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Background: The direct membrane feeding assay (DMFA), whereby gametocyte-infected blood is collected from human donors and from which mosquitoes feed through a membrane, is proving essential for assessing parameters influencing Plasmodium transmission potential in endemic countries. The success of DMFAs is closely tied to gametocyte density in the blood, with relatively high gametocytaemia ensuring optimal infection levels in mosquitoes. As transmission intensity declines with control efforts, the occurrence of asymptomatic individuals with low gametocyte densities, who can significantly contribute to the infectious reservoir, is increasing. This poses a limitation to studies relying on the experimental infection of large numbers of mosquitoes with natural isolates of Plasmodium. A simple, field-applicable method is presented for improving parasite infectivity by concentrating Plasmodium falciparum gametocytes., Methods: Anopheles gambiae received one of the following 5 blood treatments through DMFA: (i) whole blood (WB) samples from naturally-infected donors; (ii) donor blood whose plasma was replaced with the same volume of Plasmodium-naive AB + serum (1:1 control); (iii) plasma replaced with a volume of malaria-naïve AB + serum equivalent to half (1:1/2), or to a quarter (1:1/4), of the initial plasma volume; and (v) donor blood whose plasma was fully removed (RBC). The experiment was repeated 4 times using 4 distinct wild parasite isolates. Seven days post-infection, a total of 1,095 midguts were examined for oocyst presence., Results: Substituting plasma with reduced amounts (1:1/2 and 1:1/4) of Plasmodium-naive AB + serum led to a 31% and 17% increase of the mosquito infection rate and to a 85% and 308% increase in infection intensity compared to the 1:1 control, respectively. The full removal of plasma (RBC) reduced the infection rate by 58% and the intensity by 64% compared to the 1:1 control. Reducing serum volumes (1:1/2; 1:1/4 and RBC) had no impact on mosquito feeding rate and survival when compared to the 1:1 control., Conclusions: Concentrating gametocytic blood by replacing natural plasma by lower amount of naive serum can enhance the success of mosquito infection. In an area with low gametocyte density, this simple and practical method of parasite concentration can facilitate studies on human-to-mosquito transmission such as the evaluation of transmission-blocking interventions., (© 2024. The Author(s).)

Host age variation is a striking source of heterogeneity that can shape the evolution and transmission dynamic of pathogens. Compared with vertebrate systems, our understanding of the impact of host age on invertebrate-pathogen interactions remains limited. We examined the influence of mosquito age on key life-history traits driving human malaria transmission. Females of Anopheles coluzzii , a major malaria vector, belonging to three age classes (4-, 8- and 12-day-old), were experimentally infected with Plasmodium falciparum field isolates. Our findings revealed reduced competence in 12-day-old mosquitoes, characterized by lower oocyst/sporozoite rates and intensities compared with younger mosquitoes. Despite shorter median longevities in older age classes, infected 12-day-old mosquitoes exhibited improved survival, suggesting that the infection might act as a fountain of youth for older mosquitoes specifically. The timing of sporozoite appearance in the salivary glands remained consistent across mosquito age classes, with an extrinsic incubation period of approximately 13 days. Integrating these results into an epidemiological model revealed a lower vectorial capacity for older mosquitoes compared with younger ones, albeit still substantial owing to extended longevity in the presence of infection. Considering age heterogeneity provides valuable insights for ecological and epidemiological studies, informing targeted control strategies to mitigate pathogen transmission.

