Genotyping of Anopheles mosquito blood meals reveals nonrandom human host selection: implications for human-to-mosquito Plasmodium falciparum transmission.

BACKGROUND: Control of malaria parasite transmission can be enhanced by understanding which human demographic groups serve as the infectious reservoirs. Because vector biting can be heterogeneous, some infected individuals may contribute more to human-to-mosquito transmission than others. Infection prevalence peaks in school-age children, but it is not known how often they are fed upon. Genotypic profiling of human blood permits identification of individual humans who were bitten. The present investigation used this method to estimate which human demographic groups were most responsible for transmitting malaria parasites to Anopheles mosquitoes. It was hypothesized that school-age children contribute more than other demographic groups to human-to-mosquito malaria transmission. METHODS: In a region of moderate-to-high malaria incidence in southeastern Malawi, randomly selected households were surveyed to collect human demographic information and blood samples. Blood-fed, female Anopheles mosquitoes were sampled indoors from the same houses. Genomic DNA from human blood samples and mosquito blood meals of human origin was genotyped using 24 microsatellite loci. The resultant genotypes were matched to identify which individual humans were sources of blood meals. In addition, Plasmodium falciparum DNA in mosquito abdomens was detected with polymerase chain reaction. The combined results were used to identify which humans were most frequently bitten, and the P. falciparum infection prevalence in mosquitoes that resulted from these blood meals. RESULTS: Anopheles females selected human hosts non-randomly and fed on more than one human in 9% of the blood meals. Few humans contributed most of the blood meals to the Anopheles vector population. Children ≤ 5 years old were under-represented in mosquito blood meals while older males (31-75 years old) were over-represented. However, the largest number of malaria-infected blood meals was from school age children (6-15 years old). CONCLUSIONS: The results support the hypothesis that humans aged 6-15 years are the most important demographic group contributing to the transmission of P. falciparum to the Anopheles mosquito vectors. This conclusion suggests that malaria control and prevention programmes should enhance efforts targeting school-age children and males.

Screening eaves of houses reduces indoor mosquito density in rural, western Kenya.

BACKGROUND: Despite the scale-up of insecticide-treated nets and indoor residual spraying, the bulk of malaria transmission in western Kenya still occurs indoors, late at night. House improvement is a potential long-term solution to further reduce malaria transmission in the region. METHODS: The impact of eave screening on mosquito densities was evaluated in two rural villages in western Kenya. One-hundred-and-twenty pairs of structurally similar, neighbouring houses were used in the study. In each pair, one house was randomly selected to receive eave screening at the beginning of the study while the other remained unscreened until the end of the sampling period. Mosquito sampling was performed monthly by motorized aspiration method for 4 months. The collected mosquitoes were analysed for species identification. RESULTS: Compared to unscreened houses, significantly fewer female Anopheles funestus (RR = 0.40, 95% CI 0.29-0.55), Anopheles gambiae Complex (RR = 0.46, 95% CI 0.34-0.62) and Culex species (RR = 0.53, 95% CI 0.45-0.61) were collected in screened houses. No significant differences in the densities of the mosquitoes were detected in outdoor collections. Significantly fewer Anopheles funestus were collected indoors from houses with painted walls (RR = 0.05, 95% CI 0.01-0.38) while cooking in the house was associated with significantly lower numbers of Anopheles gambiae Complex indoors (RR = 0.60, 95% CI 0.45-0.79). Nearly all house owners (99.6%) wanted their houses permanently screened, including 97.7% that indicated a willingness to use their own resources. However, 99.2% required training on house screening. The cost of screening a single house was estimated at KES6,162.38 (US$61.62). CONCLUSION: Simple house modification by eave screening has the potential to reduce the indoor occurrence of both Anopheles and Culex mosquito species. Community acceptance was very high although education and mobilization may be needed for community uptake of house modification for vector control. Intersectoral collaboration and favourable government policies on housing are important links towards the adoption of house improvements for malaria control.

Vector composition, abundance, biting patterns and malaria transmission intensity in Madang, Papua New Guinea: assessment after 7 years of an LLIN-based malaria control programme.

