Considerations for first field trials of low-threshold gene drive for malaria vector control.

Sustainable reductions in African malaria transmission require innovative tools for mosquito control. One proposal involves the use of low-threshold gene drive in Anopheles vector species, where a 'causal pathway' would be initiated by (i) the release of a gene drive system in target mosquito vector species, leading to (ii) its transmission to subsequent generations, (iii) its increase in frequency and spread in target mosquito populations, (iv) its simultaneous propagation of a linked genetic trait aimed at reducing vectorial capacity for Plasmodium, and (v) reduced vectorial capacity for parasites in target mosquito populations as the gene drive system reaches fixation in target mosquito populations, causing (vi) decreased malaria incidence and prevalence. Here the scope, objectives, trial design elements, and approaches to monitoring for initial field releases of such gene dive systems are considered, informed by the successful implementation of field trials of biological control agents, as well as other vector control tools, including insecticides, Wolbachia, larvicides, and attractive-toxic sugar bait systems. Specific research questions to be addressed in initial gene drive field trials are identified, and adaptive trial design is explored as a potentially constructive and flexible approach to facilitate testing of the causal pathway. A fundamental question for decision-makers for the first field trials will be whether there should be a selective focus on earlier points of the pathway, such as genetic efficacy via measurement of the increase in frequency and spread of the gene drive system in target populations, or on wider interrogation of the entire pathway including entomological and epidemiological efficacy. How and when epidemiological efficacy will eventually be assessed will be an essential consideration before decisions on any field trial protocols are finalized and implemented, regardless of whether initial field trials focus exclusively on the measurement of genetic efficacy, or on broader aspects of the causal pathway. Statistical and modelling tools are currently under active development and will inform such decisions on initial trial design, locations, and endpoints. Collectively, the considerations here advance the realization of developer ambitions for the first field trials of low-threshold gene drive for malaria vector control within the next 5 years.

Effect of Ivermectin® on survivorship and fertility of Anopheles arabiensis in Ethiopia: an in vitro study.

BACKGROUND: Innovative vector control tools are needed to counteract insecticide resistance and residual malaria transmission. One of such innovative methods is an ivermectin (IVM) treatment to reduce vector survival. In this study, a laboratory experiment was conducted to investigate the effect of ivermectin on survivorship, fertility and egg hatchability rate of Anopheles arabiensis in Ethiopia. METHODS: An in vitro experiment was conducted using 3-5 days old An. arabiensis adults from a colony maintained at insectary of Tropical and Infectious Diseases Research Center, Jimma University (laboratory population) and Anopheles mosquitoes reared from larvae collected from natural mosquito breeding sites (wild population). The mosquitoes were allowed to feed on cattle blood treated with different doses of ivermectin (0 ng/ml, 5 ng/ml, 10 ng/ml, 20 ng/ml, 40 ng/ml and 80 ng/ml). During each feeding experiment, the mosquitoes were held in cages and blood-fed using a Hemotek feeder. Mortality and egg production were then recorded daily for up to 9 days. Time to death was analysed by a Cox frailty model with replicate as frailty term and source of mosquito (wild versus laboratory), treatment type (ivermectin vs control) and their interaction as categorical fixed effects. Kaplan Meier curves were plotted separately for wild and laboratory populations for a visual interpretation of mosquito survival as a function of treatment. RESULTS: Both mosquito source and treatment had a significant effect on survival (P < 0.001), but their interaction was not significant (P = 0.197). Compared to the controls, the death hazard of An. arabiensis that fed on ivermectin-treated blood was 2.3, 3.5, 6.5, 11.5 and 17.9 times that of the control for the 5 ng/ml, 10 ng/ml, 20 ng/ml, 40 ng/ml, and 80 ng/ml dose, respectively. With respect to the number of hatched larvae, hatched pupae and emerged adults per fed mosquitoes, a significant difference was found between the control and the 5 ng/ml dose group (P < 0.001). The number of hatched larvae and pupae, and emerged adults decreased further for the 10 ng/ml dose group and falls to zero for the higher doses. CONCLUSION: Treating cattle blood with ivermectin reduced mosquito survival, fertility, egg hatchability, larval development and adult emergence of An. arabiensis in all tested concentrations of ivermectin in both the wild and laboratory populations. Thus, ivermectin application in cattle could be used as a supplementary vector control method to tackle residual malaria transmission and ultimately achieve malaria elimination in Ethiopia.

