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The most widespread arboviral diseases such as Dengue, Chikungunya, and Zika are transmitted mainly by Aedes mosquitoes. Due to the lack of effective therapeutics for most of these diseases, vector control remains the most effective preventative and control measure. This study investigated and compared the species composition, insecticide susceptibility, and resistance mechanisms in Aedes mosquito populations from a forest reserve converted to an eco-park and a peri-domestic sites in urban Accra, Ghana. Immature Aedes were sampled from the study sites, raised to adults, and exposed to deltamethrin, permethrin, DDT, fenitrothion, bendiocarb, permethrin + PBO, and deltamethrin + PBO using WHO tube assays. Melting curve analyses were performed for F1536C, V1016I, and V410L genetic mutations in surviving and dead mosquitoes following exposure to deltamethrin and permethrin. Microplate assay was used to access enzyme activity levels in adult mosquitoes from both populations. Aedes aegypti was found to be the dominant species from both study populations. The susceptibility test results revealed a high frequency of resistance to all the insecticides except fenitrothion. F1534C mutations were observed in 100% and 97% of mosquitoes from the peri-domestic and forest population, respectively but were associated with pyrethroid resistance only in the forest population (P < 0.0001). For the first time in Aedes mosquitoes in Ghana, we report the existence V410L mutations, mostly under selection only in the forest population (HWE P < 0.0001) and conclude that Aedes vectors in urban Accra have developed resistance to many commonly used insecticides. This information is important for the formulation of vector control strategies for Aedes control in Ghana.

The Noguchi Memorial Institute for Medical Research (NMIMR) is a semi-autonomous institute of the College of Health Sciences, University of Ghana, Legon. Founded in 1979, the Institute was built with a grant aid from the Government of Japan as a gift to the people of Ghana in memory of the renowned Japanese medical scientist, Dr. Hideyo Noguchi, who died from Yellow fever infection while conducting research on the disease in Ghana. The Institute has a three-pronged mandate to conduct health related research, build human capacity and provide specialized diagnostic and disease monitoring services in support of the Ghana Health Service. Over the past 40 years, the Institute has grown to be a leading biomedical research institute in the African region. It has strong and long-standing collaborations with scientists and institutions in Africa, Japan, Europe, Australia and North America on several projects on diseases of public health importance. The Institute also hosts several regional and national centres such as Regional Influenza laboratory. The Institute’s research activities are relevant to the control and prevention of infectious diseases in Ghana, particularly, HIV/AIDS, Tuberculosis, Buruli ulcer, Polio, Malaria and emerging infectious diseases. The Institute also plays a technical/advisory role to government through collaborations with disease control programmes and has since inception provided the country with needed critical evidence in support of health policy as well as laboratory diagnostic services among others. Going forward, the Institute seeks to expand and consolidate its activities in areas of antimicrobial resistance (AMR), clinical trials, genomic surveillance and academic programs and in the next 25 years, NMIMR hopes to approach every research area using the one health approach.

Microbes play an important role in human gut homeostasis, metabolic, immunologic and physiopathology of the body. A longitudinal study conducted during 2018–2021 in the Kintampo North Municipality in Ghana demonstrated low hookworm infection cure rates following treatment with a single dose of 400 mg albendazole in some communities. To investigate associations between hookworm infection and the gut microbiome, we examined faecal samples from consented participants who were either cured or remained infected after treatment. At each time point, fecal samples were collected prior to and 10–14 days after albendazole treatment of those who were infected. We used 16S rRNA amplicon sequencing of DNA extracted from stool samples to investigate the composition and biodiversity of the gut microbiota and to identify potential microbial biomarkers associated with treatment outcomes. Results of the study showed an association between hookworm infection and increased species richness. It also demonstrated a significant variation in microbiota composition at 10–14 days following single-dose albendazole treatment. Individuals cured of hookworm infection after treatment showed a significant reduction in microbiota composition when compared to their pre-treatment state (ANOSIM; p = 0.02), whilst individuals who failed to clear the infection showed no significant change in microbiota composition (ANOSIM; p = 0.35). Both uninfected individuals and individuals who were successfully treated were similar in terms of microbial composition and structure. We also found that the abundance of Clostridia spp. was increased in positive individuals before treatment as well as in those who were not cured. Predictive functional profiling revealed the enrichment of two pyruvate ferredoxin oxidoreductase subunit pathways in individuals who remained infected after treatment (p Clostridia spp. This study suggests a relationship between human gut microbiome dysbiosis and albendazole therapy outcomes of hookworm infection. Future studies will further characterize specific biomarkers identified within this study to establish their potential for assessment of pharmacological responses to anthelminthic therapies, as well as explore the possibility of using probiotic supplementation as an adjunct treatment to increase albendazole effectiveness against hookworm.