BACKGROUND: A malaria control programme based on distribution of long-lasting insecticidal bed nets (LLINs) and artemisinin combination therapy began in Papua New Guinea in 2009. After implementation of the programme, substantial reductions in vector abundance and malaria transmission intensity occurred. The research reported here investigated whether these reductions remained after seven years of sustained effort. METHODS: All-night (18:00 to 06:00) mosquito collections were conducted using human landing catches and barrier screen methods in four villages of Madang Province between September 2016 and March 2017. Anopheles species identification and sporozoite infection with Plasmodium vivax and Plasmodium falciparum were determined with molecular methods. Vector composition was expressed as the relative proportion of different species in villages, and vector abundance was quantified as the number of mosquitoes per barrier screen-night and per person-night. Transmission intensity was quantified as the number of sporozoite-infective vector bites per person-night. RESULTS: Five Anopheles species were present, but vector composition varied greatly among villages. Anopheles koliensis, a strongly anthropophilic species was the most prevalent in Bulal, Matukar and Wasab villages, constituting 63.7-73.8% of all Anopheles, but in Megiar Anopheles farauti was the most prevalent species (97.6%). Vector abundance varied among villages (ranging from 2.8 to 72.3 Anopheles per screen-night and 2.2-31.1 Anopheles per person-night), and spatially within villages. Malaria transmission intensity varied among the villages, with values ranging from 0.03 to 0.5 infective Anopheles bites per person-night. Most (54.1-75.1%) of the Anopheles bites occurred outdoors, with a substantial proportion (25.5-50.8%) occurring before 22:00. CONCLUSION: The estimates of vector abundance and transmission intensity in the current study were comparable to or higher than estimates in the same villages in 2010-2012, indicating impeded programme effectiveness. Outdoor and early biting behaviours of vectors are some of the likely explanatory factors. Heterogeneity in vector composition, abundance and distribution among and within villages challenge malaria control programmes and must be considered when planning them.

Blood-feeding patterns of Anopheles vectors of human malaria in Malawi: implications for malaria transmission and effectiveness of LLIN interventions.

BACKGROUND: Access to human hosts by Anopheles mosquitoes is a key determinant of vectorial capacity for malaria, but it can be limited by use of long-lasting insecticidal nets (LLINs). In Malawi, pyrethroid-treated LLINs with and without the synergist piperonyl butoxide (PBO) were distributed to control malaria. This study investigated the blood-feeding patterns of malaria vectors and whether LLINs containing pyrethroid and PBO led to a reduction of human blood feeding than those containing only pyrethroids. METHODS: Mosquitoes were sampled inside houses from May 2019 through April 2020 by aspiration, pyrethrum spray catch, and light trap methods in two sites. One site (Namanolo, Balaka district) had LLINs containing only pyrethroids whereas the other (Ntaja, Machinga district) had LLINs with both pyrethroids and PBO. Anopheles species, their blood-meal host, and infection with Plasmodium falciparum were determined using PCR methods. RESULTS: A total of 6585 female Anopheles were sampled in 203 houses. Of these, 633 (9.6%) were blood-fed mosquitoes comprising of 279 (44.1%) Anopheles arabiensis, 103 (16.3%) Anopheles gambiae 212 (33.5), Anopheles funestus, 2 (0.3%), Anopheles parensis and 37 (5.8%) were unidentified Anopheles spp. Blood meal hosts were successfully identified for 85.5% (n = 541) of the blood-fed mosquitoes, of which 436 (81.0%) were human blood meals, 28 (5.2%) were goats, 11 (2.0%) were dogs, 60 (11.1%) were mixed goat-human blood meals, 5 (0.9%) were dog-human, and 1 was a mixed dog-goat. Human blood index (fraction of blood meals that were humans) was significantly higher in Namanolo (0.96) than Ntaja (0.89). Even though human blood index was high, goats were over-selected than humans after accounting for relative abundance of both hosts. The number of infectious Anopheles bites per person-year was 44 in Namanolo and 22 in Ntaja. CONCLUSION: Although LLINs with PBO PBO may have reduced human blood feeding, access to humans was extremely high despite high LLIN ownership and usage rates in both sites. This finding could explain persistently high rates of malaria infections in Malawi. However, this study had one village for each net type, thus the observed differences may have been a result of other factors present in each village.

Temporal trends in molecular markers of drug resistance in Plasmodium falciparum in human blood and profiles of corresponding resistant markers in mosquito oocysts in Asembo, western Kenya.

