K. Owusu-Edusei et al.

Background: Environmental factors such as temperature, nutrient availability, and larval density determine the outcome of postembryonic development in mosquitoes. Suboptimal temperatures, crowding, and starvation during the larval phase reduce adult mosquito size, nutrient stores and affect vectorial capacity. Methods: In this study we compared adult female Aedes aegypti, Rockefeller strain, raised under standard laboratory conditions (Large) with those raised under crowded and nutritionally deprived conditions (Small). To compare the gene expression and nutritional state of the major energy storage and metabolic organ, the fat body, we performed transcriptomics using Illumina based RNA-seq and metabolomics using GC/MS on females before and 24 hours following blood feeding. Results: Analysis of fat body gene expression between the experimental groups revealed a large number of significantly differentially expressed genes. Transcripts related to immunity, reproduction, autophagy, several metabolic pathways; including amino acid degradation and metabolism; and membrane transport were differentially expressed. Metabolite profiling identified 60 metabolites within the fat body to be significantly affected between small and large mosquitoes, with the majority of detected free amino acids at a higher level in small mosquitoes compared to large. Conclusions: Gene expression and metabolites in the adult fat body reflect the individual post-embryonic developmental history of a mosquito larva. These changes affect nutritional storage and utilization, immunity, and reproduction. Therefore, it is apparent that changes in larval environment due to weather conditions, nutrition availability, vector control efforts, and other factors can affect adult vectorial capacity in the field.

Background: Seroepidemiology provides robust estimates for tracking malaria transmission when intensity is low and useful when there is no baseline entomological data. Serological evidence of exposure to malaria vectors and parasite contribute to our understanding of the risk pathogen transmission, and facilitates implementation of targeted interventions. Ab to Anopheles gambiae salivary peptide (gSG6-P1) and merozoite surface protein one (MSP-119) reflect human exposure to malaria vectors and parasites. This study estimated malaria transmission dynamics using serological evidence of vector and parasite exposure in southern Ghana. Methods: Total IgG responses to both antigens in an age stratified cohort (<5, 5–14, >14) were measured from South-eastern Ghana. 295 randomly selected sera were analyzed from archived samples belonging to a cohort study that were followed at 3 consecutive survey months (n = 885); February, May and August 2009. Temporal variations in seroprevalence of both antigens as well as differences between the age-stratified cohorts were determined by χ 2 test with p < 0.05 statistically significant. Non-parametric repeated ANOVA – Friedman’s test was used to test differences in antibody levels. Seroprevalence data were fitted to reversible catalytic model to estimate sero-conversion rates. Results: Whereas parasite prevalence was generally low 2.4%, 2.7% and 2.4% with no apparent trends with season, seroprevalence to both gSG6-P1 and MSP119 were high (59%, 50.9%, 52.2%) and 57.6%, 52.3% and 43.6% in respective order from Feb. to August. Repeated measures ANOVA showed differences in median antibody levels across surveys with specific significant differences between February and May but not August by post hoc Dunn’s multiple comparison tests for gSG6-P1. For MSP119, no differences were observed in antibody levels between February and May but a significant decline was observed from May to August. Seroconversion rates for gSG6-P1 increased by 1.5 folds from February to August and 3 folds for MSP119. Conclusion: Data suggests exposure to infectious bites may be declining whereas mosquito bites remains high. Sustained malaria control efforts and surveillance are needed to drive malaria further down and to prevent catastrophic rebound. Operational factors foe scaling up have been discussed.