The geographic and evolutionary origins of the SARS-CoV-2 Omicron variant (BA.1), which was first detected mid-November 2021 in Southern Africa, remain unknown. We tested 13,097 COVID-19 patients sampled between mid-2021 to early 2022 from 22 African countries for BA.1 by real-time RT-PCR. By November-December 2021, BA.1 had replaced the Delta variant in all African sub-regions following a South-North gradient, with a peak Rt of 4.1. Polymerase chain reaction and near-full genome sequencing data revealed genetically diverse Omicron ancestors already existed across Africa by August 2021. Mutations, altering viral tropism, replication and immune escape, gradually accumulated in the spike gene. Omicron ancestors were therefore present in several African countries months before Omicron dominated transmission. These data also indicate that travel bans are ineffective in the face of undetected and widespread infection.

The globally invasive mosquito subspecies Aedes aegypti aegypti is a highly effective vector of human arboviruses because it specializes in biting humans and breeding in human habitats. Recent work suggests that specialization first arose as an adaptation to long, hot dry seasons in the West African Sahel, where Ae. aegypti is forced to rely on human-stored water for breeding. However, rainfall patterns in this region have changed dramatically over the past 10-20 thousand years, and we do not yet know exactly when specialization occurred. Here we use whole-genome cross-coalescent analysis to date the emergence of human specialist populations in the Sahel and thus further probe the climate hypothesis. Importantly, we take advantage of the known migration of human-specialist populations out of Africa during the Atlantic Slave Trade to calibrate the coalescent clock and thus obtain a more precise estimate of the older evolutionary event than would otherwise be possible. We find that human-specialist mosquitoes diverged rapidly from ecological generalists approximately 5,000 years ago, which corresponds to the end of the African Humid Period—a time when the Sahara dried and water stored by humans became a uniquely stable, aquatic niche in the Sahel. We also use population genomic analyses to date a previously observed influx of human-specialist alleles into major West African cities, where mosquitoes tend to be more attracted to humans than in nearby rural populations regardless of climate. In this case, the characteristic length of tracts of human-specialist ancestry present on a generalist genetic background in Kumasi, Ghana and Ouagadougou, Burkina Faso suggests the change in behavior occurred during rapid urbanization over the last 20-40 years. Taken together, we show that the timing and ecological context of two previously observed shifts towards human biting in Ae. aegypti differ; climate was likely the original driver, but urbanization has become increasingly important in recent decades. Understanding the changing relationship between mosquitoes and humans over time is critical for predicting and managing burdens of mosquito-borne disease.

The presence of mosquitoes in an area is dependent on the availability of suitable breeding sites that are influenced by several environmental factors. Identification of breeding habitats for vector surveillance and larval source management is key to disease control programs. We investigated water quality parameters and microbial composition in selected mosquito breeding sites in urban Accra, Ghana and associated these with abundance of Anopheles (Diptera: Culicidae) and Culex (Diptera: Culicidae) larvae. Physicochemical parameters and microbial composition explained up to 72% variance among the breeding sites and separated Anopheles and Culex habitats (P < 0.05). Anopheles and Culex abundances were commonly influenced by water temperature, pH, nitrate, and total hardness with contrasting impacts on the two mosquito species. In addition, total dissolved solids, biochemical oxygen demand, and alkalinity uniquely influenced Anopheles abundance, while total suspended solids, phosphate, sulphate, ammonium, and salinity were significant determinants for Culex. The correlation of these multiple parameters with the occurrence of each mosquito species was high (R2 = 0.99, P < 0.0001). Bacterial content assessment of the breeding ponds revealed that the most abundant bacterial phyla were Patescibacteria, Cyanobacteria, and Proteobacteria, constituting >70% of the total bacterial richness. The oligotrophic Patescibacteria was strongly associated with Anopheles suggestive of the mosquito’s adaptation to environments with less nutrients, while predominance of Cyanobacteria, indicative of rich nutritional source was associated with Culex larval ponds. We propose further evaluation of these significant abiotic and biotic parameters in field identification of larval sources and how knowledge of these can be harnessed effectively to reduce conducive breeding sites for mosquitoes.