BACKGROUND: Over the last two decades, the scale-up of vector control and changes in the first-line anti-malarial, from chloroquine (CQ) to sulfadoxine-pyrimethamine (SP) and then to artemether-lumefantrine (AL), have resulted in significant decreases in malaria burden in western Kenya. This study evaluated the long-term effects of control interventions on molecular markers of Plasmodium falciparum drug resistance using parasites obtained from humans and mosquitoes at discrete time points. METHODS: Dried blood spot samples collected in 2012 and 2017 community surveys in Asembo, Kenya were genotyped by Sanger sequencing for markers associated with resistance to SP (Pfdhfr, Pfdhps), CQ, AQ, lumefantrine (Pfcrt, Pfmdr1) and artemisinin (Pfk13). Temporal trends in the prevalence of these markers, including data from 2012 to 2017 as well as published data from 1996, 2001, 2007 from same area, were analysed. The same markers from mosquito oocysts collected in 2012 were compared with results from human blood samples. RESULTS: The prevalence of SP dhfr/dhps quintuple mutant haplotype C50I51R59N108I164/S436G437E540A581A613 increased from 19.7% in 1996 to 86.0% in 2012, while an increase in the sextuple mutant haplotype C50I51R59N108I164/H436G437E540A581A613 containing Pfdhps-436H was found from 10.5% in 2012 to 34.6% in 2017. Resistant Pfcrt-76 T declined from 94.6% in 2007 to 18.3% in 2012 and 0.9% in 2017. Mutant Pfmdr1-86Y decreased across years from 74.8% in 1996 to zero in 2017, mutant Pfmdr1-184F and wild Pfmdr1-D1246 increased from 17.9% to 58.9% in 2007 to 55.9% and 90.1% in 2017, respectively. Pfmdr1 haplotype N86F184S1034N1042D1246 increased from 11.0% in 2007 to 49.6% in 2017. No resistant mutations in Pfk13 were found. Prevalence of Pfdhps-436H was lower while prevalence of Pfcrt-76 T was higher in mosquitoes than in human blood samples. CONCLUSION: This study showed an increased prevalence of dhfr/dhps resistant markers over 20 years with the emergence of Pfdhps-436H mutant a decade ago in Asembo. The reversal of Pfcrt from CQ-resistant to CQ-sensitive genotype occurred following 19 years of CQ withdrawal. No Pfk13 markers associated with artemisinin resistance were detected, but the increased haplotype of Pfmdr1 N86F184S1034N1042D1246 was observed. The differences in prevalence of Pfdhps-436H and Pfcrt-76 T SNPs between two hosts and the role of mosquitoes in the transmission of drug resistant parasites require further investigation.

Eastern Equine Encephalitis Virus in Mexican Wolf Pups at Zoo, Michigan, USA.

During the 2019 Eastern equine encephalitis virus (EEEV) outbreak in Michigan, two 2-month old Mexican wolf pups experienced neurologic signs, lymphohistiocytic neutrophilic meningoencephalitis with neuronal necrosis and neuronophagia, and acute death. We identified EEEV by reverse transcription real-time PCR and in situ hybridization. Vector mosquitoes were trapped at the zoo.

Estimating spatio-temporal distributions of mosquito breeding pools in irrigated agricultural schemes: a case study at the Bwanje Valley Irrigation Scheme.

BACKGROUND: The association between irrigation and the proliferation of adult mosquitoes including malaria vectors is well known; however, irrigation schemes are treated as homogenous spatio-temporal units, with little consideration for how larval breeding varies across space and time. The objective of this study was to estimate the spatio-temporal distribution of pools of water facilitating breeding at the Bwanje Valley Irrigation Scheme (BVIS) in Malawi, Africa as a function of environmental and anthropogenic characteristics. METHODS: Irrigation structure and land cover were quantified during the dry and rainy seasons of 2016 and 2017, respectively. These data were combined with soil type, irrigation scheduling, drainage, and maintenance to model suitability for mosquito breeding across the landscape under three scenarios: rainy season, dry season with limited water resources, and a dry season with abundant water resources. RESULTS: Results demonstrate seasonal, asymmetrical breeding potential and areas of maximum breeding potential as a function of environmental characteristics and anthropogenic influence in each scenario. The highest percentage of suitable area for breeding occurs during the rainy season; however, findings show that it is not merely the amount of water in an irrigated landscape, but the management of water resources that determines the aggregation of water bodies. In each scenario, timing and direction of irrigation along with inefficient drainage render the westernmost portion of BVIS the area of highest breeding opportunity, which expands and contracts seasonally in response to water resource availability and management decisions. CONCLUSIONS: Changes in the geography of breeding potential across irrigated spaces can have profound effects on the distribution of malaria risk for those living in close proximity to irrigated agricultural schemes. The methods presented are generalizable across geographies for estimating spatio-temporal distributions of breeding risk for mosquitoes in irrigated schemes, presenting an opportunity for greater geographically targeted strategies for management.

Efficacy of extended release formulations of Natular™ (spinosad) against larvae and adults of Anopheles mosquitoes in western Kenya.