Background: There is a dearth of information on malaria endemicity in the islands of Lake Victoria in western Kenya. In this study malaria prevalence and Plasmodium sporozoite rates on Rusinga Island were investigated. The contribution of different Anopheles species to indoor and outdoor transmission of malaria was also determined. Methods: Active case detection through microscopy was used to diagnose malaria in a 10% random sample of the human population on Rusinga Island and a longitudinal entomological survey conducted in Gunda village in 2012. Nocturnally active host-seeking mosquitoes were captured indoors and outdoors using odour-baited traps. Anopheles species were tested for the presence of Plasmodium parasites using an enzyme linked immunosorbent assay. All data were analyzed using generalized linear models. Results: Single infections of Plasmodium falciparum (88.1%), P. malariae (3.96%) and P. ovale (0.79%) as well as multiple infections (7.14%) of these parasites were found on Rusinga Island. The overall malaria prevalence was 10.9%. The risk of contracting malaria was higher among dwellers of Rusinga West than Rusinga East locations (Odds Ratio [OR] = 1.5, 95% Confidence Interval [CI] 1.14 – 1.97, P = 0.003). Parasite positivity was significantly associated with individuals who did not use malaria protective measures (OR = 2.65, 95% CI 1.76 – 3.91, p < 0.001). A total of 1,684 mosquitoes, including 74 anophelines, were captured. Unlike Culex species, more of which were collected indoors than outdoors (P < 0.001), the females of An. gambiae s.l. (P = 0.477), An. funestus s.l. (P = 0.153) and Mansonia species captured indoors versus outdoors were not different. The 46 An. gambiae s.l. collected were mainly An. arabiensis (92.3%). Of the 62 malaria mosquitoes tested, 4, including 2 indoor and 2 outdoor-collected individuals had Plasmodium. Conclusion: The rather significant and unexpected contribution of P. malariae and P. ovale to the overall malaria prevalence on Rusinga Island underscores the epidemiological importance of these species in the big push towards eliminating malaria. Although current entomological interventions mainly target indoor environments, additional strategies should be considered to prevent outdoor transmission of malaria.

Background: Tick-borne pathogens experience a conflict of interest when the tick vector chooses a vertebrate host that is incompetent for pathogen transmission. The qualitative manipulation hypothesis suggests that tick-borne pathogens can resolve this conflict in their favour by manipulating the host choice behaviour of the tick vector. Methods: European Lyme disease is a model system for studying this conflict because Ixodes ricinus is a generalist tick species that vectors Borrelia pathogens that are specialized on different classes of vertebrate hosts. Avian specialists like B. garinii cannot survive in rodent reservoir hosts and vice versa for rodent specialists like B. afzelii. The present study tested whether Borrelia genospecies influenced the attraction of field-collected I. ricinus nymphs to rodent odours. Results: Nymphs were significantly attracted to questing perches that had been scented with mouse odours. However, there was no difference in questing behaviour between nymphs infected with rodent- versus bird-specialized Borrelia genospecies. Conclusion: Our study suggests that the tick, and not the pathogen, controls the early stages of host choice behaviour.

T. G. Nyenswah et al.

Background: Visceral leishmaniasis (VL) is a neglected tropical disease, which is strongly associated with poverty. VL caused by Leishmania donovani and transmitted by Phlebotomus orientalis is endemic in various remote areas of north and north-west Ethiopia. The present study was designed to determine the sand fly fauna and bionomics of P. orientalis in the VL endemic focus of Tahtay Adiyabo district. Methods: Sand flies were collected using CDC light traps (n = 602), sticky traps (n = 9,350) and indoor pyrethrum spray catches (n = 578 house visits) from indoor, peri-domestic and agricultural field habitats between May 2011 to April 2012. All sand fly specimens collected were identified to species level and counted. Results: In total, 100,772 sand fly specimens, belonging to 25 sand fly species (nine Phlebotomus and sixteen Sergentomyia) were collected and identified. S. africana and P. orientalis made up 59.1% and 23.5% of the collected sand flies, respectively. As it could be determined from the proportion of collections from outdoor (peri-domestic and agricultural fields) and indoor locations, P. orientalis appears to exhibit increased exophilic behavior. The outdoor to indoor index was 79:1 on m2 of sticky traps. Mean density of P. orientalis caught was significantly higher on horizontally placed sticky traps (mean = 60 ± 14.56/m2/night) than vertical deployed sticky traps (12 ± 3.57/m2/night). The highest abundance of P. orientalis occurred between March and April. Through July to September, there was a sharp decline in abundance of P. orientalis population. Regarding climatic variables, P. orientalis density in light traps and on sticky traps showed a significant positive and negative association with temperature and relative humidity, respectively. However, non-significant negative correlation was observed with rainfall pattern. Conclusions: Overall, P. orientalis was found to be the most abundant Phlebotomus species, showing marked seasonal abundance that mainly peaks during the dry season (March to April). Likewise, the people in the area usually sleep in compounds during these months that potentially expose them to a high risk of peri-domestic VL transmission.