Anopheles arabiensis hotspots along intermittent rivers drive malaria dynamics in semi-arid areas of Central Ethiopia.

BACKGROUND: Understanding malaria vector's population dynamics and their spatial distribution is important to define when and where the largest infection risks occur and implement appropriate control strategies. In this study, the seasonal spatio-temporal dynamics of the malaria vector population and transmission intensity along intermittent rivers in a semi-arid area of central Ethiopia were investigated. METHODS: Mosquitoes were collected monthly from five clusters, 2 close to a river and 3 away from a river, using pyrethrum spray catches from November 2014 to July 2016. Mosquito abundance was analysed by the mixed Poisson regression model. The human blood index and sporozoite rate was compared between seasons by a logistic regression model. RESULTS: A total of 2784 adult female Anopheles gambiae sensu lato (s.l.) were collected during the data collection period. All tested mosquitoes (n = 696) were identified as Anopheles arabiensis by polymerase chain reaction. The average daily household count was significantly higher (P = 0.037) in the clusters close to the river at 5.35 (95% CI 2.41-11.85) compared to the clusters away from the river at 0.033 (95% CI 0.02-0.05). Comparing the effect of vicinity of the river by season, a significant effect of closeness to the river was found during the dry season (P = 0.027) and transition from dry to wet season (P = 0.032). Overall, An. arabiensis had higher bovine blood index (62.8%) as compared to human blood index (23.8%), ovine blood index (9.2%) and canine blood index (0.1%). The overall sporozoite rate was 3.9% and 0% for clusters close to and away from the river, respectively. The overall Plasmodium falciparum and Plasmodium vivax entomologic inoculation rates for An. arabiensis in clusters close to the river were 0.8 and 2.2 infective bites per person/year, respectively. CONCLUSION: Mosquito abundance and malaria transmission intensity in clusters close to the river were higher which could be attributed to the riverine breeding sites. Thus, vector control interventions including targeted larval source management should be implemented to reduce the risk of malaria infection in the area.

Ugandan stakeholder hopes and concerns about gene drive mosquitoes for malaria control: new directions for gene drive risk governance.

BACKGROUND: The African Union's High-Level Panel on Emerging Technologies identified gene drive mosquitoes as a priority technology for malaria elimination. The first field trials are expected in 5-10 years in Uganda, Mali or Burkina Faso. In preparation, regional and international actors are developing risk governance guidelines which will delineate the framework for identifying and evaluating risks. Scientists and bioethicists have called for African stakeholder involvement in these developments, arguing the knowledge and perspectives of those people living in malaria-afflicted countries is currently missing. However, few African stakeholders have been involved to date, leaving a knowledge gap about the local social-cultural as well as ecological context in which gene drive mosquitoes will be tested and deployed. This study investigates and analyses Ugandan stakeholders' hopes and concerns about gene drive mosquitoes for malaria control and explores the new directions needed for risk governance. METHODS: This qualitative study draws on 19 in-depth semi-structured interviews with Ugandan stakeholders in 2019. It explores their hopes for the technology and the risks they believed pertinent. Coding began at a workshop and continued through thematic analysis. RESULTS: Participants' hopes and concerns for gene drive mosquitoes to address malaria fell into three themes: (1) ability of gene drive mosquitoes to prevent malaria infection; (2) impacts of gene drive testing and deployment; and, (3) governance. Stakeholder hopes fell almost exclusively into the first theme while concerns were spread across all three. The study demonstrates that local stakeholders are able and willing to contribute relevant and important knowledge to the development of risk frameworks. CONCLUSIONS: International processes can provide high-level guidelines, but risk decision-making must be grounded in the local context if it is to be robust, meaningful and legitimate. Decisions about whether or not to release gene drive mosquitoes as part of a malaria control programme will need to consider the assessment of both the risks and the benefits of gene drive mosquitoes within a particular social, political, ecological, and technological context. Just as with risks, benefits-and importantly, the conditions that are necessary to realize them-must be identified and debated in Uganda and its neighbouring countries.

