Is that a real oocyst? Insectary establishment and identification of Plasmodium falciparum oocysts in midguts of Anopheles mosquitoes fed on infected human blood in Tororo, Uganda

Abstract

Background The human infectious reservoir for malaria consists of individuals capable of infecting mosquitoes. Oocyst prevalence and density are typical indicators of human infectivity to mosquitoes. However, identification of oocysts is challenging, particularly in areas of low malaria transmission intensity where few individuals may infect mosquitoes, and infected mosquitoes tend to have few oocysts. Here, features that differentiate oocysts from other oocyst-like in mosquito midguts are explained and illustrated. In addition, the establishment and maintenance of infrastructure to perform malaria transmission experiments is described. This work may support other initiatives to set up membrane feeding infrastructure and guide oocyst detection in low transmission settings. Methods In 2014, an insectary was developed and equipped in Tororo district, Uganda. A colony of Anopheles gambiae s.s. mosquitoes (Kisumu strain) was initiated to support infectivity experiments from participants enrolled in a large cohort study. Venous blood drawn from participants who were naturally infected with malaria parasites was used for membrane feeding assays, using 60–80 mosquitoes per experiment. Approximately 9–10 days after feeding, mosquitoes were dissected, and midguts were stained in mercurochrome and examined by light microscopy for Plasmodium falciparum oocysts and similar structures. In supportive experiments, different staining procedures were compared using in vitro cultured parasites. Results A stable colony of the Kisumu strain of An. gambiae s.s. was achieved, producing 5000–10,000 adult mosquitoes on a weekly basis. Challenges due to temperature fluctuations, mosquito pathogens and pests were successfully overcome. Oocysts were characterized by: presence of malaria pigment, clearly defined edge, round shape within the mosquito midgut or on the peripheral tissue and always attached to the epithelium. The main distinguishing feature between artifacts and mature oocysts was the presence of defined pigment within the oocysts. Conclusions Oocysts may be mistaken for other structures in mosquito midguts. Distinguishing real oocysts from oocyst-like structures may be challenging for inexperienced microscopists due to overlapping features. The characteristics and guidelines outlined here support identification of oocysts and reliable detection at low oocyst densities. Practical advice on sustaining a healthy mosquito colony for feeding experiments is provided. Following the reported optimization, the established infrastructure in Tororo allows assessments of infectivity of naturally infected parasite carriers.

A microsporidian impairs Plasmodium falciparum transmission in Anopheles arabiensis mosquitoes

“…We note that 2 midguts were found to have a low number of oocysts by microscopy (1,3) and were not found to have Plasmodium based on the qPCR assay. This finding suggests that either, (a) qPCR was not able to detect some lowdensity midgut oocyst infections or, (b) as noted by others 33 , there is potential for oocyst misidentification by microscopy in a field laboratory setting. Altogether, these results indicate that Microsporidia MB reduces the establishment of Plasmodium oocysts in the Anopheles midguts and impairs the colonization of Anopheles salivary glands by Plasmodium sporozoites.…”

Comparison of infectivity of Plasmodium vivax to wild-caught and laboratory-adapted (colonized) Anopheles arabiensis mosquitoes in Ethiopia

“…We have not observed any evidence for melanization in the present study. In addition, we also examined mosquito guts for pathogens that may influence parasite development such as microsporidia [43] and found no evidence for this. Future studies may nevertheless benefit from examining sporozoites, a limitation of the present study.…”

“…More and more laboratories are establishing mosquito colonies to examine infectivity among natural infections [42]. Whilst established colonies offer some advantage in terms of feeding efficiency [43], it is generally assumed that locally relevant mosquitoes are important to allow inference to the local transmission situation. We evaluated the permissiveness of An.…”

A Plasmodium vivax experimental human infection model for evaluating efficacy of interventions

“…This is potentially due to conditions during membrane feeding being suboptimal for efficient transmission, or because gametocytes may localize to subdermal capillaries for accurate evaluation of midgut infection (22). It was also followed by a study detailing the difficulty with oocyst identification by microscopy (23). Moreover, similar structures identified later in the same QIMR laboratory were confirmed PCR negative.…”