E. Lukianova-Hleb et al.

Background: The endosymbiosis in trypanosomatids is characterized by co-evolution between one bacterium and its host protozoan in a mutualistic relationship, thus constituting an excellent model to study organelle origin in the eukaryotic cell. In this association, an intense metabolic exchange is observed between both partners: the host provides energetic molecules and a stable environment to a reduced wall symbiont, while the bacterium is able to interfere in host metabolism by enhancing phospholipid production and completing essential biosynthesis pathways, such as amino acids and hemin production. The bacterium envelope presents a reduced cell wall which is mainly composed of cardiolipin and phosphatidylcholine, being the latter only common in intracellular prokaryotes. Phosphatidylinositol (PI) is also present in the symbiont and host cell membranes. This phospholipid is usually related to cellular signaling and to anchor surface molecules, which represents important events for cellular interactions. Methods: In order to investigate the production of PI and its derivatives in symbiont bearing trypanosomatids, aposymbiotic and wild type strains of Angomonas deanei, as well as isolated symbionts, were incubated with [3H]myo-inositol and the incorporation of this tracer was analyzed into inositol-containing molecules, mainly phosphoinositides and lipoproteins. Gene searches and their phylogenies were also performed in order to investigate the PI synthesis in symbiontbearing trypanosomatids. Results: Our results showed that the bacterium did not incorporate the tracer and that both strains produced similar quantities of PI and its derivatives, indicating that the symbiont does not influence the production of these metabolites. Gene searches related to PI synthesis revealed that the trypanosomatid genome contains an inositol transporter, PI synthase and the myo-inositol synthase. Thus, the host is able to produce PI either from exogenous myo-inositol (inositol transporter) or from myo-inositol synthesized de novo. Phylogenetic analysis using other organisms as references indicated that, in trypanosomatids, the genes involved in PI synthesis have a monophyletic origin. In accordance with experimental data, sequences for myo-inositol transport or for myo-inositol and PI biosynthesis were not found in the symbiont. Conclusions: Altogether, our results indicate that the bacterium depends on the host to obtain PI.

Background: The introduction of the endosymbiotic bacterium, Wolbachia into Aedes aegypti populations is a novel approach to reduce disease transmission. The presence of Wolbachia limits the ability of the mosquito to transmit dengue virus (DENV) and the strength of this effect appears to correlate with Wolbachia densities in the mosquito. There is also some evidence that Wolbachia densities may increase following the consumption of a bloodmeal. Here we have examined whether multiple blood feeds lead to increases in density or associated changes in Wolbachia-mediated blocking of DENV. Methods: The Wolbachia infected Aedes aegypti mosquito line was used for the study. There were three treatment groups; a non-blood fed control, a second group fed once and a third group fed twice on human blood. All groups were orally infected with DENV-2 and then their midguts and salivary glands were dissected 10–11 days post infection. RNA/DNA was simultaneously extracted from each tissue and subsequently used for DENV RNA copies and Wolbachia density quantification respectively. Results: We found variation between replicate vector competence experiments and no clear evidence that Wolbachia numbers increased in either the salivary glands or remainder of the body with feeding and hence saw no corresponding improvements in DENV blocking. Conclusions: Aedes aegypti are “sip” feeders returning often to obtain bloodmeals and hence it is important to assess whether repeat blood feeding improved the efficacy of Wolbachia-based DENV blocking. Our work suggests in the laboratory context when Wolbachia densities are high that repeat feeding does not improve blocking and hence this ability should likely be stable with respect to feeding cycle in the field.

