This could be a consequence of an increase in the parasite: vector RNA ratio as the parasite progresses developmentally through the mosquito. can be used to distinguish early versus late stage oocyst development in the mosquito. Currently, circumsporozoite protein (CSP), which is usually detectable only after day 7 post-infection, is the only marker used for detection of contamination in mosquitoes. Our results open the prospect to develop a non-CSP based detection assay for assessment of contamination in mosquitoes and evaluate the effect of intervention measures on malaria transmission in an endemic setting. Introduction Malaria remains one of the most serious global public health challenges. Nonetheless, between 2000 and 2014, malaria incidence rates have decreased Oxytetracycline (Terramycin) by 37% and mortality rates by 60%1. However, more recent years have seen a decline in the success of malaria control efforts. In the year 2015, 211 million malaria cases and in 2016, 216 million cases of malaria were reported (WHO Report 2017). Several adversarial factors such as population growth and climate change and a need for sustained intervention efforts by Oxytetracycline (Terramycin) international aid groups and government agencies in endemic countries may be accountable for the stalling of progress in the fight against malaria. These results also indicate that more novel approaches and tools to assess the effect of intervention measures may be needed to cause a further reduction in malaria contamination rates in areas of transmission. The life cycle relies on a mammalian host for asexual replication to generate intra-erythrocytic forms and a mosquito vector for further transmission which includes sexual reproduction and exchange of genetic materials. The sexual stage in the mosquito commences when an mosquito bites a gametocyte-carrying infected host and then the parasite undergoes the following developmental phases: gametogenesis (differentiation of male and female gametocytes into male and female gametes); fertilization leading to formation of a diploid zygote; motile ookinete; and the oocyst2. The duration of the mosquito cycle varies between species; for it lasts for approximately 21 days. The oocyst stage begins at day 2 post-fertilization, when the ookinete traverses the peritrophic membrane and develops into oocysts in the basal lamina of the midgut3. The oocyst phase lasts for approximately 10C12 days and is characterized by extensive sporogony resulting in the production of several hundreds to thousands of sporozoites which eventually leave the midgut and travel to the salivary gland of the mosquito and attain their infectious form4,5. During the past decades, there has been a significant improvement in our knowledge of the molecular processes and parasite molecules involved in gametogenesis, fertilization, and ookinete formation in mosquitoes6C8. However, the molecular events and parasite molecules that govern developmental progression of the midgut oocysts are poorly understood. Circumsporozoite protein (CSP), a sporozoite surface protein that is currently the only marker available for use in mosquito-stage immunological assays9, can first be detected in oocysts on day 7 Oxytetracycline (Terramycin) post-blood meal10. The earliest microscopic detection of developing oocysts is only feasible after day 6 post blood-meal3. Thus, there is an unmet need for the discovery of novel parasite markers that can be used for detection during the early stages of parasite development in infected mosquitoes. Earlier detection of a contamination during the developmental cycle in the mosquito midgut would provide a more accurate picture of the mosquito infectivity rate and help measure the effectiveness of intervention tools such as seasonal malaria chemoprevention programs, vaccines, bed-nets, and insecticides in endemic areas. In this study, we characterized the transcriptional profile of developmental progression through the midgut on days 2, 4, 6 and 8 Rabbit Polyclonal to MAPK1/3 post-blood meal, identified a genetic signature that detects presence of the parasite in the mosquito, and developed an immunological assay based on the detection of individual or a combination of four antigens. To our knowledge, this is the first study to demonstrate detection of in mosquitoes as early as day 2 through day 8 post-blood meal. These results support development of multiplex genetic and immunological assays that can detect at all developmental stages within infected mosquitoes for surveillance and drug and vaccine efficacy studies. Results Identification of parasite biomarkers of malaria transmission by microarray analysis of the transcriptome as the parasite developmentally progresses through the mosquito Microarray analysis was performed in four mechanical replicates using RNA purified from uninfected mosquito midguts versus infected mosquito midguts (2, 4, 6, and 8 days post-infection). Twenty midguts were pooled per time point and the average oocyst burden of mosquito midguts at day 8 was 64 oocysts/midgut. Importantly, equivalent molar amounts of cy3 (uninfected mosquito midguts) and cy5 (infected mosquito midguts) Oxytetracycline (Terramycin) labeled cRNA was hybridized to a microarray chip (Agilent Technologies,.