Global control of hookworm infections relies on periodic Mass Drug Administration of benzimidazole drugs to high-risk groups, regardless of infection status. Mutations in the isotype-1 β-tubulin gene have been identified in veterinary nematodes, resulting in structural changes and reduced drug-binding. In Ghana, previous studies have demonstrated significant variability in albendazole effectiveness among people infected with the hookworm Necator americanus, although the mechanisms underlying deworming response have not been defined. Using hookworm egg samples from a cross-sectional study in Ghana, we developed a multiplex amplicon deep sequencing (MAD-seq) method to screen genomic regions encapsulating putative drug-resistance markers in N. americanus isotype-1 β-tubulin gene. Three single nucleotide polymorphisms (SNPs) corresponding to resistance-associated mutations (F167Y, E198A, F200Y) within the coding region of the isotype-1 β-tubulin gene were characterized using MAD-seq in 30 matched pre- and post-treatment samples from individuals with persistent infection following therapy. Post-sequence analysis showed that the highest mean alternative nucleotide allele at each PCR amplicon was 0.034% (167amplicon) and 0.025% (198/200amplicon), suggesting minimal allelic variation. No samples contained the F167Y SNP, while one contained low-frequency reads associated with E198A (3.15%) and F200Y (3.13%). This MAD-seq method provides a highly sensitive tool to monitor the three putative benzimidazole resistance markers at individual and community levels. Further work is required to understand the association of these polymorphisms to treatment response.

Estimates of SARS-CoV-2 transmission rates have significant public health policy implications since they shed light on the severity of illness in various groups and aid in strategic deployment of diagnostics, treatment and vaccination. Population-based investigations have not been conducted in Ghana to identify the seroprevalence of SARS-CoV-2. We conducted an age stratified nationally representative household study to determine the seroprevalence of SARS-CoV-2 and identify risk factors between February and December 2021. Study participants, 5 years and older regardless of prior or current infection COVID-19 infection from across Ghana were included in the study. Data on sociodemographic characteristics, contact with an individual with COVID-19-related symptoms, history of COVID-19-related illness, and adherence to infection prevention measures were collected. Serum obtained was tested for total antibodies with the WANTAI ELISA kit. The presence of antibodies against SAR-COV-2 was detected in 3,476 of 5,348 participants, indicating a seroprevalence of 67.10% (95% CI: 63.71–66.26). Males had lower seroprevalence (65.8% [95% CI: 63.5–68.04]) than females (68.4% [95% CI: 66.10–69.92]). Seroprevalence was lowest in >20 years (64.8% [95% CI: 62.36–67.19]) and highest among young adults; 20–39 years (71.1% [95% CI 68.83,73.39]). Seropositivity was associated with education, employment status and geographic location. Vaccination status in the study population was 10%. Exposure is more likely in urban than rural areas thus infection prevention protocols must be encouraged and maintained. Also, promoting vaccination in target groups and in rural areas is necessary to curb transmission of the virus.

