Thermal tolerance of mosquito eggs is associated with urban adaptation and human interactions.

Climate change is expected to profoundly affect mosquito distributions and their ability to serve as vectors for disease, specifically with the anticipated increase in heat waves. The rising temperature and frequent heat waves can accelerate mosquito life cycles, facilitating higher disease transmission. Conversely, higher temperatures could increase mosquito mortality as a negative consequence. Warmer temperatures are associated with increased human density, suggesting a need for anthropophilic mosquitoes to adapt to be more hardy to heat stress. Mosquito eggs provide an opportunity to study the biological impact of climate warming as this stage is stationary and must tolerate temperatures at the site of female oviposition. As such, egg thermotolerance is critical for survival in a specific habitat. In nature, Aedes mosquitoes exhibit different behavioral phenotypes, where specific populations prefer depositing eggs in tree holes and prefer feeding non-human vertebrates. In contrast, others, particularly human-biting specialists, favor laying eggs in artificial containers near human dwellings. This study examined the thermotolerance of eggs, along with adult stages, for Aedes aegypti and Ae. albopictus lineages associated with known ancestry and shifts in their relationship with humans. Mosquitoes collected from areas with higher human population density, displaying increased human preference, and having a human-associated ancestry profile have increased egg viability following high-temperature stress. Unlike eggs, thermal tolerance among adults showed no significant correlation based on the area of collection or human-associated ancestry. This study highlights that the egg stage is likely critical to mosquito survival when associated with humans and needs to be accounted when predicting future mosquito distribution.

Extensive variation and strain-specificity in dengue virus susceptibility among African Aedes aegypti populations.

African populations of the mosquito Aedes aegypti are usually considered less susceptible to infection by human-pathogenic flaviviruses than globally invasive populations found outside Africa. Although this contrast has been well documented for Zika virus (ZIKV), it is unclear to what extent it is true for dengue virus (DENV), the most prevalent flavivirus of humans. Addressing this question is complicated by substantial genetic diversity among DENV strains, most notably in the form of four genetic types (DENV1 to DENV4), that can lead to genetically specific interactions with mosquito populations. Here, we carried out a survey of DENV susceptibility using a panel of seven field-derived Ae. aegypti colonies from across the African range of the species and a colony from Guadeloupe, French West Indies as non-African reference. We found considerable variation in the ability of African Ae. aegypti populations to acquire and replicate a panel of six DENV strains spanning the four DENV types. Although African Ae. aegypti populations were generally less susceptible than the reference non-African population from Guadeloupe, in several instances some African populations were equally or more susceptible than the Guadeloupe population. Moreover, the relative level of susceptibility between African mosquito populations depended on the DENV strain, indicating genetically specific interactions. We conclude that unlike ZIKV susceptibility, there is no clear-cut dichotomy in DENV susceptibility between African and non-African Ae. aegypti. DENV susceptibility of African Ae. aegypti populations is highly heterogeneous and largely governed by the specific pairing of mosquito population and DENV strain.

Two promising candidates for paratransgenesis, Elizabethkingia and Asaia, increase in both sexes of Anopheles gambiae mosquitoes after feeding.

BACKGROUND: The male mosquito microbiome may be important for identifying ideal candidates for disease control. Among other criteria, mosquito-associated symbionts that have high localization in both male and female mosquitoes and are transmissible through both vertical and sexual routes are desirable. However, mosquito microbiome studies have mainly been female-focused. In this study, the microbiota of male and female Anopheles gambiae sensu lato (s.l.) were compared to identify shared or unique bacteria. METHODS: Late larval instars of Anopheles mosquitoes were collected from the field and raised to adults. Equal numbers of males and females of 1-day-old non-sugar-fed, 4-5-day-old sugar-fed and post-blood-fed females were randomly selected for whole-body analyses of bacteria 16S rRNA. RESULTS: Results revealed that male and female mosquitoes generally share similar microbiota except when females were blood-fed. Compared to newly emerged unfed mosquitoes, feeding on sugar and/or blood increased variability in microbial composition (⍺-diversity), with a higher disparity among females (39% P = 0.01) than in males (29% P = 0.03). Elizabethkingia meningoseptica and Asaia siamensis were common discriminants between feeding statuses in both males and females. While E. meningoseptica was particularly associated with sugar-fed mosquitoes of both sexes and sustained after blood feeding in females, A. siamensis was also increased in sugar-fed mosquitoes but decreased significantly in blood-fed females (LDA score > 4.0, P < 0.05). Among males, A. siamensis did not differ significantly after sugar meals. CONCLUSIONS: Results indicate the opportunities for stable infection in mosquitoes should these species be used in bacteria-mediated disease control. Further studies are recommended to investigate possible host-specific tissue tropism of bacteria species which will inform selection of the most appropriate microbes for effective transmission-blocking strategies.