Background: Chagas disease is caused by Trypanosoma cruzi, which is transmitted by triatomine vectors. The northeastern region of Brazil is endemic for Chagas disease and has the largest diversity of triatomine species. T. cruzi development in its triatomine vector depends on diverse factors, including the composition of bacterial gut microbiota. Methods: We characterized the triatomines captured in the municipality of Russas (Ceará) by sequencing the cytochrome c oxidase subunit I (COI) gene. The composition of the bacterial community in the gut of peridomestic Triatoma brasiliensis and Triatoma pseudomaculata was investigated using culture independent methods based on the amplification of the 16S rRNA gene by polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), DNA fragment cloning, Sanger sequencing and 454 pyrosequencing. Additionally, we identified TcI and TcII types of T. cruzi by sequencing amplicons from the gut metagenomic DNA with primers for the mini-exon gene. Results: Triatomines collected in the peridomestic ecotopes were diagnosed as T. pseudomaculata and T. brasiliensis by comparing their COI sequence with GenBank. The rate of infection by T. cruzi in adult triatomines reached 80% for T. pseudomaculata and 90% for T. brasiliensis. According to the DNA sequences from the DGGE bands, the triatomine gut microbiota was primarily composed of Proteobacteria and Actinobacteria. However, Firmicutes and Bacteroidetes were also detected, although in much lower proportions. Serratia was the main genus, as it was encountered in all samples analyzed by DGGE and 454 pyrosequencing. Members of Corynebacterinae, a suborder of the Actinomycetales, formed the next most important group. The cloning and sequencing of full-length 16S rRNA genes confirmed the presence of Serratia marcescens, Dietzia sp., Gordonia terrae, Corynebacterium stationis and Corynebacterium glutamicum. Conclusions: The study of the bacterial microbiota in the triatomine gut has gained increased attention because of the possible role it may play in the epidemiology of Chagas disease by competing with T. cruzi. Culture independent methods have shown that the bacterial composition of the microbiota in the guts of peridomestic triatomines is made up by only few bacterial species.

Background: Bacillus sphaericus is a mosquito-larvae pathogen which proliferates in the host cadavers, spreading and preserving the infection within the larval habitats for prolonged periods. In this pilot field study, we presented B. sphaericus-containing attractive sugar baits (ASB) to wild Anopheles sergentii adults, with the assumption that bait-fed, B. sphaericus-carrying mosquitoes are able to efficiently transmit the pathogen to the larval habitats, causing larval mortality and leading to a decrease in the subsequent adult population. Methods: The experiment was conducted over 75 days in two desert-surrounded streamlets. Blooming Ochradenus baccatus bushes were sprayed with bait solutions consisting of sugar and food dye marker solutions, with or without B. sphaericus at the experimental and control streamlets, respectively. Adult mosquito and larvae numbers were monitored before and after the treatment application, and the proportion of bait-fed adults was determined by visual inspection for dye presence. Results: Presence of food dye confirmed a large fraction of the adult mosquito population (70%-75%) readily ingested Bacillus sphaericus- containing bait. By the end of the study period, the larval population at the experimental site was six-fold smaller than the concurrent larval population at the control site. The ensuing adult An. sergentii population was also reduced to about 60% at the experimental site, while the adult mosquito population at the control site had increased 2.4 fold over the same time-frame. The reduction in adult mosquito numbers became apparent after a lag time (10 days), suggesting the treatment had minimal effect on adult longevity, also indicated by the post-treatment increase in the proportion of old mosquitoes and concomitant decrease in the proportion of young mosquitoes. Conclusions: Presentation of B. sphaericus-containing ASB substantially impacts the larval population, which in turn leads to a significant reduction of the ensuing numbers of adult mosquitoes. Although such outcomes may be the result of other causes, these preliminary results raise the possibility that adult mosquitoes can efficiently transmit the pathogen into the larval habitats. The transfer of B. sphaericus via contaminated adult mosquito carriers may allow introduction of pathogens to breeding places which are dispersed, hard to find, or difficult to access.

J. R. Kugelman et al.

A. I. Khalafalla et al.

J. S. Hall et al.

S. Medcalf et al.

A. Filia et al.

Background: Quantitative reverse transcription PCR (qRT-PCR) is a robust and accessible method to assay gene expression and to infer gene regulation. Being a chain of procedures, this technique is subject to systematic error due to biological and technical limitations mainly set by the starting material and downstream procedures. Thus, rigorous data normalization is critical to grant reliability and repeatability of gene expression quantification by qRT-PCR. A number of ‘housekeeping genes’, involved in basic cellular functions, have been commonly used as internal controls for this normalization process. However, these genes could themselves be regulated and must therefore be tested a priori. Results: We evaluated eight reference genes for their stability as internal controls of olfactory gene expression in the antennae of Rhodnius prolixus, a Chagas disease vector. Five experimental conditions, including changes in age, developmental stage and feeding status were tested in both sexes. We show that the evaluation of candidate reference genes is necessary for each combination of sex, tissue and physiological condition analyzed in order to avoid inconsistent results and conclusions. Although, Normfinder and geNorm software yielded different results between males and females, five genes (SDH, Tub, GAPDH, Act and G6PDH) appeared in the first positions in all rankings obtained. By using gene expression data of a single olfactory co-receptor gene as an example, we demonstrated the extent of changes expected using different internal standards. Conclusions: This work underlines the need for a rigorous selection of internal standards to grant the reliability of normalization processes in qRT-PCR studies. Furthermore, it is shown that particular physiological or developmental conditions require independent evaluation of a diverse set of potential reference genes.

