Population Dynamics of <i>Anopheles albimanus</i> (Diptera: Culicidae) at Ipetí-Guna, a Village in a Region Targeted for Malaria Elimination in Panamá

Anopheles albimanus Wiedemann is a major malaria vector in Mesoamerica and the Caribbean whose population dynamics, in response to changing environments, has been relatively poorly studied. Here, we present monthly adult and larvae data collected from May 2016 to December 2017 in Ipet&#237;-Guna, a village within an area targeted for malaria elimination in the Rep&#250;blica de Panam&#225;. During the study period we collected a total of 1678 Anopheles spp. mosquitoes (1602 adults and 76 larvae). Over 95% of the collected Anopheles spp. mosquitoes were An. albimanus. Using time series analysis techniques, we found that population dynamics of larvae and adults were not significantly correlated with each other at any time lag, though correlations were highest at one month lag between larvae and adults and four months lag between adults and larvae. Larvae population dynamics had cycles of three months and were sensitive to changes in temperature with 5 months lag, while adult abundance was correlated with itself (1 month lag) and with the normalized difference vegetation index (NDVI) with three months lag. A key observation from our study is the absence of both larvae and adults of An. albimanus between January and April from environments associated with Guna population&#8217;s daily activities, which suggests this time window could be the best time to implement elimination campaigns aimed at clearing Plasmodium spp. parasites from Guna populations using, for example, mass drug administration

Insights into an Optimization of Plasmodium vivax Sal-1 In Vitro Culture: The Aotus Primate Model

Malaria is one of the most significant tropical diseases, and of the Plasmodium species that cause human malaria, P. vivax is the most geographically widespread. However, P. vivax remains a relatively neglected human parasite since research is typically limited to laboratories with direct access to parasite isolates from endemic field settings or from non-human primate models. This restricted research capacity is in large part due to the lack of a continuous P. vivax in vitro culture system, which has hampered the ability for experimental research needed to gain biological knowledge and develop new therapies. Consequently, efforts to establish a long-term P. vivax culture system are confounded by our poor knowledge of the preferred host cell and essential nutrients needed for in vitro propagation. Reliance on very heterogeneous P. vivax field isolates makes it difficult to benchmark parasite characteristics and further complicates development of a robust and reliable culture method. In an effort to eliminate parasite variability as a complication, we used a well-defined Aotus-adapted P. vivax Sal-1 strain to empirically evaluate different short-term in vitro culture conditions and compare them with previous reported attempts at P. vivax in vitro culture Most importantly, we suggest that reticulocyte enrichment methods affect invasion efficiency and we identify stabilized forms of nutrients that appear beneficial for parasite growth, indicating that P. vivax may be extremely sensitive to waste products. Leuko-depletion methods did not significantly affect parasite development. Formatting changes such as shaking and static cultures did not seem to have a major impact while; in contrast, the starting haematocrit affected both parasite invasion and growth. These results support the continued use of Aotus-adapted Sal-1 for development of P. vivax laboratory methods; however, further experiments are needed to optimize culture conditions to support long-term parasite development

Insights into an Optimization of <i>Plasmodium vivax</i> Sal-1 <i>In Vitro</i> Culture: The <i>Aotus</i> Primate Model

<div><p>Malaria is one of the most significant tropical diseases, and of the <i>Plasmodium</i> species that cause human malaria, <i>P</i>. <i>vivax</i> is the most geographically widespread. However, <i>P</i>. <i>vivax</i> remains a relatively neglected human parasite since research is typically limited to laboratories with direct access to parasite isolates from endemic field settings or from non-human primate models. This restricted research capacity is in large part due to the lack of a continuous <i>P</i>. <i>vivax in vitro</i> culture system, which has hampered the ability for experimental research needed to gain biological knowledge and develop new therapies. Consequently, efforts to establish a long-term <i>P</i>. <i>vivax</i> culture system are confounded by our poor knowledge of the preferred host cell and essential nutrients needed for <i>in vitro</i> propagation. Reliance on very heterogeneous <i>P</i>. <i>vivax</i> field isolates makes it difficult to benchmark parasite characteristics and further complicates development of a robust and reliable culture method. In an effort to eliminate parasite variability as a complication, we used a well-defined <i>Aotus</i>-adapted <i>P</i>. <i>vivax</i> Sal-1 strain to empirically evaluate different short-term <i>in vitro</i> culture conditions and compare them with previous reported attempts at <i>P</i>. <i>vivax in vitro</i> culture Most importantly, we suggest that reticulocyte enrichment methods affect invasion efficiency and we identify stabilized forms of nutrients that appear beneficial for parasite growth, indicating that <i>P</i>. <i>vivax</i> may be extremely sensitive to waste products. Leuko-depletion methods did not significantly affect parasite development. Formatting changes such as shaking and static cultures did not seem to have a major impact while; in contrast, the starting haematocrit affected both parasite invasion and growth. These results support the continued use of <i>Aotus</i>-adapted Sal-1 for development of <i>P</i>. <i>vivax</i> laboratory methods; however, further experiments are needed to optimize culture conditions to support long-term parasite development.</p></div

Shaking and static format support cultures similarly.

