DOI: 10.5061/dryad.6djh9w0z5
title: Data from: Different distribution of malaria parasite in left and right extremities of vertebrate hosts translates into differences in parasite transmission

description: Malaria, a vector-borne disease caused by Plasmodium spp., remains a major
 global cause of mortality. Optimization of disease control strategies
 requires a thorough understanding of the processes underlying parasite
 transmission. While the number of transmissible stages (gametocytes) of
 Plasmodium in blood is frequently used as an indicator of host-to-mosquito
 transmission potential, this relationship is not always clear. Significant
 effort has been made in developing molecular tools that improve gametocyte
 density estimation and therefore prediction of mosquito infection rates.
 However a significant level of uncertainty around estimates remains. The
 weakness in the relationship between gametocyte burden, measured from a
 blood sample, and the mosquito infection rate could be explained by a
 non-homogeneous distribution of gametocytes in the bloodstream. The
 estimated gametocyte density would then only be a single snapshot that
 does not reflect the host infectivity. This aspect of Plasmodium
 infection, however, remains largely neglected. In both humans and birds,
 we found here that the gametocyte densities differed depending on which
 side of the body the sample was taken, suggesting that gametocytes are not
 homogeneously distributed within the vertebrate host. We observed a
 fluctuating asymmetry, in other words, the extremity of the body with the
 highest density of parasites is not always the same from one individual to
 another. An estimation of gametocyte density from only one blood sample,
 as is commonly measured, could, therefore, over- or underestimated the
 infectivity of gametocyte carriers. This might have important consequences
 on the epidemiology of the disease since we show that this variation
 influences host-to-mosquito transmission. Vectors fed on the least
 infected body part had a lower parasite burden than those fed on the most
 infected part. The heterogeneous distribution of gametocytes in
 bloodstream should be considered to improve diagnosis and test new malaria
 control strategies.
description: <b>Human malaria</b> The study was conducted at the Institut de Recherche en Sciences de la Santé in Bobo Dioulasso, South-Western Burkina Faso. The intensity of malaria transmission is high and perennial in this area, with a peak from August to November. Blood slides were collected from December 2018 to July 2019 from 42 asymptomatic children aged 5-12 years attending the elementary schools of Dandé, Soumousso, Klesso, Samandeni - four villages located in the surroundings of Bobo Dioulasso. <i>P. falciparum</i> is the predominant parasite species in these villages, accounting for more than 95% of malaria cases. Separate finger-prick blood samples from the right and left hand of each volunteer were collected, Giemsa-stained and screened for asexual parasites and gametocytes. Gametocyte densities were determined from each slide as the number of gametocytes per 1000 leucocytes. Each slide was read twice by two independent qualified microscopists. Slides were declared negative after a minimum reading of 100 fields. We discovered 8 gametocyte-free individuals leaving 34 individuals for analysis.   <b>Avian malaria</b> <i>Parasite strain  </i> <i>Plasmodium relictum</i> is the most prevalent form of avian malaria in Europe. The lineage used in these experiments (lineage SGS1) was isolated from infected great tits (<i>Parus major</i>) caught in the region of Lausanne (Switzerland) in 2015. The strain has since been maintained by regular passage between infected and naïve canaries (<i>Serinus canaria</i>) via intraperitoneal injection. Twenty three uninfected canaries were split into three experimental blocks (Block 1: 8, Block 2: 8, Block 3: 7) and inoculated by means of an intraperitoneal injection of 150-200μL of a blood mixture collected from five chronically infected canaries. Birds in the same experimental block were infected with a blood mixture from an independent group of infected birds. For each block infected birds were then either “exposed” (block 1, 2, 3 = 5) or “unexposed” (block 1, 2 = 3, block 3 = 2) to mosquito bites (Fig 1B). <i>Mosquito rearing </i> <i>Culex pipiens</i> mosquitos used in the experiment were from a population collected from the field (Lausanne, 46°31′25.607″N 6°34′40.714″E, altitude: 380 m) in August 2017 and since maintained under laboratory conditions. Mosquitoes were reared as described by Vézilier et al. 2010 in an insectary at 25°C ± 1°C, 70 ± 5% RH and with 12L:12D photoperiod. On the day prior to mosquito exposure, 500 7-10 day old female mosquitoes were haphazardly chosen from different emergence cages and placed inside new cages (100 females per cage). During this time females were deprived of sugar solution to increase hunger levels and maximize the biting rate. Water was provided to prevent dehydration, but removed 6 hours prior to the start of the experiment. <i>Experimental design </i> The three experimental blocks were carried out in February, March and April 2018 respectively. Twelve days after infection, coinciding with the acute phase of the <i>P. relictum</i> infection in canaries<sup>55</sup>, birds were placed individually into compartmentalized cages designed for physically separating their two legs. For birds in the “exposed” group, at 6:00 pm, 45-50 uninfected female mosquitoes were added to each compartment (left and right) for 180 minutes. Unexposed birds were placed under the same experimental conditions but without mosquitoes. At the end of the mosquito exposure period (9:00 pm), a red lamp was used to capture mosquitoes and five microliters of blood was collected from the medial metatarsal vein of each leg. Three independent drops of blood were then smeared onto three different microscope slides for each of the samples. Blood fed mosquitoes were placed individually into a numbered plastic tube covered with a mesh. Food was provided in the form of a cotton pad soaked in a 10% sugar solution placed on top of each tube. Mosquitoes were dissected 7 to 8 days later and the number of <i>Plasmodium</i> oocysts in their midgut counted with the aid of a binocular microscope. Haematin excreted at the bottom of each plastic tube was quantified as an estimate of the female’s blood meal size. The intensity of bird infection (parasitaemia) was determined visually by counting the number of infected red blood cells per 3000 erythrocytes in randomly chosen fields on the blood smears. The three replicate were used to calculate an average parasitaemia (mean ± SE) for each leg of each bird. The legs of each bird were then classified as either the lower infected leg (LIL) or higher infected leg (HIL). All slides were examined by the same experimenter, and parasitaemia was used as a proxy for transmissible stage (gametocytes) production because parasitaemia and gametocytaemia are strongly positively correlated in this system.