DOI: 10.5061/dryad.ns1rn8pps
title: Last-come, best served? Mosquito biting order and Plasmodium transmission

description: A pervasive characteristic of parasite infections is their tendency to be
 overdispersed. Understanding the mechanisms underlying this overdispersed
 distribution is of key importance as it may impact the transmission
 dynamics of the pathogen. Although multiple factors ranging from
 environmental stochasticity to inter-individual heterogeneity may explain
 parasite overdispersion, parasite infection is also observed to be
 overdispersed in inbred host population maintained under laboratory
 conditions, suggesting that other mechanisms at play. Here, we show that
 the aggregated distribution of malaria parasite within mosquito vectors is
 partially explained by a temporal heterogeneity in parasite infectivity
 triggered by the bites of mosquitoes. The transmission of the parasite was
 tripled between the first and the last blood fed mosquito in a period of
 only three hours. Surprisingly the increase in transmission is not
 associated with an increase in parasite investment in the production of
 the transmissible stage. Overall, we highlight that Plasmodium is capable
 of responding to the bites of mosquitoes to increase its own transmission
 at a much faster pace than initially thought and that this is partly
 responsible for overdispersed distribution of infection. We discuss the
 underlying mechanisms as well as the broader implications of this plastic
 response for the epidemiology of malaria.
description: To investigate the impact of mosquito bite-driven plasticity on
 <i>Plasmodium</i> transmission, three experiments were carried
 out in which infected birds were exposed to mosquitoes for 3 hours (6 – 9
 p.m.) and mosquitoes were sampled at regular intervals thereafter
 (different protocols for the three experiments, see below). To investigate
 the impact of vector bites on parasite population growth, the parasitaemia
 (number of parasites in the blood) and gametocytaemia (number of
 gametocytes in the blood) of vertebrate hosts exposed or not (control) to
 mosquitoes were measured just before and just after the mosquito exposure
 period using blood smears. All experiments were carried
 out using domestic canaries (<i>Serinus
 canaria</i>). <b>Experiment 1_ Oocyst burden and mosquito biting order: batch experiment</b> Two experimental blocks were carried out with 14 and 5 infected birds respectively with two different isolates of Plasmodium relictum. Day 11-13 post-infection, corresponding to the acute phase of infection, blood was sampled from each bird at 5:45 p.m. Straight afterwards blood sampling, birds were placed individually in an experimental cage (L40 x W40 x H40 cm). At 6:00 p.m., 8 and 3 haphazardly chosen birds, for block 1 and 2 respectively, were exposed to four successive batches of 25 ± 3 uninfected females’ mosquitoes. Each mosquito batch was left in the cage for 45 minutes before being taken out and replaced by a new batch (i.e. batch 1 (T0min), batch 2 (T45min), batch 3 (T90min) and batch 4 (T135min)). Blood fed mosquitoes in each batch were counted and individually placed in numbered plastic tubes (30 ml) covered with a mesh with a cotton pad soaked in a 10% glucose solution. At the end of the last mosquito exposure session (9:00 p. m.) a second blood sample was taken from each bird. A red lamp was used to capture blood fed mosquitoes without disturbing the birds and the mosquitoes. Unexposed birds (control) were placed in the same experimental condition but without mosquitoes. Tubes containing the blood fed mosquitoes were kept in standard insectary conditions to obtain an estimate of the blood meal size and the success of the infection (infection prevalence and oocyst burden). For this purpose, 7-8 days post blood meal, the females were taken out of the tubes and the amount of haematin excreted at the bottom of each tube was quantified as an estimate of the blood meal size. Females were then dissected and the number of Plasmodium oocysts in their midgut counted with the aid of a binocular microscope. <b>Experiment 2_ Oocyst burden and mosquito biting order: individual monitoring</b> To obtain a finer measurement of the impact of mosquito biting order on the oocyst burden, a second experiment was carried out with the same protocol as described above, except that exposed birds (4 of the 8 infected birds) were exposed to a single batch of 100 uninfected mosquitoes for 3h (6:00-9:00 p.m.). Female mosquitoes were continuously observed and individually removed from the cages immediately after blood feeding in order to record the order of biting of each female. <b>Experiment 3_ Mosquito biting order and density of parasites ingested</b> The third experiment was carried out to investigate whether the total number of parasites in the blood meal, immediately after the blood feeding, fluctuated during the feeding bout. Two infected birds were exposed to a single batch of 100 mosquitoes for 3h (6.00 pm – 9.00 pm) and mosquitoes were removed from the cages immediately after blood feeding. The order of biting of each female was recorded and every second mosquito collected was either immersed immediately in liquid nitrogen to quantify the number of parasites ingested by qPCR or stored in plastic tubes and dissected one week later to count the number of oocysts in the midgut.
description: NA = missing values Batch_ID = 1: batch 1
 (T0<sub>min</sub>), 2: batch 2
 (T45<sub>min</sub>), 3: batch 3
 (T90<sub>min</sub>) and 4: batch 4
 (T135<sub>min</sub>) Haematin =
 ng Bird status = Exposed (Exp) or not (Unexp) to
 mosquito bites