Streamlined SMFA and mosquito dark-feeding regime significantly improve malaria transmission-blocking assay robustness and sensitivity.

BACKGROUND: The development of malaria transmission-blocking strategies including the generation of malaria refractory mosquitoes to replace the wild populations through means of gene drives hold great promise. The standard membrane feeding assay (SMFA) that involves mosquito feeding on parasitized blood through an artificial membrane system is a vital tool for evaluating the efficacy of transmission-blocking interventions. However, despite the availability of several published protocols, the SMFA remains highly variable and broadly insensitive. METHODS: The SMFA protocol was optimized through coordinated culturing of Anopheles coluzzii mosquitoes and Plasmodium falciparum parasite coupled with placing mosquitoes under a strict dark regime before, during, and after the gametocyte feed. RESULTS: A detailed description of essential steps is provided toward synchronized generation of highly fit An. coluzzii mosquitoes and P. falciparum gametocytes in preparation for an SMFA. A dark-infection regime that emulates the natural vector-parasite interaction system is described, which results in a significant increase in the infection intensity and prevalence. Using this optimal SMFA pipeline, a series of putative transmission-blocking antimicrobial peptides (AMPs) were screened, confirming that melittin and magainin can interfere with P. falciparum development in the vector. CONCLUSION: A robust SMFA protocol that enhances the evaluation of interventions targeting human malaria transmission in laboratory setting is reported. Melittin and magainin are identified as highly potent antiparasitic AMPs that can be used for the generation of refractory Anopheles gambiae mosquitoes.

A chromosomal reference genome sequence for the malaria mosquito, Anopheles gambiae, Giles, 1902, Ifakara strain.

We present a genome assembly from an individual female Anopheles gambiae (the malaria mosquito; Arthropoda; Insecta; Diptera; Culicidae), Ifakara strain. The genome sequence is 264 megabases in span. Most of the assembly is scaffolded into three chromosomal pseudomolecules with the X sex chromosome assembled. The complete mitochondrial genome was also assembled and is 15.4 kilobases in length.

The circadian clock modulates Anopheles gambiae infection with Plasmodium falciparum.

Key behaviours, physiologies and gene expressions in Anopheles mosquitoes impact the transmission of Plasmodium. Such mosquito factors are rhythmic to closely follow diel rhythms. Here, we set to explore the impact of the mosquito circadian rhythm on the tripartite interaction between the vector, the parasite and the midgut microbiota, and investigate how this may affect the parasite infection outcomes. We assess Plasmodium falciparum infection prevalence and intensity, as a proxy for gametocyte infectivity, in Anopheles gambiae mosquitoes that received a gametocyte-containing bloodfeed and measure the abundance of the midgut microbiota at different times of the mosquito rearing light-dark cycle. Gametocyte infectivity is also compared in mosquitoes reared and maintained under a reversed light-dark regime. The effect of the circadian clock on the infection outcome is also investigated through silencing of the CLOCK gene that is central in the regulation of animal circadian rhythms. The results reveal that the A. gambiae circadian cycle plays a key role in the intensity of infection of P. falciparum gametocytes. We show that parasite gametocytes are more infectious during the night-time, where standard membrane feeding assays (SMFAs) at different time points in the mosquito natural circadian rhythm demonstrate that gametocytes are more infectious when ingested at midnight than midday. When mosquitoes were cultured under a reversed light/dark regime, disrupting their natural physiological homeostasis, and infected with P. falciparum at evening hours, the infection intensity and prevalence were significantly decreased. Similar results were obtained in mosquitoes reared under the standard light/dark regime upon silencing of CLOCK, a key regulator of the circadian rhythm, highlighting the importance of the circadian rhythm for the mosquito vectorial capacity. At that time, the mosquito midgut microbiota load is significantly reduced, while the expression of lysozyme C-1 (LYSC-1) is elevated, which is involved in both the immune response and microbiota digestion. We conclude that the tripartite interactions between the mosquito vector, the malaria parasite and the mosquito gut microbiota are finely tuned to support and maintain malaria transmission. Our data add to the knowledge framework required for designing appropriate and biologically relevant SMFA protocols.

