, Equatorial Guinea.

BACKGROUND: The human biting rate (HBR), an important parameter for assessing malaria transmission and evaluating vector control interventions, is commonly estimated by human landing collections (HLC). Although intense efforts have been made to find alternative non-exposure mosquito collection methods, HLC remains the standard for providing reliable and consistent HBRs. The aim of this study was to assess the relationship between human landing and light trap collections (LTC), in an attempt to estimate operationally feasible conversion factors between the two. The study was conducted as part of the operational research component of the Bioko Island Malaria Control Project (BIMCP), Equatorial Guinea. METHODS: Malaria mosquitoes were collected indoors and outdoors by HLCs and LTCs in three villages on Bioko Island, Equatorial Guinea during five bimonthly collections in 2009. Indoor light traps were suspended adjacent to occupied long-lasting, insecticide-treated bed nets. Outdoor light traps were placed close to the outer wall under the roof of the collection house. Collected specimens were subjected to DNA extraction and diagnostic PCR to identify species within the Anopheles gambiae complex. Data were analysed by simple regression of log-transformed values and by Bayesian regression analysis. RESULTS: There was a poor correlation between the two collection methods. Results varied by location, venue, month, house, but also by the statistical method used. The more robust Bayesian analyses indicated non-linear relationships and relative sampling efficiencies being density dependent for the indoor collections, implying that straight-forward and simple conversion factors could not be calculated for any of the locations. Outdoor LTC:HLC relationships were weak, but could be estimated at 0.10 and 0.07 for each of two locations. CONCLUSIONS: Light trap collections in combination with bed nets are not recommended as a reliable method to assess human biting rates on Bioko Island. Different statistical analyses methods give variable and inconsistent results. Substantial variation in collection methods prevents the determination of reliable and operationally feasible conversion factors for both indoor and outdoor data. Until improved mosquito collection methods are developed that can provide reliable and unbiased HBR estimates, HLCs should continue to serve as the reference method for HBR estimation.

Limited usefulness of microsatellite markers from the malaria vector Anopheles gambiae when applied to the closely related species Anopheles melas.

Anopheles melas is a brackish water mosquito found in coastal West Africa where it is a dominant malaria vector locally. In order to facilitate genetic studies of this species, 45 microsatellite loci originally developed for Anopheles gambiae were sequenced in An. melas. Those that were suitable based on repeat number and flanking regions were examined in 2 natural populations from Equatorial Guinea. Only 15 loci were eventually deemed suitable as polymorphic markers in An. melas populations. These loci were screened in 4 populations from a wider geographic range. Heterozygosity estimates ranged from 0.18 to 0.79, and 2.5-15 average alleles were observed per locus, yielding 13 highly polymorphic markers and 2 loci with lower variability. To examine the usefulness of microsatellite markers when applied in a sibling species, the original An. gambiae specific markers were used to amplify 5 loci in An. melas. Null alleles were found for 1 An. gambiae marker. We discuss the pitfalls of using microsatellite loci across closely related species and conclude that in addition to the problem of null alleles associated with this practice, many loci may prove to be of very limited use as polymorphic markers even when used in a sibling species.

Impact of disabled circadian clock on yellow fever mosquito Aedes aegypti fitness and behaviors.

Like other insects, Aedes aegypti displays strong daily patterns in host seeking and mating. Much of these behaviors are believed to be under the control of a circadian clock, an endogenous timekeeping mechanism relying on transcriptional/translational negative feedback loops that drive rhythmic physiology and behavior. To examine the connection between the circadian clock and various Ae. aegypti behaviors, we knocked out the core clock gene cycle using CRISPR/Cas9. We found that the rhythmic pattern and intensity of mRNA expression of seven circadian genes, including AeCyc-/-, were altered across the day/night cycle as well as in constant darkness conditions. We further show that the mutant CYC protein is incapable of forming a dimer with CLK to stimulate per expression and that the endogenous clock is disabled in AeCyc-/- mosquitoes. AeCyc-/- do not display the bimodal locomotor activity pattern of wild type, have a significantly reduced response to host odor, reduced egg hatching rates, delayed embryonic development and reduced adult survival and mating success. Surprisingly however, the propensity to blood feed in AeCyc-/- females is significantly higher than in wildtype females. Together with other recent work on the circadian clock control of key aspects of mosquito biology, our data on how cycle KO affects mosquito behavior and fitness provides a basis for further work into the pathways that connect the mosquito endogenous clock to its vector competence.