BackgroundDengue, Zika and Chikungunya are Aedes-borne viral diseases that have risen to become great global health concerns in the past years. Several countries in Africa have reported outbreaks of these diseases and despite Ghana sharing borders with some of such countries, it remains free of these outbreaks.Recent studies in Ghana have revealed that there are antibodies and viral RNA of the Dengue virus serotype-2 among individuals in some localities in the Greater Accra Region. This is an indication of a possible silent transmission ongoing in the population, hence the need to assess the risk of transmission of these viruses within the country. This cross-sectional study, therefore, assessed the risk of transmission of Dengue, Zika and Chikungunya viruses in a domestic/peri-domestic (Madina) and a forest (Achimota Forest) population in the Greater Accra Region, Ghana.Methodology/FindingsAll stages of the Aedes mosquito (egg, larvae, pupae and adults) were collected around homes and in the forest area for estimation of risk indices. All eggs and immature stages were reared to adults and morphologically identified. The predominant species of Aedes mosquitoes identified from both sites were Aedes aegypti (98 % in Madina and 98.1% in Achimota forest). Aedes albopictus, an important arbovirus vector, was identified only in Madina at a prevalence of 1.5% but Achimota forest had the higher species diversity. Both study sites recorded high risk indices; Madina: Positive Ovitrap Index = 26.6%, Container Index = 36.8%, House Index = 19.8%, Breteau Index = 70.4%; Achimota: Positive Ovitrap Index = 34.2% and Container Index = 67.9%. RT-PCR to detect the presence of Dengue, Chikungunya and Zika viruses was negative for all pools tested.ConclusionAll entomological risk indicators estimated showed that both sites had a high potential of an outbreak of arboviral diseases following the introduction of these viruses.Author SummaryThe detection of antibodies and viral RNA of the dengue virus serotype 2 in some communities in the urban city of Accra, suggested the possibility of silent transmission of arboviral disease within the city. We assessed the risk of arboviral disease transmission using entomological risk indices. The study was a cross-sectional study conducted in a forest and peri domestic setting located in the southern urban city of Accra.The different stages of the Aedes mosquito were collected and, houses and containers positive for Aedes mosquitoes were also noted. The Breteau (BI), House (HI), Container (CI) and Positive ovitrap (POI) indices were determined. Real Time-PCR was conducted to determine the presence of Dengue, Zika and Chikungunya viruses in the larvae and adults collected.Aedes aegypti was the most common species identified from both sites. Aedes albopictus another competent arbovirus vector was identified in the peri-domestic site. Almost all risk indices recorded for both sites were higher than the WHO thresholds allowed for these indices. However, real time-PCR to detect the presence of Dengue, Chikungunya and Zika viruses was negative.The high entomological risk indicators estimated showed that both sites had a great potential of an outbreak following the introduction of these viruses, and a well-structured surveillance for these vectors is highly recommended. The detection of the presence of Ae. albopictus, an invasive species is also of great concern.

A vertically transmitted microsporidian, Microsporidia MB, with the ability to disrupt Plasmodium development was reported in Anopheles arabiensis from Kenya, East Africa. To demonstrate its range of incidence, archived DNA samples from 7575 Anopheles mosquitoes collected from Ghana were screened. MB prevalence was observed at 1.8%. An. gambiae s.s constituted 87% of positive mosquitoes while the remaining were from An. coluzzii. Both sibling species had similar positivity rates (24% and 19%; p = 0.42) despite the significantly higher number of An. gambiae s.s analysed (An. gambiae s.s = 487; An. coluzzii = 94; p = 0.0005). The microsporidian was also more prevalent in emerged adults from field-collected larvae than field-caught adults (p Microsporidia MB in Anopheles mosquitoes in West Africa. It indicates possible widespread among malaria vector species and warrants investigations into the symbiont’s diversity across sub-Saharan Africa.

A vertically transmitted microsporidian, Microsporidia MB, with the ability to disrupt Plasmodium development was reported in Anopheles arabiensis from Kenya, East Africa. To demonstrate its range of incidence, archived DNA samples from 7575 Anopheles mosquitoes collected from Ghana were screened. MB prevalence was observed at 1.8%. An. gambiae s.s constituted 87% of positive mosquitoes while the remaining were from An. coluzzii. Both sibling species had similar positivity rates (24% and 19%; p= 0.42) despite the significantly higher number of An. gambiae s.s analysed (An. gambiae s.s= 487; An. coluzzii= 94; p= 0.0005). The microsporidian was also more prevalent in field-collected larvae than adults (p< 0.0001) suggestive of an efficient vertical transmission and/or horizontal transfer among larvae. This is the first report of Microsporidia MB in Anopheles mosquitoes in West Africa. It indicates possible widespread among malaria vector species and warrants investigations into the symbiont’s diversity across the sub-Saharan Africa.