Gene drive mosquitoes can aid malaria elimination by retarding Plasmodium sporogonic development.

Gene drives hold promise for the genetic control of malaria vectors. The development of vector population modification strategies hinges on the availability of effector mechanisms impeding parasite development in transgenic mosquitoes. We augmented a midgut gene of the malaria mosquito Anopheles gambiae to secrete two exogenous antimicrobial peptides, magainin 2 and melittin. This small genetic modification, capable of efficient nonautonomous gene drive, hampers oocyst development in both Plasmodium falciparum and Plasmodium berghei. It delays the release of infectious sporozoites, while it simultaneously reduces the life span of homozygous female transgenic mosquitoes. Modeling the spread of this modification using a large-scale agent-based model of malaria epidemiology reveals that it can break the cycle of disease transmission across a range of transmission intensities.

Plasmodium oocysts respond with dormancy to crowding and nutritional stress.

Malaria parasites develop as oocysts in the mosquito for several days before they are able to infect a human host. During this time, mosquitoes take bloodmeals to replenish their nutrient and energy reserves needed for flight and reproduction. We hypothesized that these bloodmeals are critical for oocyst growth and that experimental infection protocols, typically involving a single bloodmeal at the time of infection, cause nutritional stress to the developing oocysts. Therefore, enumerating oocysts disregarding their growth and differentiation state may lead to erroneous conclusions about the efficacy of transmission blocking interventions. Here, we examine this hypothesis in Anopheles coluzzii mosquitoes infected with the human and rodent parasites Plasmodium falciparum and Plasmodium berghei, respectively. We show that oocyst growth and maturation rates decrease at late developmental stages as infection intensities increase; an effect exacerbated at very high infection intensities but fully restored with post infection bloodmeals. High infection intensities and starvation conditions reduce RNA Polymerase III activity in oocysts unless supplemental bloodmeals are provided. Our results suggest that oocysts respond to crowding and nutritional stress with a dormancy-like strategy, which urges the development of alternative methods to assess the efficacy of transmission blocking interventions.

Converting endogenous genes of the malaria mosquito into simple non-autonomous gene drives for population replacement.

Gene drives for mosquito population replacement are promising tools for malaria control. However, there is currently no clear pathway for safely testing such tools in endemic countries. The lack of well-characterized promoters for infection-relevant tissues and regulatory hurdles are further obstacles for their design and use. Here we explore how minimal genetic modifications of endogenous mosquito genes can convert them directly into non-autonomous gene drives without disrupting their expression. We co-opted the native regulatory sequences of three midgut-specific loci of the malaria vector Anopheles gambiae to host a prototypical antimalarial molecule and guide-RNAs encoded within artificial introns that support efficient gene drive. We assess the propensity of these modifications to interfere with the development of Plasmodium falciparum and their effect on fitness. Because of their inherent simplicity and passive mode of drive such traits could form part of an acceptable testing pathway of gene drives for malaria eradication.

Transmission blocking immunity in the malaria non-vector mosquito Anopheles quadriannulatus species A.

Despite being phylogenetically very close to Anopheles gambiae, the major mosquito vector of human malaria in Africa, Anopheles quadriannulatus is thought to be a non-vector. Understanding the difference between vector and non-vector mosquitoes can facilitate development of novel malaria control strategies. We demonstrate that An. quadriannulatus is largely resistant to infections by the human parasite Plasmodium falciparum, as well as by the rodent parasite Plasmodium berghei. By using genetics and reverse genetics, we show that resistance is controlled by quantitative heritable traits and manifested by lysis or melanization of ookinetes in the mosquito midgut, as well as by killing of parasites at subsequent stages of their development in the mosquito. Genes encoding two leucine-rich repeat proteins, LRIM1 and LRIM2, and the thioester-containing protein, TEP1, are identified as essential in these immune reactions. Their silencing completely abolishes P. berghei melanization and dramatically increases the number of oocysts, thus transforming An. quadriannulatus into a highly permissive parasite host. We hypothesize that the mosquito immune system is an important cause of natural refractoriness to malaria and that utilization of this innate capacity of mosquitoes could lead to new methods to control transmission of the disease.