Outdoor host seeking behaviour of Anopheles gambiae mosquitoes following initiation of malaria vector control on Bioko Island, Equatorial Guinea.

BACKGROUND: Indoor-based anti-vector interventions remain the preferred means of reducing risk of malaria transmission in malaria endemic areas around the world. Despite demonstrated success in reducing human-mosquito interactions, these methods are effective solely against endophilic vectors. It may be that outdoor locations serve as an important venue of host seeking by Anopheles gambiae sensu lato (s.l.) mosquitoes where indoor vector suppression measures are employed. This paper describes the host seeking activity of anopheline mosquito vectors in the Punta Europa region of Bioko Island, Equatorial Guinea. In this area, An. gambiae sensu stricto (s.s.) is the primary malaria vector. The goal of the paper is to evaluate the importance of An gambiae s.l. outdoor host seeking behaviour and discuss its implications for anti-vector interventions. METHODS: The venue and temporal characteristics of host seeking by anopheline vectors in a hyperendemic setting was evaluated using human landing collections conducted inside and outside homes in three villages during both the wet and dry seasons in 2007 and 2008. Additionally, five bi-monthly human landing collections were conducted throughout 2009. Collections were segregated hourly to provide a time distribution of host-seeking behaviour. RESULTS: Surprisingly high levels of outdoor biting by An. gambiae senso stricto and An. melas vectors were observed throughout the night, including during the early evening and morning hours when human hosts are often outdoors. As reported previously, An. gambiae s.s. is the primary malaria vector in the Punta Europa region, where it seeks hosts outdoors at least as much as it does indoors. Further, approximately 40% of An. gambiae s.l. are feeding at times when people are often outdoors, where they are not protected by IRS or LLINs. Repeated sampling over two consecutive dry-wet season cycles indicates that this result is independent of seasonality. CONCLUSIONS: An. gambiae s.l. mosquitoes currently seek hosts in outdoor venues as much as indoors in the Punta Europa region of Bioko Island. This contrasts with an earlier pre-intervention observation of exclusive endophagy of An. gambiae in this region. In light of this finding, it is proposed that the long term indoor application of insecticides may have resulted in an adaptive shift toward outdoor host seeking in An. gambiae s.s. on Bioko Island.

The Expression of Chemosensory Genes in Male Maxillary Palps of Anopheles coluzzii (Diptera: Culicidae) and An. quadriannulatus.

Because of its importance as a malaria vector, Anopheles coluzzii's Coetzee & Wilkerson olfactory system has been studied extensively. Among this work is a series of studies comparing the expression of chemosensory genes in olfactory organs in females and/or males of these species. These have identified species- and female-biased chemosensory gene expression patterns. However, many questions remain about the role of chemosensation in male anopheline biology. To pave the way for future work we used RNAseq to compare chemosensory gene expression in the male maxillary palps of An. coluzzii and its sibling species An. quadriannulatus Theobald. As expected, the chemosensory gene repertoire is small in the male maxillary palps. Both species express the tuning receptors Or8 and Or28 at relatively high levels. The CO2 receptor genes Gr22-Gr24 are present in both species as well, although at much lower level than in females. Additionally, several chemoreceptors are species-specific. Gr37 and Gr52 are exclusive to An. coluzzii, whereas Or9 and Gr60 were detected only in An. quadriannulatus. Furthermore, several chemosensory genes show differential expression between the two species. Finally, several Irs, Grs, and Obps that show strong differential expression in the female palps, are absent or lowly expressed in the male palps. While many questions remain about the role of chemosensation in anopheline male biology, these results suggest that the male maxillary palps could have both a sex- and species-specific role in the perception of chemical stimuli. This work may guide future studies on the role of the male maxillary palp in these species.