In the past decade, there has been increasing interest in mosquito microbiome research, leading to large amounts of data on different mosquito species, with various underlying physiological characteristics, and from diverse geographical locations. However, guidelines and standardized methods for conducting mosquito microbiome research are lacking. To streamline methods in mosquito microbiome research and optimize data quality, reproducibility, and comparability, as well as facilitate data curation in a centralized location, we are establishing the Mosquito Microbiome Consortium, a collaborative initiative for the advancement of mosquito microbiome research. Our overall goal is to collectively work on unraveling the role of the mosquito microbiome in mosquito biology, while critically evaluating its potential for mosquito-borne disease control. This perspective serves to introduce the consortium and invite broader participation. It highlights the issues we view as most pressing to the community and proposes guidelines for conducting mosquito microbiome research. We focus on four broad areas in this piece: (1) sampling/experimental design for field, semi-field, or laboratory studies; (2) metadata collection; (3) sample processing, sequencing, and use of appropriate controls; and (4) data handling and analysis. We finally summarize current challenges and highlight future directions in mosquito microbiome research. We hope that this piece will spark discussions around this area of disease vector biology, as well as encourage careful considerations in the design and implementation of mosquito microbiome research. Video Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s40168-020-00987-7.

The drivers and patterns of zoonotic virus emergence in the human population are poorly understood. The mosquito

The main malaria vectors in sub-Saharan Africa, the Anopheles gambiae (Giles; Diptera: Culicidae), normally breed in clean water sources. However, evidence suggests an on-going adaptation of Anopheline species to polluted breeding habitats in urban settings. This study aimed at understanding the adaptation to breeding in water bodies with different qualities, in five selected mosquito breeding sites in urban Accra, Ghana. The study sites were also evaluated for the WHO water-quality parameters as a measure of pollution, and insecticide residues. Field mosquitoes were evaluated for five genes; CYP6P3, CYP4H19, CYP4H24, GSTD1-4, and ABCC11-associated with insecticide detoxification-using quantitative RT-PCR, as well as Mono-oxygenase, Alpha Esterase, Glutathione S-transferase, and insensitive acetylcholinesterase (AChE) using biochemical enzyme assays. The lab-reared, insecticide susceptible An. gambiae Kisumu strain was bred in the most polluted water source for 10 generations and evaluated for the same genes and enzymes. The results revealed that the fold expression of the genes was higher in the larvae compared with the adults. The results also suggest that detoxification enzymes could be involved in the adaptation of An. gambiae to polluted breeding sites. Correlation analysis revealed a highly positive significant correlation between calcium levels and all five genes (P < 0.05). Stepwise linear regression to understand which of the variables predicted the expression of the genes revealed that sulphate was responsible for ABCC11 and CYP4H24, alkalinity for GSTD1-4, and calcium for CYP4H19 and CYP6P3. The detailed genetic basis of this adaptation need to be further investigated. A further understanding of this adaptation may provide outlooks for controlling malaria and other disease vectors adapted to polluted breeding water sources.

Background Malaria remains an important public health threat claiming many lives particularly in Sub-Saharan Africa. Light microscopy which is a blood-based test is the Gold standard for laboratory diagnosis of malaria in the clinical settings. The lack of sensitivity of Microscopy coupled with the challenges associated with blood sampling necessitates exploring alternative methods of identifying malaria cases. Aims and Objectives The aim of this study was to detect the presence of Plasmodium Lactate Dehydrogenase (pLDH) and Plasmodium falciparum Histidine-Rich Protein 2 (PfHRP2) in the saliva of suspected malaria patients and to compare the diagnostic accuracy of these saliva-borne biomarkers with the results of blood film microscopy using Nested PCR as the reference test. Methods The research was a comparative study. Matched saliva and blood samples of suspected malaria patients were collected. The blood samples were aliquoted into 2 smaller volumes and used to run blood film microscopy (thick and thin) and Nested PCR to detect DNA of the 5 Plasmodium species known to clinically infect man. Sand-wiched ELISA was separately run to qualitatively detect pLDH and PfHRP2 in the saliva samples. Accuracy indices of the saliva-based assays (saliva pLDH and PfHRP2) were compared with the conventional blood-based Microscopy. Results Of the participating 188 subjects, malaria prevalence rates of 51 (27.13%), 80(42.55%), 117 (62.23%) and 95 (50.53%) were detected by the Microscopy, saliva pLDH, saliva PfHRP2 and the PCR respectively. The sensitivity of the saliva PfHRP2 ELISA, 78.95% (95% CI, 69.38-86.64%) and saliva pLDH ELISA, 64.21% (95% CI 53.72 -73.79) were better than those obtained for the blood film Microscopy. Conclusions The saliva pLDH and the PfHRP2 ELISAs were found to be more sensitive (64.21% and 78.95% respectively) in defining malaria cases than the results obtained for the conventional blood film microscopy. Both the saliva pLDH and the PfHRP2 ELISAs showed moderate agreement with the results of the Nested PCR with Kappa co-efficient of 0.44 and 0.5 respectively.