BACKGROUND: Larval source management is recommended as a supplementary vector control measure for the prevention of malaria. Among the concerns related to larviciding is the feasibility of implementation in tropical areas with large numbers of habitats and the need for frequent application. Formulated products of spinosad that are designed to be effective for several weeks may mitigate some of these concerns. METHODS: In a semi-field study, three formulations of spinosad (emulsifiable concentrate, extended release granules and tablet formulations) were tested in naturalistic habitats in comparison to an untreated control. Cohorts of third instar Anopheles gambiae (Diptera: Culicidae) were introduced into the habitats in screened cages every week up to four weeks after application and monitored for survivorship over three days. A small-scale field trial was then conducted in two villages. Two of the spinosad formulations were applied in one village over the course of 18 months. Immature mosquito populations were monitored with standard dippers in sentinel sites and adult populations were monitored by pyrethrum spray catches. RESULTS: In the semi-field study, the efficacy of the emulsifiable concentrate of spinosad waned 1 week after treatment. Mortality in habitats treated with the extended release granular formulation of spinosad was initially high but declined gradually over 4 weeks while mortality in habitats treated with the dispersable tablet formulation was low immediately after treatment but rose to 100% through four weeks. In the field study, immature and adult Anopheles mosquito populations were significantly lower in the intervention village compared to the control village during the larviciding period. Numbers of collected mosquitoes were lower in the intervention village compared to the control village during the post-intervention period but the difference was not statistically significant. CONCLUSIONS: The extended release granular formulation and the dispersible tablet formulations of spinosad are effective against larval Anopheles mosquitoes for up to four weeks and may be an effective tool as part of larval source management programmes for reducing adult mosquito density and malaria transmission.

Species abundance, composition, and nocturnal activity of female Anopheles (Diptera: Culicidae) in malaria-endemic villages of Papua New Guinea: assessment with barrier screen sampling.

BACKGROUND: Community composition of Anopheles mosquitoes, and their host-seeking and peridomestic behaviour, are important factors affecting malaria transmission. In this study, barrier screen sampling was used to investigate species composition, abundance, and nocturnal activity of Anopheles populations in villages of Papua New Guinea. METHODS: Mosquitoes were sampled from 6 pm to 6 am in five villages from 2012 to 2016. The barrier screens were positioned between the village houses and the perimeter of villages where cultivated and wild vegetation ("the bush") grew thickly. Female Anopheles that rested on either village or bush side of the barrier screens, as they commuted into and out of the villages, were captured. Similarity in species composition among villages was assessed. Mosquitoes captured on village and bush sides of the barrier screens were sorted by feeding status and by hour of collection, and their numbers were compared using negative binomial generalized linear models. RESULTS: Females of seven Anopheles species were present in the sample. Species richness ranged from four to six species per village, but relative abundance was highly uneven within and between villages, and community composition was similar for two pairs of villages and highly dissimilar in a fifth. For most Anopheles populations, more unfed than blood-fed mosquitoes were collected from the barrier screens. More blood-fed mosquitoes were found on the side of the barrier screens facing the village and relatively more unfed ones on the bush side, suggesting commuting behaviour of unfed host-seeking females into the villages from nearby bush and commuting of blood-fed females away from villages towards the bush. For most populations, the majority of host-seeking mosquitoes arrived in the village before midnight when people were active and unprotected from the mosquitoes by bed nets. CONCLUSION: The uneven distribution of Anopheles species among villages, with each site dominated by different species, even among nearby villages, emphasizes the importance of vector heterogeneity in local malaria transmission and control. Yet, for most species, nocturnal activity patterns of village entry and host seeking predominantly occurred before midnight indicating common behaviours across species and populations relative to human risk of exposure to Anopheles bites.

Household-level and surrounding peri-domestic environmental characteristics associated with malaria vectors Anopheles arabiensis and Anopheles funestus along an urban-rural continuum in Blantyre, Malawi.