Stability of the effect of silencing fibronectin type III domain-protein 1 (FN3D1) gene on Anopheles arabiensis reared under different breeding site conditions.

BACKGROUND: Malaria vector mosquitoes acquire midgut microbiota primarily from their habitat. The homeostasis of these microbial communities plays an essential role in the mosquito longevity, the most essential factor in the mosquito vectorial capacity. Our recent study revealed that silencing genes involved in regulation of the midgut homeostasis including FN3D1, FN3D3 and GPRGr9 reduced the survival of female adult Anopheles arabiensis mosquitoes. In the present study, we investigate the stability of the gene silencing efficiency of mosquitoes reared in three different breeding conditions representing distinct larval habitat types: town brick pits in Jimma, flood pools in the rural land of Asendabo and roadside pools in Wolkite. METHODS: First-instar larvae of An. arabiensis mosquitoes were reared separately using water collected from the three breeding sites. The resulting adult females were micro-injected with dsRNA targeting the FN3D1 gene (AARA003032) and their survival was monitored. Control mosquitoes were injected with dsRNA Lacz. In addition, the load of midgut microbiota of these mosquitoes was determined using flow cytometry. RESULTS: Survival of naïve adult female mosquitoes differed between the three sites. Mosquitoes reared using water collected from brick pits and flood pools survived longer than mosquitoes reared using water collected from roadside. However, the FN3D1 gene silencing effect on survival did not differ between the three sites. CONCLUSIONS: The present study revealed that the efficacy of FN3D1 gene silencing is not affected by variation in the larval habitat. Thus, silencing this gene has potential for application throughout sub-Saharan Africa.

The effect of silencing immunity related genes on longevity in a naturally occurring Anopheles arabiensis mosquito population from southwest Ethiopia.

BACKGROUND: Vector control remains the most important tool to prevent malaria transmission. However, it is now severely constrained by the appearance of physiological and behavioral insecticide resistance. Therefore, the development of new vector control tools is warranted. Such tools could include immunization of blood hosts of vector mosquitoes with mosquito proteins involved in midgut homeostasis (anti-mosquito vaccines) or genetic engineering of mosquitoes that can drive population-wide knockout of genes producing such proteins to reduce mosquito lifespan and malaria transmission probability. METHODS: To achieve this, candidate genes related to midgut homeostasis regulation need to be assessed for their effect on mosquito survival. Here, different such candidate genes were silenced through dsRNA injection in the naturally occurring Anopheles arabiensis mosquitoes and the effect on mosquito survival was evaluated. RESULTS: Significantly higher mortality rates were observed in the mosquitoes silenced for FN3D1 (AARA003032), FN3D3 (AARA007751) and GPRGr9 (AARA003963) genes as compared to the control group injected with dsRNA against a non-related bacterial gene (LacZ). This observed difference in mortality rate between the candidate genes and the control disappeared when gene-silenced mosquitoes were treated with antibiotic mixtures, suggesting that gut microbiota play a key role in the observed reduction of mosquito survival. CONCLUSIONS: We demonstrated that interference with the expression of the FN3D1, FN3D3 or GPRGr9 genes causes a significant reduction of the longevity of An. arabiensis mosquito in the wild.

Immune resistance and tolerance strategies in malaria vector and non-vector mosquitoes.