Molecular identification of cattle ticks in the Greater Accra Region of Ghana: a high occurrence of Rhipicephalus microplus.

Ticks are competent vectors of a wide range of pathogens. They are of veterinary and public health importance as they affect both animal and human health. Transhumance and the transboundary movements of cattle within the West African Sub-region have facilitated the spread of ticks which threatens the introduction of invasive species. Currently, Rhipicephalus microplus have been identified in the Upper East Region of Ghana which could mean a wider distribution of the species across the country due to livestock trade. This study focused on three sites in the Greater Accra Region, which serves as the gateway to receiving most of the cattle transported from the northern regions of Ghana. Ticks were sampled from August 2022 in the wet season to January 2023 in the dry season. Three tick genera were identified: Amblyomma (19.5%), Hyalomma (1.1%), and Rhipicephalus (79.3%) from the 1,489 feeding ticks collected from cattle. Furthermore, Rhipicephalus microplus, Hyalomma rufipes and Amblyomma variegatum were identified molecularly using primers that target the mitochondrial COI gene. There was a significant association between the tick species and seasons (p < 0.001). Finding R. microplus in this study indicates the extent of the spread of this invasive tick species in Ghana and highlights the need for efficient surveillance systems and control measures within the country.

Entomological risk assessment for transmission of arboviral diseases by Aedes mosquitoes in a domestic and forest site in Accra, Ghana.

Dengue, Zika and chikungunya are Aedes-borne viral diseases that have 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 these countries, such outbreaks are yet to be detected. Viral RNA and antibodies against dengue serotype-2 have recently been reported among individuals in some localities in the regional capital of Ghana. This is an indication of a possible silent transmission ongoing in the population. This study, therefore, investigated the entomological transmission risk of dengue, Zika and chikungunya viruses in a forest and domestic population in the Greater Accra Region, Ghana. All 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 were hatched and reared to larvae or adults for morphological identification together with larvae and adults collected from the field. The forest population had higher species richness with 7 Aedes species. The predominant species of Aedes mosquitoes identified from both sites was Aedes aegypti (98%). Aedes albopictus, an important arbovirus vector, was identified only in the peri-domestic population at a prevalence of 1.5%, significantly higher than previously reported. All risk indices were above the WHO threshold except the House Index for the domestic site which was moderate (19.8). The forest population recorded higher Positive Ovitrap (34.2% vs 26.6%) and Container (67.9% vs 36.8%) Indices than the peri-domestic population. Although none of the mosquito pools showed the presence of dengue, chikungunya or Zika viruses, all entomological risk indicators showed that both sites had a high potential arboviral disease transmission risk should any of these viruses be introduced. Continuous surveillance is recommended in these and other sites in the Metropolis to properly map transmission risk areas to inform outbreak preparedness strategies.

Assessing the performance of regular surgical nose masks as a sampling method for SARS-CoV-2 detection in a cross-sectional study.