BACKGROUND: Malaria is increasing in some recently urbanized areas that historically were considered lower risk. Understanding what drives urban transmission is hampered by inconsistencies in how "urban" contexts are defined. A dichotomized "urban-rural" approach, based on political boundaries may misclassify environments or fail to capture local drivers of risk. Small-scale agriculture in urban or peri-urban settings has been shown to be a major risk determinant. METHODS: Household-level Anopheles abundance patterns in and around Malawi's commercial capital of Blantyre (~ 1.9 M pop.) were analysed. Clusters (N = 64) of five houses each located at 2.5 km intervals along eight transects radiating out from Blantyre city centre were sampled during rainy and dry seasons of 2015 and 2016. Mosquito densities were measured inside houses using aspirators to sample resting mosquitoes, and un-baited CDC light traps to sample host seeking mosquitoes. RESULTS: Of 38,895 mosquitoes captured, 91% were female and 87% were Culex spp. Anopheles females (N = 5058) were primarily captured in light traps (97%). Anopheles abundance was greater during rainy seasons. Anopheles funestus was more abundant than Anopheles arabiensis, but both were found on all transects, and had similar associations with environmental risk factors. Anopheles funestus and An. arabiensis females significantly increased with distance from the urban centre, but this trend was not consistent across all transects. Presence of small-scale agriculture was predictive of greater Anopheles spp. abundance, even after controlling for urbanicity, number of nets per person, number of under-5-year olds, years of education, and season. CONCLUSIONS: This study revealed how small-scale agriculture along a rural-to-urban transition was associated with An. arabiensis and An. funestus indoor abundances, and that indoor Anopheles density can be high within Blantyre city limits, particularly where agriculture is present. Typical rural areas with lower house density and greater distance from urban centres reflected landscapes more suitable for Anopheles reproduction and house invasion. However, similar characteristics and elevated Anopheles abundances were also found around some houses within the city limits. Thus, dichotomous designations of "urban" or "rural" can obscure important heterogeneity in the landscape of Plasmodium transmission, suggesting the need for more nuanced assessment of urban malaria risk and prevention efforts.

Explaining variation in adult Anopheles indoor resting abundance: the relative effects of larval habitat proximity and insecticide-treated bed net use.

BACKGROUND: Spatial determinants of malaria risk within communities are associated with heterogeneity of exposure to vector mosquitoes. The abundance of adult malaria vectors inside people's houses, where most transmission takes place, should be associated with several factors: proximity of houses to larval habitats, structural characteristics of houses, indoor use of vector control tools containing insecticides, and human behavioural and environmental factors in and near houses. While most previous studies have assessed the association of larval habitat proximity in landscapes with relatively low densities of larval habitats, in this study these relationships were analysed in a region of rural, lowland western Kenya with high larval habitat density. METHODS: 525 houses were sampled for indoor-resting mosquitoes across an 8 by 8 km study area using the pyrethrum spray catch method. A predictive model of larval habitat location in this landscape, previously verified, provided derivations of indices of larval habitat proximity to houses. Using geostatistical regression models, the association of larval habitat proximity, long-lasting insecticidal nets (LLIN) use, house structural characteristics (wall type, roof type), and peridomestic variables (cooking in the house, cattle near the house, number of people sleeping in the house) with mosquito abundance in houses was quantified. RESULTS: Vector abundance was low (mean, 1.1 adult Anopheles per house). Proximity of larval habitats was a strong predictor of Anopheles abundance. Houses without an LLIN had more female Anopheles gambiae s.s., Anopheles arabiensis and Anopheles funestus than houses where some people used an LLIN (rate ratios, 95% CI 0.87, 0.85-0.89; 0.84, 0.82-0.86; 0.38, 0.37-0.40) and houses where everyone used an LLIN (RR, 95% CI 0.49, 0.48-0.50; 0.39, 0.39-0.40; 0.60, 0.58-0.61). Cooking in the house also reduced Anopheles abundance across all species. The number of people sleeping in the house, presence of cattle near the house, and house structure modulated Anopheles abundance, but the effect varied with Anopheles species and sex. CONCLUSIONS: Variation in the abundance of indoor-resting Anopheles in rural houses of western Kenya varies with clearly identifiable factors. Results suggest that LLIN use continues to function in reducing vector abundance, and that larval source management in this region could lead to further reductions in malaria risk by reducing the amount of an obligatory resource for mosquitoes near people's homes.

Patterns and determinants of malaria risk in urban and peri-urban areas of Blantyre, Malawi.

BACKGROUND: Although malaria disease in urban and peri-urban areas of sub-Saharan Africa is a growing concern, the epidemiologic patterns and drivers of transmission in these settings remain poorly understood. Factors associated with variation in malaria risk in urban and peri-urban areas were evaluated in this study. METHODS: A health facility-based, age and location-matched, case-control study of children 6-59 months of age was conducted in four urban and two peri-urban health facilities (HF) of Blantyre city, Malawi. Children with fever who sought care from the same HF were tested for malaria parasites by microscopy and PCR. Those testing positive or negative on both were defined as malaria cases or controls, respectively. RESULTS: A total of 187 cases and 286 controls were studied. In univariate analyses, higher level of education, possession of TV, and electricity in the house were negatively associated with malaria illness; these associations were similar in urban and peri-urban zones. Having travelled in the month before testing was strongly associated with clinical malaria, but only for participants living in the urban zones (OR = 5.1; 95% CI = 1.62, 15.8). Use of long-lasting insecticide nets (LLINs) the previous night was not associated with protection from malaria disease in any setting. In multivariate analyses, electricity in the house, travel within the previous month, and a higher level of education were all associated with decreased odds of malaria disease. Only a limited number of Anopheles mosquitoes were found by aspiration inside the households in the peri-urban areas, and none was collected from the urban households. CONCLUSION: Travel was the main factor influencing the incidence of malaria illness among residents of urban Blantyre compared with peri-urban areas. Identification and understanding of key mobile demographic groups, their behaviours, and the pattern of parasite dispersal is critical to the design of more targeted interventions for the urban setting.