In the fight against malaria, transmission blocking interventions (TBIs) such as transmission blocking vaccines or drugs, are promising approaches to complement conventional tools. They aim to prevent the infection of vectors and thereby reduce the subsequent exposure of a human population to infectious mosquitoes. The effectiveness of these approaches has been shown to depend on the initial intensity of infection in mosquitoes, often measured as the mean number of oocysts resulting from an infectious blood meal in absence of intervention. In mosquitoes exposed to a high intensity of infection, current TBI candidates are expected to be ineffective at completely blocking infection but will decrease parasite load and therefore, potentially also affect key parameters of vector transmission. The present study investigated the consequences of changes in oocyst intensity on subsequent parasite development and mosquito survival. To address this, we experimentally produced different intensities of infection for Anopheles gambiae females from Burkina Faso by diluting gametocytes from three natural Plasmodium falciparum local isolates and used a newly developed non-destructive method based on the exploitation of mosquito sugar feeding to track parasite and mosquito life history traits throughout sporogonic development. Our results indicate the extrinsic incubation period (EIP) of P. falciparum and mosquito survival did not vary with parasite density but differed significantly between parasite isolates with estimated EIP50 of 16 (95% CI: 15-18), 14 (95% CI: 12-16) and 12 (95% CI: 12-13) days and median longevity of 25 (95% CI: 22-29), 15 (95% CI: 13-15) and 18 (95% CI: 17-19) days for the three isolates respectively. Our results here do not identify unintended consequences of the decrease of parasite loads in mosquitoes on the parasite incubation period or on mosquito survival, two key parameters of vectorial capacity, and hence support the use of transmission blocking strategies to control malaria., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Guissou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Success in reducing malaria transmission through vector control is threatened by insecticide resistance in mosquitoes. Although the proximal molecular mechanisms and genetic determinants involved are well documented, little is known about the influence of the environment on mosquito resistance to insecticides. The aim of this study was to assess the effect of plant sugar feeding on the response of Anopheles gambiae sensu lato to insecticides. Adults were fed with one of four treatments, namely a 5% glucose control solution, nectariferous flowers of Barleria lupulina, of Cascabela thevetia and a combination of both B. lupulina + C. thevetia. WHO tube tests were performed with 0.05% and 0.5% deltamethrin, and knockdown rate (KD) and the 24 h mosquito mortality were measured. Plant diet significantly influenced mosquito KD rate at both concentrations of deltamethrin. Following exposure to 0.05% deltamethrin, the B. lupulina diet induced a 2.5 fold-increase in mosquito mortality compared to 5% glucose. Species molecular identification confirmed the predominance of An. gambiae (60% of the samples) over An. coluzzii and An. arabiensis in our study area. The kdr mutation L1014F displayed an allelic frequency of 0.75 and was positively associated with increased phenotypic resistance to deltamethrin. Plant diet, particularly B. lupulina, increased the susceptibility of mosquitoes to insecticides. The finding that B. lupulina-fed control individuals (i.e. not exposed to deltamethrin) also displayed increased 24 h mortality suggests that plant-mediated effects may be driven by a direct effect of plant diet on mosquito survival rather than indirect effects through interference with insecticide-resistance mechanisms. Thus, some plant species may weaken mosquitoes, making them less vigorous and more vulnerable to the insecticide. There is a need for further investigation, using a wider range of plant species and insecticides, in combination with other relevant environmental factors, to better understand the expression and evolution of insecticide resistance., (© 2022. The Author(s).)

Background: Besides feeding on blood, females of the malaria vector Anopheles gambiae sensu lato readily feed on natural sources of plant sugars. The impact of toxic secondary phytochemicals contained in plant-derived sugars on mosquito physiology and the development of Plasmodium parasites remains elusive. The focus of this study was to explore the influence of the alkaloid ricinine, found in the nectar of the castor bean Ricinus communis, on the ability of mosquitoes to transmit Plasmodium falciparum., Methods: Females of Anopheles gambiae and its sibling species Anopheles coluzzii were exposed to ricinine through sugar feeding assays to assess the effect of this phytochemical on mosquito survival, level of P. falciparum infection and growth rate of the parasite., Results: Ricinine induced a significant reduction in the longevity of both Anopheles species. Ricinine caused acceleration in the parasite growth rate with an earlier invasion of the salivary glands in both species. At a concentration of 0.04 g l -1 in An. coluzzii, ricinine had no effect on mosquito infection, while 0.08 g l -1 ricinine-5% glucose solution induced a 14% increase in An. gambiae infection rate., Conclusions: Overall, our findings reveal that consumption of certain nectar phytochemicals can have unexpected and contrasting effects on key phenotypic traits that govern the intensity of malaria transmission. Further studies will be required before concluding on the putative role of ricinine as a novel control agent, including the development of ricinine-based toxic and transmission-blocking sugar baits. Testing other secondary phytochemicals in plant nectar will provide a broader understanding of the impact which plants can have on the transmission of vector-borne diseases., (© 2021. The Author(s).)