<p>(<b>A</b>) Glass T flask used for shaking conditions to replicate the conditions used in [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004870#pntd.0004870.ref014" target="_blank">14</a>]. (<b>B</b>) Parasitemias of two biological replicates (MN23026b and MN23009b) grown in shaking or static conditions at equal haematocrits. (<b>C</b>) Conversion rates between rings to trophozoites or trophozoites to schizonts in the two biological replicates (<b>D</b>) Parasitized erythrocyte multiplication rate (PEMR) of the two biological replicates in either shaking or static conditions using reticulocytes from hemochromatosis patients enriched by density. Error bars represent the standard error. Results are not statically significant.</p

Experimental design using <i>Aotus lemurinus lemurinus</i> monkeys.

<p>Diagram showing the inoculations of the <i>Aotus</i> monkeys with <i>P</i>. <i>vivax</i> Sal-1. The percentages shown are for staging of the parasites at bleed (R, rings; T, trophozoites; S, schizonts; G, gametocytes). Table shows the experiments that each bleed was used for.</p

Starting haematocrit (hct) can influence <i>in vitro</i> outcome of <i>P</i>.<i>vivax</i> Sal-1.

<p>Cultures from two independent biological replicates, MN23009a and MN23009b, were initiated at different hcts. Reticulocytes enriched by density from patients with hemochromatosis were used. Cultures were initiated with the initial draw at 6% or 12% hct (iRBC, infected RBC) or were initiated with the 6% initial draw (iRBC) and hct was doubled to 12% hct with uninfected enriched reticulocytes (density hemochromatosis) (12% iRBC + RBC) to account for any differences due to the overall total parasite biomass. (<b>A</b>). Parasitemia over time from the two biological replicates. Survival was affected by the conversion differences in B as the two cultures were initiated at majority rings (MN23009a) or mixed rings and trophozoites (MN23009b) (<b>B</b>) Conversion rate differences in the two biological replicates in A between rings to trophozoites and trophozoites to schizonts. (<b>C</b>) Parasitized erythrocyte multiplication rate was only possible to measure in one biological replicate, MN23009a. Error bars represent the standard error. To set up a culture at 12% hct, we first set up the culture at 6% hct and doubled the hct with uninfected fresh, hemochromatosis human blood (12% iRBC + RBC). Results were not statically significant.</p

Media supplementation can influence the health and growth of the parasite.

<p>(<b>A</b>) Parasitemia graph showing the effect of GlutaMAX on parasitemia at different time points using three independent biologicals <i>P</i>. <i>vivax</i> Sal-1 parasites from MN23062a and b and MN23009a. Reticulocytes from hemochromatosis patients enriched by density were used for these experiments. (<b>B</b>) Histogram comparing parasite stages at different time points from the parasites in A: (<b>C</b>) Conversion percentages of rings to trophozoites and trophozoites to schizonts in media supplemented with or without GlutaMAX. Data from the three independent biological replicates in A. Error bars represent the standard error. While the parasitemia differences are not statically significant, we did observed a longer persistence of parasites in the cultures supplemented with GlutaMAX and we did observed reinvasion only in the GlutaMAX culture.</p

Reticulocyte enrichment method can affect invasion of <i>P</i>. <i>vivax</i> Sal-1.

<p>(<b>A</b>) Table showing methods used to enrich reticulocytes from peripheral blood derived either from hemochromatosis patients and Buffy Coat blood packs. The enrichment was performed according to either published studies or manufacturer’s instructions detailed in the Materials and Methods. The resulting total reticulocyte percentage was determined by staining with NMB. (<b>B</b>) The final fold change in parasitemia following invasion can be normalized to compare different invasion assays using the parasitized erythrocyte multiplication rate (PEMR) by dividing the final number of rings by the input number of schizonts [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004870#pntd.0004870.ref018" target="_blank">18</a>]. (<b>C</b>) PEMR demonstrates that the hemochromatosis reticulocytes enriched by density using the [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004870#pntd.0004870.ref014" target="_blank">14</a>] method resulted in the best invasion in two independent biological replicates. This invasion was better than the invasion in the same blood enriched by Percoll and significantly better than the reticulocytes from Buffy Coat enriched by density or CD71-microbeads. Parasites were from <i>Aotus</i> MN21014 and MN28016. Statistical significance was determined using Dunnett’s multiple comparison test. P value <0.05 (<b>D</b>) Final ring parasitemias of the invasion assays in B. Error bars represent the standard error.</p