Assessment of submicroscopic infections and gametocyte carriage of Plasmodium falciparum during peak malaria transmission season in a community-based cross-sectional survey in western Kenya, 2012.

BACKGROUND: Although malaria control intervention has greatly decreased malaria morbidity and mortality in many African countries, further decline in parasite prevalence has stagnated in western Kenya. In order to assess if malaria transmission reservoir is associated with this stagnation, submicroscopic infection and gametocyte carriage was estimated. Risk factors and associations between malaria control interventions and gametocyte carriage were further investigated in this study. METHODS: A total of 996 dried blood spot samples were used from two strata, all smear-positives (516 samples) and randomly selected smear-negatives (480 samples), from a community cross-sectional survey conducted at peak transmission season in 2012 in Siaya County, western Kenya. Plasmodium falciparum parasite presence and density were determined by stained blood smear and by 18S mRNA transcripts using nucleic acid sequence-based amplification assay (NASBA), gametocyte presence and density were determined by blood smear and by Pfs25 mRNA-NASBA, and gametocyte diversity by Pfg377 mRNA RT-PCR and RT-qPCR. RESULTS: Of the randomly selected smear-negative samples, 69.6 % (334/480) were positive by 18S-NASBA while 18S-NASBA detected 99.6 % (514/516) smear positive samples. Overall, 80.2 % of the weighted population was parasite positive by 18S-NASBA vs 30.6 % by smear diagnosis and 44.0 % of the weighted population was gametocyte positive by Pfs25-NASBA vs 2.6 % by smear diagnosis. Children 5-15 years old were more likely to be parasitaemic and gametocytaemic by NASBA than individuals >15 years old or children <5 years old while gametocyte density decreased with age. Anaemia and self-reported fever within the past 24 h were associated with increased odds of gametocytaemia. Fever was also positively associated with parasite density, but not with gametocyte density. Anti-malarial use within the past 2 weeks decreased the odds of gametocytaemia, but not the odds of parasitaemia. In contrast, recent anti-malarial use was associated with lowered parasite density, but not the gametocyte density. Use of ITNs was associated with lower odds for parasitaemia in part of the study area with a longer history of ITN interventions. In the same part of study area, the odds of having multiple gametocyte alleles were also lower in individuals using ITNs than in those not using ITNs and parasite density was positively associated with gametocyte diversity. CONCLUSION: A large proportion of submicroscopic parasites and gametocytes in western Kenya might contribute to the stagnation in malaria prevalence, suggesting that additional interventions targeting the infectious reservoir are needed. As school aged children and persons with anaemia and fever were major sources for gametocyte reservoir, these groups should be targeted for intervention and prevention to reduce malaria transmission. Anti-malarial use was associated with lower parasite density and odds of gametocytaemia, but not the gametocyte density, indicating a limitation of anti-malarial impact on the transmission reservoir. ITN use had a protective role against parasitaemia and gametocyte diversity in western Kenya.

Lagenidium giganteum pathogenicity in mammals.

Infections of mammals by species in the phylum Oomycota taxonomically and molecularly similar to known Lagenidium giganteum strains have increased. During 2013-2014, we conducted a phylogenetic study of 21 mammalian Lagenidium isolates; we found that 11 cannot be differentiated from L. giganteum strains that the US Environmental Protection Agency approved for biological control of mosquitoes; these strains were later unregistered and are no longer available. L. giganteum strains pathogenic to mammals formed a strongly supported clade with the biological control isolates, and both types experimentally infected mosquito larvae. However, the strains from mammals grew well at 25°C and 37°C, whereas the biological control strains developed normally at 25°C but poorly at higher temperatures. The emergence of heat-tolerant strains of L. giganteum pathogenic to lower animals and humans is of environmental and public health concern.

Elizabethkingia anophelis: molecular manipulation and interactions with mosquito hosts.