SummaryThe majority of mosquito-borne illness is spread by a few mosquito species that have evolved to specialize in biting humans, yet the precise causes of this behavioral shift are poorly understood. We address this gap in the arboviral vector Aedes aegypti. We first characterize the behaviour of mosquitoes from 27 sites scattered across the species’ ancestral range in sub-Saharan Africa, revealing previously unrecognized diversity in female preference for human versus animal odor. We then use modelling to show that this diversity can be almost fully predicted by two ecological factors – dry season intensity and human population density. Finally we integrate this information with whole genome sequence data from 345 individual mosquitoes to identify a single underlying ancestry component linked to human preference, with genetic changes concentrated in a few key chromosomal regions. Our findings strongly suggest that human-biting in this important disease vector originally evolved as a by-product of breeding in human-stored water in areas where doing so provided the only means to survive the long, hot dry season. Our model also predicts that changes in human population density are likely to drive future mosquito evolution. Rapid urbanization may drive a shift to human-biting in many cities across Africa by 2050.

Dried filter blood spots have become a significant blood collection method for screening individuals for clinical purposes. When used for ELISAs, they are normally discarded after the blood has been eluted. However, they may still be useful for extraction of DNA for molecular-based assays. The aim of this work was to determine the integrity of DNA extracted from filter paper spots from which blood has initially been eluted for ELISA with sample dilution buffer (SDB) and phosphate buffered saline (PBS). DNA was extracted from the eluted filter spots, the eluate, and dried blood filter spots (controls) using spin column extraction. The quality and quantity of the extracted DNA was assessed and used for PCR to further evaluate their usefulness in molecular assays. Concentration of DNA obtained was dependent on the buffer used for processing the filter blood blots. Accounting for the DNA concentration obtained from dried blood spots, which were used as controls, DNA extracted from the already eluted blood spots were 32 times higher in PBS than SDB processed filter paper. The ratio was even higher for the eluates, which were 57 times higher in PBS than SDS eluates. SDB eluates had significantly higher average DNA concentration than their eluted filter paper, but their purity ratios were similar. 85% PCR success rate was achieved with the DNA samples. Useful DNA can be extracted from blood spots after it has been eluted with SDB. Although the DNA concentration and purity may be low, the DNA could be useful for rather simple PCR assays.Author SummaryCollection of blood onto filter paper has become an accepted method for screening individuals for clinical and public health purposes since the 1960s. This method of blood collection has become increasingly popular due to its ease and convenience in collection and transportation. The use of dried blood spots for clinical evaluations and research has become very significant. For research purposes, DBS when used for ELISAs are discarded after single use. DNA may however be extracted from the used filter blots and used for molecular assays. The concentration of DNA obtained may be low but simple assays like PCR could be done using the DNA extracted from the eluted filter spot.

Background The gut microbiota is known to play a role in a mosquito vector’s life history, a subject of increasing research. Laboratory experiments are essential for such studies and require laboratory colonies. In this study, the conservation of field-obtained midgut microbiota was evaluated in laboratory-reared Anopheles gambiae (s.l.) mosquitoes continuously hatched in water from field breeding habitats. Methods Pupae and late instars were obtained from the field and reared, and the emerged adults were blood-fed. The eggs obtained from them were hatched in either water from the field or in dechlorinated tap water. The mosquito colonies were maintained for 10 generations. Midguts of female adults from unfed F0 (emerging from field-caught pupae and larvae), F5 and F10 were dissected out and genomic DNA was extracted for 16S metagenomic sequencing. The sequences were compared to investigate the diversity and bacterial compositional differences using ANCOM and correlation clustering methods. Results Less than 10% of the bacterial families identified had differential relative abundances between generational groups and accounted for 46% of the variation observed. Although diversity reduced in F10 mosquitoes during laboratory colonization (Shannon-Weaver; P-value < 0.05), 50% of bacterial genera were conserved in those bred continuously in field-water compared to 38% in those bred in dechlorinated tap water. Conclusions To our knowledge, this study is the first report on the assessment of gut bacterial community of mosquitoes during laboratory colonization and recommends the use of water from the natural breeding habitats if they are intended for microbiota research. Electronic supplementary material The online version of this article (10.1186/s13071-019-3287-0) contains supplementary material, which is available to authorized users.