BACKGROUND: The Anopheles gambiae complex consists of species that vary greatly in their capacity to transmit malaria. The mosquito immune system has been identified as a key factor that can influence whether Plasmodium infection establishes within the mosquito vector. This study was designed to investigate the immune responses of An. coluzzii, An. arabiensis and An. quadriannulatus mosquitoes. The first two mosquito species are major vectors of malaria in sub-Saharan Africa, while the third is thought to be a non-vector. METHODS: All three mosquito species were reared in mixed cultures. Their capacity to eliminate P. berghei and regulate midgut bacteria was examined. RESULTS: Our results revealed large differences in mosquito resistance to P. berghei. In all three mosquito species, immune reactions involving the complement system were triggered when the number of parasites that mosquitoes were challenged with exceeded a certain level, i.e. immune tolerance threshold. This threshold was markedly lower in An. quadriannulatus compared to An. coluzzii and An. arabiensis. We also demonstrated that the level of immune tolerance to P. berghei infection in the haemolymph is inversely correlated with the level of immune tolerance to microbiota observed in the midgut lumen after a blood meal. The malaria non-vector mosquito species, An. quadriannulatus was shown to have a much higher level of tolerance to microbiota in the midgut than An. coluzzii. CONCLUSIONS: We propose a model whereby an increased tolerance to microbiota in the mosquito midgut results in lower tolerance to Plasmodium infection. In this model, malaria non-vector mosquito species are expected to have increased immune resistance in the haemocoel, possibly due to complement priming by microbiota elicitors. We propose that this strategy is employed by the malaria non-vector mosquito, An. quadriannulatus, while An. coluzzii has reduced tolerance to bacterial infection in the midgut and consequently reduced immune resistance to Plasmodium infection at the haemocoel level. An in-depth understanding of the molecular mechanisms regulating immune tolerance versus resistance in different mosquito vectors of malaria could guide the design of new vector and disease control strategies.

Flow cytometry analysis of the microbiota associated with the midguts of vector mosquitoes.

BACKGROUND: The scientific interest to understand the function and structure of the microbiota associated with the midgut of mosquito disease vectors is increasing. The advancement of such a knowledge has encountered challenges and limitations associated with conventional culture-based and PCR techniques. METHODS: Flow cytometry (FCM) combined with various cell marking dyes have been successfully applied in the field of ecological microbiology to circumvent the above shortcomings. Here, we describe FCM technique coupled with live/dead differential staining dyes SYBR Green I (SGI) and Propidium Iodide (PI) to quantify and study other essential characteristics of the mosquito gut microbiota. RESULTS: A clear discrimination between cells and debris, as well as between live and dead cells was achieved when the midgut homogenate was subjected to staining with 5 × 103 dilution of the SGI and 30 µM concentration of the PI. Reproducibly, FCM event collections produced discrete populations including non-fluorescent cells, SYBR positive cells, PI fluorescing cells and cells that fluoresce both in SYBR and PI, all these cell populations representing, respectively, background noise, live bacterial, dead cells and inactive cells with partial permeability to PI. The FCM produced a strong linear relationship between cell counts and their corresponding dilution factors (R (2) = 0.987), and the technique has a better precision compared to qRT-PCR. The FCM count of the microbiota reached a peak load at 18 h post-feeding and started declining at 24 h. The present FCM technique also successfully applied to quantify bacterial cells in fixed midgut samples that were homogenized in 4 % PFA. CONCLUSION: The FCM technique described here offers enormous potential and possibilities of integration with advanced molecular biochemical techniques for the study of the microbiota community in disease vector mosquitoes.

Detection of viable plasmodium ookinetes in the midguts of anopheles coluzzi using PMA-qrtPCR.

BACKGROUND: Mosquito infection with malaria parasites depends on complex interactions between the mosquito immune response, the parasite developmental program and the midgut microbiota. Simultaneous monitoring of the parasite and bacterial dynamics is important when studying these interactions. PCR based methods of genomic DNA (gDNA) have been widely used, but their inability to discriminate between live and dead cells compromises their application. The alternative method of quantification of mRNA mainly reports on cell activity rather than density. METHOD: Quantitative real-time (qrt) PCR in combination with Propidium Monoazide (PMA) treatment (PMA-qrtPCR) has been previously used for selectively enumerating viable microbial cells. PMA penetrates damaged cell membranes and intercalates in the DNA inhibiting its PCR amplification. Here, we tested the potential of PMA-qrtPCR to discriminate between and quantify live and dead Plasmodium berghei malarial parasites and commensal bacteria in the midgut of Anopheles coluzzii Coetzee & Wilkerson 2013 (formerly An. gambiae M-form). RESULTS: By combining microscopic observations with reverse transcriptase PCR (RT-PCR) we reveal that, in addition to gDNA, mRNA from dead parasites also persists inside the mosquito midgut, therefore its quantification cannot accurately reflect live-only parasites at the time of monitoring. In contrast, pre-treating the samples with PMA selectively inhibited qrtPCR amplification of parasite gDNA, with about 15 cycles (Ct-value) difference between PMA-treated and control samples. The limit of detection corresponds to 10 Plasmodium ookinetes. Finally, we show that the PMA-qrtPCR method can be used to quantify bacteria that are present in the mosquito midgut. CONCLUSION: The PMA-qrtPCR is a suitable method for quantification of viable parasites and bacteria in the midgut of Anopheles mosquitoes. The method will be valuable when studying the molecular interactions between the mosquito, the malaria parasite and midgut microbiota.