Flavobacteria (members of the family Flavobacteriaceae) dominate the bacterial community in the Anopheles mosquito midgut. One such commensal, Elizabethkingia anophelis, is closely associated with Anopheles mosquitoes through transstadial persistence (i.e., from one life stage to the next); these and other properties favor its development for paratransgenic applications in control of malaria parasite transmission. However, the physiological requirements of E. anophelis have not been investigated, nor has its capacity to perpetuate despite digestion pressure in the gut been quantified. To this end, we first developed techniques for genetic manipulation of E. anophelis, including selectable markers, reporter systems (green fluorescent protein [GFP] and NanoLuc), and transposons that function in E. anophelis. A flavobacterial expression system based on the promoter PompA was integrated into the E. anophelis chromosome and showed strong promoter activity to drive GFP and NanoLuc reporter production. Introduced, GFP-tagged E. anophelis associated with mosquitoes at successive developmental stages and propagated in Anopheles gambiae and Anopheles stephensi but not in Aedes triseriatus mosquitoes. Feeding NanoLuc-tagged cells to A. gambiae and A. stephensi in the larval stage led to infection rates of 71% and 82%, respectively. In contrast, a very low infection rate (3%) was detected in Aedes triseriatus mosquitoes under the same conditions. Of the initial E. anophelis cells provided to larvae, 23%, 71%, and 85% were digested in A. stephensi, A. gambiae, and Aedes triseriatus, respectively, demonstrating that E. anophelis adapted to various mosquito midgut environments differently. Bacterial cell growth increased up to 3-fold when arginine was supplemented in the defined medium. Furthermore, the number of NanoLuc-tagged cells in A. stephensi significantly increased when arginine was added to a sugar diet, showing it to be an important amino acid for E. anophelis. Animal erythrocytes promoted E. anophelis growth in vivo and in vitro, indicating that this bacterium could obtain nutrients by participating in erythrocyte lysis in the mosquito midgut.

Identification of Avian and Hemoparasite DNA in Blood-Engorged Abdomens of Culex pipiens (Diptera; Culicidae) from a West Nile Virus Epidemic region in Suburban Chicago, Illinois.

Multiple mosquito-borne parasites cocirculate in nature and potentially interact. To understand the community of parasites cocirculating with West Nile virus (WNV), we screened the bloodmeal content of Culex pipiens L. mosquitoes for three common types of hemoparasites. Blood-fed Cx. pipiens were collected from a WNV-epidemic area in suburban Chicago, IL, from May to September 2005 through 2010. DNA was extracted from dissected abdomens and subject to PCR and direct sequencing to identify the vertebrate host. RNA was extracted from the head or thorax and screened for WNV using quantitative reverse transcriptase PCR. Seventy-nine engorged females with avian host origin were screened using PCR and amplicon sequencing for filarioid nematodes, Haemosporida, and trypanosomatids. Filarioid nematodes were identified in 3.8% of the blooded abdomens, Plasmodium sp. in 8.9%, Haemoproteus in 31.6%, and Trypanosoma sp. in 6.3%. The sequences from these hemoparasite lineages were highly similar to sequences from birds in prior studies in suburban Chicago. Overall, 50.6% of blood-fed Culex pipiens contained hemoparasite DNA in their abdomen, presumably from current or prior bloodmeals. Additionally, we detected hemoparasite DNA in the blooded abdomen of three of 10 Cx. pipiens infected with WNV.

Host group formation decreases exposure to vector-borne disease: a field experiment in a 'hotspot' of West Nile virus transmission.

Animals can decrease their individual risk of predation by forming groups. The encounter-dilution hypothesis extends the potential benefits of gregariousness to biting insects and vector-borne disease by predicting that the per capita number of insect bites should decrease within larger host groups. Although vector-borne diseases are common and can exert strong selective pressures on hosts, there have been few tests of the encounter-dilution effect in natural systems. We conducted an experimental test of the encounter-dilution hypothesis using the American robin (Turdus migratorius), a common host species for the West Nile virus (WNV), a mosquito-borne pathogen. By using sentinel hosts (house sparrows, Passer domesticus) caged in naturally occurring communal roosts in the suburbs of Chicago, we assessed sentinel host risk of WNV exposure inside and outside of roosts. We also estimated per capita host exposure to infected vectors inside roosts and outside of roosts. Sentinel birds caged inside roosts seroconverted to WNV more slowly than those outside of roosts, suggesting that social groups decrease per capita exposure to infected mosquitoes. These results therefore support the encounter-dilution hypothesis in a vector-borne disease system. Our results suggest that disease-related selective pressures on sociality may depend on the mode of disease transmission.

Ingestibility, digestibility, and engineered biological control potential of Flavobacterium hibernum, isolated from larval mosquito habitats.