Nose masks are widely worn for protection against respiratory pathogens, including SARS-CoV-2. They have been reported as possible substrates for viral sampling and testing for COVID-19 but, evaluations have so far been purposive; involving individuals known to have the infection and using improved materials on the nose masks to trap the virus. We investigated the feasibility of using the regular 3-ply surgical masks and, voluntary coughing as a mode of particle expulsion for detecting SARS-CoV-2 infections in a cross-sectional study at Ghana's first COVID-19 testing reference laboratory, the Noguchi Memorial Institute for Medical Research, University of Ghana. Paired samples of naso-oropharyngeal swabs and nose masks already worn by 103 consenting adult participants (retro masks) were collected. Participants were also required to produce three strong coughs into a newly supplied sterile surgical nose mask. Pre-wetted swabs in Viral Transport Media (VTM) were used in swabbing the inner lining of each nose mask. The swabs used were then stored in VTM to maintain the integrity of the samples. PCR results of SARS-CoV-2 detection from the nose masks were compared to those from naso-oropharyngeal swabs ('gold-standard'). Out of the 103 participants tested with all three methods, 66 individuals sampled with naso-oropharyngeal swabs were detected as positive, and the retro and new masks matched 9 and 4, respectively. Only 3 individuals were positive across all three sampling methods accessed. The retro nose masks performed better in matching the gold-standard results than the new mask + coughing method, with 90% vs 80% sensitivity, positive predictive value of 13.6% vs 6%, and a weak but significant linear relationship (adj. R2 = 0.1; P = 0.0004). Importantly, we also show that the nose masks would work for sampling whether individuals are symptomatic or asymptomatic since gold-standard PCR cycling threshold (Ct) values for positive individuals did not differ between the two groups (P< 0.05). We recommend including features such as talking during participant engagement, use of a spontaneous cough inducer and increased coughing bouts > 3, to improve the performance of sterile nose masks for SARS-CoV-2 detection.

Investigating Environmental Determinants of Soil-Transmitted Helminths Transmission using GPS Tracking and Metagenomics Technologies.

Background: The Global Health community aims to eliminate soil-transmitted helminth (STH) infections by 2030. Current preventive methods such as Mass Drug Administration, WASH practices, and health education needs to be complimented to halt transmission. We tracked the movement of hookworm-infected and non-infected persons and investigated soil factors in the places they frequented within an endemic community to further understand the role of human movement and sources of infections. Methods: 59 positive and negative participants wore GPS tracking devices for 10 consecutive days and their movement data captured in real time. The data was overlaid on the community map to determine where each group differentially spent most of their time. Soil samples were collected from these identified sites and other communal places. Physical and chemical properties were determined for each sample using standard methods and helminth eggs cultured into larvae using the Baermann technique. Bivariate and multivariate analyses were used to determine associations between larvae counts and soil factors. Helminth species were identified with metagenomic sequencing and their distributions mapped to sampling sites in the community. Results: The study found that there was no significant difference in the average larvae counts in soil between sites assessed by infected and non-infected participants (P=0.59). However, soil factors, such as pH, carbon and sandy-loamy texture were associated with high larvae counts (P<0.001) while nitrogen and clay content were associated with low counts(P<0.001). The dominant helminth species identified were Panagrolaimus superbus (an anhydrobiotic helminth), Parastrongyloides trichosuri (a parasite of small mammals), Trichuris trichuria (whipworm), and Ancylostoma caninum (dog hookworm). Notably, no Necator americanus was identified in any soil sample. Conclusion: This study provides important insights into the association between soil factors and soil-transmitted helminths. These findings contribute to our understanding of STH epidemiology and support evidence-based decision-making for elimination strategies.

The effect of single dose albendazole (400 mg) treatment on the human gut microbiome of hookworm-infected Ghanaian individuals.

Microbes play a key 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 stool samples from consented participants who were either cured or remained infected after treatment. At each time point, stool was collected prior to and 10-14 days after albendazole treatment. We used 16S rRNA amplicon sequencing of DNA extracted from stool samples to investigate the composition and diversity of the gut microbiota and to identify potential microbial biomarkers associated with treatment outcomes. Hookworm infection was associated with increased species richness (p = 0.0093). Among treated individuals, there was also 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 change in microbiota composition (ANOSIM; p = 0.35). Uninfected individuals and those who were successfully treated were similar in their microbial composition and structure. We also found that the abundance of Clostridia spp. was increased in infected individuals pre- or post-treatment. Predictive functional profiling revealed the enrichment of two pyruvate ferredoxin oxidoreductase subunit pathways in individuals who remained infected after treatment (p < 0.05), alluding to an upturn of strictly anaerobic commensal bacteria such as 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.

Development of an Affordable ELISA Targeting the SARS-CoV-2 Nucleocapsid and Its Application to Samples from the Ongoing COVID-19 Epidemic in Ghana.