Spatial distribution of Glossina sp. and Trypanosoma sp. in south-western Ethiopia.

BACKGROUND: Accurate information on the distribution of the tsetse fly is of paramount importance to better control animal trypanosomosis. Entomological and parasitological surveys were conducted in the tsetse belt of south-western Ethiopia to describe the prevalence of trypanosomosis (PoT), the abundance of tsetse flies (AT) and to evaluate the association with potential risk factors. METHODS: The study was conducted between 2009 and 2012. The parasitological survey data were analysed by a random effects logistic regression model, whereas the entomological survey data were analysed by a Poisson regression model. The percentage of animals with trypanosomosis was regressed on the tsetse fly count using a random effects logistic regression model. RESULTS: The following six risk factors were evaluated for PoT (i) altitude: significant and inverse correlation with trypanosomosis, (ii) annual variation of PoT: no significant difference between years, (iii) regional state: compared to Benishangul-Gumuz (18.0%), the three remaining regional states showed significantly lower PoT, (iv) river system: the PoT differed significantly between the river systems, (iv) sex: male animals (11.0%) were more affected than females (9.0%), and finally (vi) age at sampling: no difference between the considered classes. Observed trypanosome species were T. congolense (76.0%), T. vivax (18.1%), T. b. brucei (3.6%), and mixed T. congolense/vivax (2.4%). The first four risk factors listed above were also evaluated for AT, and all have a significant effect on AT. In the multivariable model only altitude was retained with AT decreasing with increasing altitude. Four different Glossina species were identified i.e. G. tachinoides (52.0%), G. pallidipes (26.0%), G.morsitans submorsitans (15.0%) and G. fuscipes fuscipes (7.0 %). Significant differences in catches/trap/day between districts were observed for each species. No association could be found between the tsetse fly counts and trypanosomosis prevalence. CONCLUSIONS: Trypanosomosis remains a constraint to livestock production in south-western Ethiopia. Four Glossina and three Trypanosoma species were observed. Altitude had a significant impact on AT and PoT. PoT is not associated with AT, which could be explained by the importance of mechanical transmission. This needs to be investigated further as it might jeopardize control strategies that target the tsetse fly population.

Melanotic pathology and vertical transmission of the gut commensal Elizabethkingia meningoseptica in the major malaria vector Anopheles gambiae.

BACKGROUND: The resident gut flora is known to have significant impacts on the life history of the host organism. Endosymbiotic bacterial species in the Anopheles mosquito gut are potent modulators of sexual development of the malaria parasite, Plasmodium, and thus proposed as potential control agents of malaria transmission. RESULTS: Here we report a melanotic pathology in the major African malaria vector Anopheles gambiae, caused by the dominant mosquito endosymbiont Elizabethkingiameningoseptica. Transfer of melanised tissues into the haemolymph of healthy adult mosquitoes or direct haemolymph inoculation with isolated E. meningoseptica bacteria were the only means for transmission and de novo formation of melanotic lesions, specifically in the fat body tissues of recipient individuals. We show that E. meningoseptica can be vertically transmitted from eggs to larvae and that E. meningoseptica-mono-associated mosquitoes display significant mortality, which is further enhanced upon Plasmodium infection, suggesting a synergistic impact of E. meningoseptica and Plasmodium on mosquito survival. CONCLUSION: The high pathogenicity and permanent association of E. meningoseptica with An. Gambiae through vertical transmission constitute attractive characteristics towards the potential design of novel mosquito/malaria biocontrol strategies.