Flavobacterium hibernum, isolated from larval habitats of the eastern tree hole mosquito, A. triseriatus, remained suspended in the larval feeding zone much longer (8 days) than other bacteria. Autofluorescent protein markers were developed for the labeling of F. hibernum with a strong flavobacterial expression system. Green fluorescent protein (GFP)-tagged F. hibernum cells were quickly consumed by larval mosquitoes at an ingestion rate of 9.5 × 10(4)/larva/h. The ingested F. hibernum cells were observed mostly in the foregut and midgut and rarely in the hindgut, suggesting that cells were digested and did not pass the gut viably. The NanoLuc luciferase reporter system was validated for quantitative larval ingestion rate and bacterial fate analyses. Larvae digested 1.87 × 10(5) cells/larva/h, and few F. hibernum cells were excreted intact. Expression of the GFP::Cry11A fusion protein with the P20 chaperone protein from Bacillus thuringiensis H-14 was successfully achieved in F. hibernum. Whole-cell bioassays of recombinant F. hibernum exhibited high larvicidal activity against A. triseriatus in microplates and in microcosms simulating tree holes. F. hibernum cells persisted in microcosms at 100, 59, 30, and 10% of the initial densities at days 1, 2, 3, and 6, respectively, when larvae were absent, while larvae consumed nearly all of the F. hibernum cells within 3 days of their addition to microcosms.

Modeling larval malaria vector habitat locations using landscape features and cumulative precipitation measures.

BACKGROUND: Predictive models of malaria vector larval habitat locations may provide a basis for understanding the spatial determinants of malaria transmission. METHODS: We used four landscape variables (topographic wetness index [TWI], soil type, land use-land cover, and distance to stream) and accumulated precipitation to model larval habitat locations in a region of western Kenya through two methods: logistic regression and random forest. Additionally, we used two separate data sets to account for variation in habitat locations across space and over time. RESULTS: Larval habitats were more likely to be present in locations with a lower slope to contributing area ratio (i.e. TWI), closer to streams, with agricultural land use relative to nonagricultural land use, and in friable clay/sandy clay loam soil and firm, silty clay/clay soil relative to friable clay soil. The probability of larval habitat presence increased with increasing accumulated precipitation. The random forest models were more accurate than the logistic regression models, especially when accumulated precipitation was included to account for seasonal differences in precipitation. The most accurate models for the two data sets had area under the curve (AUC) values of 0.864 and 0.871, respectively. TWI, distance to the nearest stream, and precipitation had the greatest mean decrease in Gini impurity criteria in these models. CONCLUSIONS: This study demonstrates the usefulness of random forest models for larval malaria vector habitat modeling. TWI and distance to the nearest stream were the two most important landscape variables in these models. Including accumulated precipitation in our models improved the accuracy of larval habitat location predictions by accounting for seasonal variation in the precipitation. Finally, the sampling strategy employed here for model parameterization could serve as a framework for creating predictive larval habitat models to assist in larval control efforts.

Daily blood feeding rhythms of laboratory-reared North American Culex pipiens.

BACKGROUND: Blood feeding by free-living insect vectors of disease is rhythmic and can be used to predict when infectious bites will occur. These daily rhythms can also be targeted by control measures, as in insecticide-treated nets. Culex pipiens form pipiens and C.p. f. molestus are two members of the Culex pipiens assemblage and vectors of West Nile Virus throughout North America. Although Culex species vector human pathogens and parasites, the daily blood feeding rhythms of C.p. f. molestus, to our knowledge, have not been studied. We described and compared the daily blood feeding rhythms of three laboratory-reared populations of Culex pipiens, one of which has confirmed molestus ancestry. We also examined the plasticity of blood feeding time for these three populations. RESULTS: For most (>70%) C.p. f. pipiens and C.p. f. molestus collected from metropolitan Chicago, IL, blood feeding took place during scotophase. Peak blood feeding occurred in mid-scotophase, 3-6 hours after lights off. For C.p. f. pipiens originating from Pennsylvania, most mosquitoes (> 90%) blood fed during late photophase and early scotophase. C.p. f. molestus denied a blood meal during scotophase were less likely to blood feed during early photophase (< 20%) than were C.p. f. pipiens from Chicago (> 50%). C.p. f. pipiens from Pennsylvania were capable of feeding readily at any hour of photo- or scotophase. CONCLUSIONS: Daily blood feeding rhythms of C.p. f. molestus are similar to those of C.p. f. pipiens, particularly when populations originate from the same geographic region. However, the timing of blood feeding is more flexible for C.p. f. pipiens populations relative to C.p. f. molestus.