The immobilisation of mosquitoes for injection experiments is a requirement that must be achieved for the necessary time duration to complete the process. Unfortunately, the use of cold anaesthesia in hot tropical countries is not very effective and the use of CO2 anaesthesia requires continuous exposure, which can be harmful to the experimenter. To circumvent this problem we evaluated the use of triethylamine (FlyNap) in the anaesthesia of Anopheles gambiae mosquitoes. FlyNap has been used widely in Drosophila studies, and is known to irreversibly anaesthetise mosquitoes. Very small volumes of the original FlyNap as well as different dilutions using absolute ethanol were evaluated to determine the dosage that will effectively anesthetise Anopheles gambiae female mosquitoes for the necessary duration. The results showed that the 1/10 (FlyNap/absolute ethanol) dilution worked well and could be used for the anaesthesia. Injecting live adults mosquitoes for scientific experiments is a delicate process that requires immobilising them for the required period of time. The immobilisation can be done using two main methods; chilling using cold anaesthesia (Garver & Dimopoulos 2007) or by chemical anaesthesia using CO2 (Lamacchia et al. 2013). In tropical climates cold anaesthesia is not very effective, and some mosquitoes can withstand some degree of chilling and hence may not be immobilised for long. Chemical anaesthesia, such as CO2 on the other hand, can result in the death of mosquitoes, and may be harmful to humans. A chemical anaesthesia commonly used is triethylamine. It is safe and poses no harmful effects to humans (Budavari 1989). Triethylamine formulated as FlyNap® (Carolina Biological Supply Co., NC, U.S.A.) is commonly available for anaesthetic purposes for insects. FlyNap is a mixture of 50 % triethylamine, 25 % ethanol and 25 % fragrance. It is 100 % volatile, water-soluble and can be stored in a cool, well-ventilated place. When exposed to FlyNap, insects are quickly incapacitated but remain alive and are unable to fly for several hours (Kramer et al. 1990). However, when in high doses, exposure causes death of the insect. It has been widely used in Drosophila anaesthesia (Paternostro et al. 2001; Babcock et al. 2008), and only used in lethal doses to kill mosquitoes (O’Guinn & Turell 2002; Goodman et al. 2003; Chen & Hillyer 2013). Our attempts to immobilise adult Anopheles gambiae mosquitoes using cold anaesthesia for injection experiments have proven unsuccessful, primarily due to the inability to immobilise mosquitoes long enough to complete the procedure. To circumvent this problem, this study aimed at evaluating the use of FlyNap and the dosage that will effectively anaesthetise An. gambiae long enough for experimental assays, and also result in high mosquito recovery rates. The experiment was conducted with 3and 5day-old Kisumu strain female An. gambiae mosquitoes. The mosquitoes were fed ad libitum on 10 % sucrose solution, following emergence. For each experiment, 10 mosquitoes were placed in a plastic cup and exposed to one of the following concentrations of FlyNap: 1 μl and 2 μl of stock, as well as 10 μl solutions of 1/10, 1/100, 1/1000 dilutions of FlyNap. The dilutions were prepared with absolute ethanol. The FlyNap to be tested was aliquoted onto small cotton balls and placed in the cups with the mosquitoes. To evaluate the additional effect of the ethanol in anaesthetising the mosquitoes, a control experiment using 10 μl absolute ethanol only was included for the 3and 5-day-old mosquito groups. Each cup was turned upside down to ensure that the fumes from the FlyNap did not escape and could effectively knock down the mosquitoes. The time taken for the first mosquito to be knocked down was recorded for each experiment, and the number of mosquitoes knocked down was recorded at 10-min intervals. After all mosquitoes were knocked down, they were transferred to new cups. The time taken for the first mosquito to revive was noted, and the number of revived mosquitoes was subsequently recorded at 30-min intervals. All experiments were replicated three times to determine the average time of knock *Author for correspondence. E-mail: ddesouza@noguchi.ug.edu.gh