Experimental Plasmodium vivax infection of key Anopheles species from the Brazilian Amazon

Abstract

BackgroundAnopheles darlingi is the major malaria vector in countries located in the Amazon region. Anopheles aquasalis and Anopheles albitarsis s.l. are also proven vectors in this region. Anopheles nuneztovari s.l. and Anopheles triannulatus s.l. were found infected with Plasmodium vivax; however, their status as vectors is not yet well defined. Knowledge of susceptibility of Amazon anopheline populations to Plasmodium infection is necessary to better understand their vector capacity. Laboratory colonization of An. darlingi, the main Amazon vector, has proven to be difficult and presently An. aquasalis is the only available autonomous colony.MethodsLarvae of An. darlingi, An. albitarsis s.l., An. nuneztovari s.l. and An. triannulatus s.l. were collected in the field and reared until adult stage. Adults of An. aquasalis were obtained from a well-established colony. Mosquitoes were blood-fed using a membrane-feeding device containing infected blood from malarial patients.The infection of the distinct Anopheles species was evaluated by the impact variance of the following parameters: (a) parasitaemia density; (b) blood serum inactivation of the infective bloodmeal; (c) influence of gametocyte number on infection rates and number of oocysts. The goal of this work was to compare the susceptibility to P. vivax of four field-collected Anopheles species with colonized An. aquasalis.ResultsAll Anopheles species tested were susceptible to P. vivax infection, nevertheless the proportion of infected mosquitoes and the infection intensity measured by oocyst number varied significantly among species. Inactivation of the blood serum prior to mosquito feeding increased infection rates in An. darlingi and An. triannulatus s.l., but was diminished in An. albitarsis s.l. and An. aquasalis. There was a positive correlation between gametocyte density and the infection rate in all tests (Z = −8.37; p < 0.001) but varied among the mosquito species. Anopheles albitarsis s.l., An. aquasalis and An. nuneztovari s.l. had higher infection rates than An. darlingi.ConclusionAll field-collected Anopheles species, as well as colonized An. aquasalis are susceptible to experimental P. vivax infections by membrane feeding assays. Anopheles darlingi, An. albitarsis s.l. and An. aquasalis are very susceptible to P. vivax infection. However, colonized An. aquasalis mosquitoes showed the higher infection intensity represented by infection rate and oocyst numbers. This study is the first to characterize experimental development of Plasmodium infections in Amazon Anopheles vectors and also to endorse that P. vivax infection of colonized An. aquasalis is a feasible laboratory model.

Infectivity of symptomatic and asymptomatic Plasmodium vivax infections to a Southeast Asian vector, Anopheles dirus

“…dirus would become infected with P. vivax from a symptomatic blood meal was approximately 50%, similar to the previous report of 43% when the mosquito was allowed to feed directly from the skin of symptomatic adults in Thailand (Sattabongkot et al, 1991), alleviating the concern that the membrane feeding assay may not represent natural infection. Similar infection rates (45-60%) were also measured in Anopheles aquasalis and Anopheles albitarsis in Brazillian Amazon and Anopheles albimanus in Colombia by membrane feeding using patients’ blood (Solarte et al, 2011; Rios-Velasquez et al, 2013; Vallejo et al, 2016). However, direct comparison between membrane feeding and skin feeding remains an important step in the future to fully assess how well the membrane feeding assay reflects natural infection, especially for asymptomatic carriers whose parasitemias are generally very low and whose parasite distributions in the skin may have great impacts on mosquito infections.…”

“…Plasmodium vivax thus poses a great challenge for malaria eradication. Due to the lack of an in vitro culture system that produces infectious gametocytes, information about P. vivax transmission efficiency is limited, and has mostly relied on direct or membrane feeding experiments using blood from human malaria infections (Sattabongkot et al, 1991, 2003; Gamage-Mendis et al, 1993; Zollner et al, 2006; Rios-Velasquez et al, 2013; Vallejo et al, 2016). …”

Purification Methodology for Viable and Infective Plasmodium vivax Gametocytes That Is Compatible with Transmission-Blocking Assays

“…aquasalis, Anopheles albitarsis, and Anopheles darlingi are the main vectors in South America and are associated with malaria transmission (11). A. aquasalis has successfully been used under laboratory conditions for P. vivax infections (10), and an infection protocol with P. vivax on A. darlingi was recently established (12). For these assays, purified gametocytes were used to prepare blood dilutions at a hematocrit level of 36% using washed uninfected fresh Oϩ blood and AB serum at a final volume of 1 ml.…”

“…For these assays, purified gametocytes were used to prepare blood dilutions at a hematocrit level of 36% using washed uninfected fresh Oϩ blood and AB serum at a final volume of 1 ml. Next, 100 mosquitoes were fed on these samples and reared accordingly (10). Prior to infection, the viability of the gametocytes was tested by exflagellation assays.…”

“…Those assays were performed using an established colony of the Anopheles aquasalis mosquito vector, as described elsewhere (10). Successful infections were determined by the presence of oocysts in the midgut of the mosquitoes.…”