INTRODUCTION: The true nature of the population spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in populations is often not fully known as most cases, particularly in Africa, are asymptomatic. Finding the true magnitude of SARS-CoV-2 spread is crucial to provide actionable data about the epidemiological progress of the disease for researchers and policymakers. This study developed and optimized an antibody enzyme-linked immunosorbent assay (ELISA) using recombinant nucleocapsid antigen expressed in-house using a simple bacterial expression system. METHODS: Nucleocapsid protein from SARS-CoV-2 was expressed and purified from Escherichia coli. Plasma samples used for the assay development were obtained from Ghanaian SARS-CoV-2 seropositive individuals during the pandemic, while seronegative controls were plasma samples collected from blood donors before the coronavirus disease 2019 (COVID-19) pandemic. Another set of seronegative controls was collected during the COVID-19 pandemic. Antibody detection and levels within the samples were validated using commercial kits and Luminex. Analyses were performed using GraphPad Prism, and the sensitivity, specificity and background cut-off were calculated. RESULTS AND DISCUSSION: This low-cost ELISA (£0.96/test) assay has a high prediction of 98.9%, and sensitivity and specificity of 97% and 99%, respectively. The assay was subsequently used to screen plasma from SARS-CoV-2 RT-PCR-positive Ghanaians. The assay showed no significant difference in nucleocapsid antibody levels between symptomatic and asymptomatic, with an increase of the levels over time. This is in line with our previous publication. CONCLUSION: This study developed a low-cost and transferable assay that enables highly sensitive and specific detection of human anti-SARS-CoV-2 IgG antibodies. This assay can be modified to include additional antigens and used for continuous monitoring of sero-exposure to SARS-CoV-2 in West Africa.

Modeling SARS-CoV-2 antibody seroprevalence and its determinants in Ghana: A nationally representative cross-sectional survey.

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.

Dating the origin and spread of specialization on human hosts in Aedes aegypti mosquitoes.

The globally invasive mosquito subspecies Aedes aegypti aegypti is an effective vector of human arboviruses, in part 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 relies on human-stored water for breeding. Here, we use whole-genome cross-coalescent analysis to date the emergence of human-specialist populationsand thus further probe the climate hypothesis. Importantly, we take advantage of the known migration of specialists 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 5000 years ago, at 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. The characteristic length of tracts of human-specialist ancestry present on a generalist genetic background in Kumasi and Ouagadougou 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.

Evolution of a Mosquito's Hatching Behavior to Match Its Human-Provided Habitat.

AbstractA subspecies of the yellow fever mosquito, Aedes aegypti, has recently evolved to specialize in biting and living alongside humans. It prefers human odor over the odor of nonhuman animals and breeds in human-provided artificial containers rather than the forest tree holes of its ancestors. Here, we report one way this human specialist has adapted to the distinct ecology of human environments. While eggs of the ancestral subspecies rarely hatch in pure water, those of the derived human specialist do so readily. We trace this novel behavior to a shift in how eggs respond to dissolved oxygen, low levels of which may signal food abundance. Moreover, we show that while tree holes are consistently low in dissolved oxygen, artificial containers often have much higher levels. There is thus a concordance between the hatching behavior of each subspecies and the aquatic habitat it uses in the wild. We find this behavioral variation is heritable, with both maternal and zygotic effects. The zygotic effect depends on dissolved oxygen concentration (i.e., a genotype-environment interaction, or G×E), pointing to potential changes in oxygen-sensitive circuits. Together, our results suggest that a shift in hatching response contributed to the pernicious success of this human-specialist mosquito and illustrate how animals may rapidly adapt to human-driven changes in the environment.

Extensive variation and strain-specificity in dengue virus susceptibility among African Aedes aegypti populations.