Background: Bendiocarb was introduced for the first time for Indoor Residual Spraying (IRS) in Tanzania in 2012 as part of the interim national insecticide resistance management plan. This move followed reports of increasingly alarming levels of pyrethroid resistance across the country. This study used the insecticide quantification kit (IQK) to investigate the intra-operational IRS coverage and quality of spraying, and decay rate of bendiocarb on different wall surfaces in Kagera region. Methods: To assess intra-operational IRS coverage and quality of spraying, 104 houses were randomly selected out of 161,414 sprayed houses. A total of 509 samples (218 in Muleba and 291 in Karagwe) were obtained by scraping the insecticide samples from wall surfaces. To investigate decay rate, 66 houses (36 in Muleba and 30 in Karagwe) were selected and samples were collected monthly for a period of five months. Laboratory testing of insecticide concentration was done using IQKTM [Innovative Vector Control Consortium]. Results: Of the 509 samples, 89.5% met the World Health Organization (WHO) recommended concentration (between 100–400 mg/m2) for IRS target dosage. The proportion of samples meeting WHO standards varied between Karagwe (84.3%) and Muleba (96.3%) (p < 0.001). Assessment of quality of spraying at house level revealed that Muleba (84.8%) had a significantly higher proportion of households that met the expected target dosage (100–400 mg/m2) compared to Karagwe (68.9%) (p < 0.001). The quality of spraying varied across different wall substrates in both districts. Evaluation of bendiocarb decay showed that the proportion of houses with recommended concentration declined from 96.9%, 93.5% and 76.2% at months one, two, and three post IRS, respectively (p-trend = 0.03). The rate of decay increased in the fourth and fifth month post spraying with only 55.9% and 26.3% houses meeting the WHO recommendations, respectively. Conclusion: IQK is an important tool for assessing IRS coverage and quality of spraying. The study found adequate coverage of IRS; however, residual life of bendiocarb was observed to be three months. Results suggest that in order to maintain the recommended concentrations with bendiocarb, a second spray cycle should be carried out after three months.

Background: Accurate determination of Schistosoma infection rates in low endemic regions to examine progress towards interruption of transmission and elimination requires highly sensitive diagnostic tools. An existing lateral flow (LF) based test demonstrating ongoing infections through detection of worm circulating anodic antigen (CAA), was improved for sensitivity through implementation of a protocol allowing increased sample input. Urine is the preferred sample as collection is non-invasive and sample volume is generally not a restriction. Methods: Centrifugal filtration devices provided a method to concentrate supernatant of urine samples extracted with trichloroacetic acid (TCA). For field trials a practical sample volume of 2 mL urine allowed detection of CAA down to 0.3 pg/mL. The method was evaluated on a set of urine samples (n = 113) from an S. mansoni endemic region (Kisumu, Kenya) and compared to stool microscopy (Kato Katz, KK). In this analysis true positivity was defined as a sample with either a positive KK or UCAA test. Results: Implementation of the concentration method increased clinical sensitivity (Sn) from 44 to 98% when moving from the standard 10 μL (UCAA10 assay) to 2000 μL (UCAA2000 assay) urine sample input. Sn for KK varied between 23 and 35% for a duplicate KK (single stool, two slides) to 52% for a six-fold KK (three consecutive day stools, two slides). The UCAA2000 assay indicated 47 positive samples with CAA concentration above 0.3 pg/mL. The six-fold KK detected 25 egg positives; 1 sample with 2 eggs detected in the 6-fold KK was not identified with the UCAA2000 assay. Conclusions: Larger sample input increased Sn of the UCAA assay to a level indicating ‘true’ infection. Only a single 2 mL urine sample is needed, but analysing larger sample volumes could still increase test accuracy. The UCAA2000 test is an appropriate candidate for accurate identification of all infected individuals in low-endemic regions. Assay materials do not require refrigeration and collected urine samples may be stored and transported to central test laboratories without the need to be frozen.