African populations of the mosquito Aedes aegypti are usually considered less susceptible to infection by human-pathogenic flaviviruses than globally invasive populations found outside Africa. Although this contrast has been well documented for Zika virus (ZIKV), it is unclear to what extent it is true for dengue virus (DENV), the most prevalent flavivirus of humans. Addressing this question is complicated by substantial genetic diversity among DENV strains, most notably in the form of four genetic types (DENV1 to DENV4), that can lead to genetically specific interactions with mosquito populations. Here, we carried out a continent-wide survey of DENV susceptibility using a panel of field-derived Ae. aegypti colonies from across the African range of the species and a colony from Guadeloupe, French West Indies as non-African reference. We found considerable variation in the ability of African Ae. aegypti populations to acquire and replicate a panel of six DENV strains spanning the four DENV types. Although African Ae. aegypti populations were generally less susceptible than the reference non-African population from Guadeloupe, in several instances some African populations were equally or more susceptible than the Guadeloupe population. Moreover, the relative level of susceptibility between African mosquito populations depended on the DENV strain, indicating genetically specific interactions. We conclude that unlike ZIKV susceptibility, there is no clear-cut dichotomy in DENV susceptibility between African and non-African Ae. aegypti. DENV susceptibility of African Ae. aegypti populations is highly heterogeneous and largely governed by the specific pairing of mosquito population and DENV strain.

RETRACTED: Gradual emergence followed by exponential spread of the SARS-CoV-2 Omicron variant in Africa.

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.

Building the capacity of West African countries in Aedes surveillance: inaugural meeting of the West African Aedes Surveillance Network (WAASuN).

Arboviral diseases such as dengue, Zika and chikungunya transmitted by Aedes mosquitoes have been reported in 34 African countries. Available data indicate that in recent years there have been dengue and chikungunya outbreaks in the West Africa subregion, in countries including Côte d'Ivoire, Burkina Faso, Gabon, Senegal, and Benin. These viral diseases are causing an increased public health burden, which impedes poverty reduction and sustainable development. Aedes surveillance and control capacity, which are key to reducing the prevalence of arboviral infections, need to be strengthened in West Africa, to provide information essential for the formulation of effective vector control strategies and the prediction of arboviral disease outbreaks. In line with these objectives, the West African Aedes Surveillance Network (WAASuN) was created in 2017 at a meeting held in Sierra Leone comprising African scientists working on Aedes mosquitoes. This manuscript describes the proceedings and discusses key highlights of the meeting.

Evidence of High Frequencies of Insecticide Resistance Mutations in Aedes aegypti (Culicidae) Mosquitoes in Urban Accra, Ghana: Implications for Insecticide-based Vector Control of Aedes-borne Arboviral Diseases.

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.

Water Physicochemical Parameters and Microbial Composition Distinguish Anopheles and Culex Mosquito Breeding Sites: Potential as Ecological Markers for Larval Source Surveillance.

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.

Application of multiplex amplicon deep-sequencing (MAD-seq) to screen for putative drug resistance markers in the Necator americanus isotype-1 ß-tubulin gene.

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 ß-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 ß-tubulin gene. Three single nucleotide polymorphisms (SNPs) corresponding to resistance-associated mutations (F167Y, E198A, F200Y) within the coding region of the isotype-1 ß-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.

Bio-products from Serratia marcescens isolated from Ghanaian Anopheles gambiae reduce Plasmodium falciparum burden in vector mosquitoes.

Novel ideas for control of mosquito-borne disease include the use of bacterial symbionts to reduce transmission. Bacteria belonging to the family Enterobacteriaceae isolated from mosquito midgut have shown promise in limiting Plasmodium intensity in the Anopheles vector. However, the mechanism of interaction between bacteria and parasite remains unclear. This study aimed at screening bio-products of two bacteria candidates for their anti-Plasmodial effects on mosquito stages of P. falciparum. Enterobacter cloacae and Serratia marcescens were isolated from field-caught Anopheles gambiae s.l. Spent media from liquid cultures of these bacteria were filtered, lyophilized and dissolved in sterile phosphate buffered saline (PBS). The re-dissolved bacterial products were added to gametocytaemic blood meals and fed to An. gambiae mosquitoes via membrane feeders. Control groups were fed on infected blood with or without lyophilized LB medium. The effect of the products on the infection prevalence and intensity of P. falciparum in mosquitoes was assessed by dissecting mosquito midguts and counting oocysts 10-11 days post-infection. S. marcescens bio-products elicited significant reduction in the number of mosquitoes infected (P=4.02 x10-5) with P. falciparum and the oocyst intensity (P<2 x 10-16) than E. cloacae products (P>0.05 for both prevalence and intensity) compared to the control (lyophilized LB medium). These data support the use of bioproducts released by S. marcescens for malaria control based on transmission blocking in the vector.