Despite its epidemiological importance, the time Plasmodium parasites take to achieve development in the vector mosquito (the extrinsic incubation period, EIP) remains poorly characterized. A novel non-destructive assay designed to estimate EIP in single mosquitoes, and more broadly to study Plasmodium-Anopheles vectors interactions, is presented. The assay uses small pieces of cotton wool soaked in sugar solution to collect malaria sporozoites from individual mosquitoes during sugar feeding to monitor infection status over time. This technique has been tested across four natural malaria mosquito species of Africa and Asia, infected with Plasmodium falciparum (six field isolates from gametocyte-infected patients in Burkina Faso and the NF54 strain) and across a range of temperatures relevant to malaria transmission in field conditions. Monitoring individual infectious mosquitoes was feasible. The estimated median EIP of P. falciparum at 27 °C was 11 to 14 days depending on mosquito species and parasite isolate. Long-term individual tracking revealed that sporozoites transfer onto cotton wool can occur at least until day 40 post-infection. Short individual EIP were associated with short mosquito lifespan. Correlations between mosquito/parasite traits often reveal trade-offs and constraints and have important implications for understanding the evolution of parasite transmission strategies.

Background: The sterile insect technique (SIT) is a vector control strategy relying on the mass release of sterile males into wild vector populations. Current sex separation techniques are not fully efficient and could lead to the release of a small proportion of females. It is therefore important to evaluate the effect of irradiation on the ability of released females to transmit pathogens. This study aimed to assess the effect of irradiation on the survival and competence of Anopheles arabiensis females for Plasmodium falciparum in laboratory conditions., Methods: Pupae were irradiated at 95 Gy of gamma-rays, and emerging females were challenged with one of 14 natural isolates of P. falciparum. Seven days post-blood meal (dpbm), irradiated and unirradiated-control females were dissected to assess the presence of oocysts, using 8 parasite isolates. On 14 dpbm, sporozoite dissemination in the head/thorax was also examined, using 10 parasites isolates including 4 in common with the 7 dpbm dissection (oocyst data). The survivorship of irradiated and unirradiated-control mosquitoes was monitored., Results: Overall, irradiation reduced the proportion of mosquitoes infected with the oocyst stages by 17% but this effect was highly inconsistent among parasite isolates. Secondly, there was no significant effect of irradiation on the number of developing oocysts. Thirdly, there was no significant difference in both the sporozoite infection rate and load between the irradiated and unirradiated-control mosquitoes. Fourthly, irradiation had varying effects on female survival with either a negative effect or no effect., Conclusions: The effect of irradiation on mosquito competence strongly varied among parasite isolates. Because of such isolate variability and, the fact that different parasite isolates were used to collect oocyst and sporozoite data, the irradiation-mediated reduction of oocyst prevalence was not confirmed for the sporozoite stages. Our data indicate that irradiated female An. arabiensis could contribute to malaria transmission, and highlight the need for perfect sexing tools, which would prevent the release of females as part of SIT programmes.

Background: The sterile insect technique (SIT) aims at suppressing or decreasing insect pest populations by introducing sterile insects into wild populations. SIT requires the mass-production of insects and their sterilization through, for example, radiation. However, both mass-rearing and radiation can affect the life history traits of insects making them less competitive than their wild counterparts. In the malaria mosquito Anopheles arabiensis, some progress has been made to improve the mating competitiveness of mass-reared irradiated males. However, to date, no study has explored the relative effects of colonization and irradiation on important reproductive traits in this species. Such data may help to focus research efforts more precisely to improve current techniques., Methods: Two strains of An. arabiensis originating from the same locality were used: one reared in the laboratory for five generations and the second collected as late larval instars in the field prior to experimentation. Pupae were irradiated with 95 Gy and some adult reproductive traits, including insemination rate, fecundity, oviposition behavior, fertility and male survivorship, were assessed in different mating combinations., Results: Our study revealed the different effects of mosquito strain and irradiation on reproductive processes. The insemination rate was higher in field (67.3%) than in laboratory (54.9%) females and was negatively affected by both female and male irradiation (un-irradiated vs irradiated: 70.2 vs 51.3% in females; 67.7 vs 53.7% in males). Irradiated females did not produce eggs and egg prevalence was lower in the field strain (75.4%) than in the laboratory strain (83.9%). The hatching rate was higher in the field strain (88.7%) than in the laboratory strain (70.6%) as well as in un-irradiated mosquitoes (96.5%) than in irradiated ones (49%). Larval viability was higher in the field strain (96.2%) than in the laboratory strain (78.5%) and in un-irradiated mosquitoes (97.6%) than irradiated ones (52%). Finally, field males lived longer than laboratory males (25.1 vs 20.5 days, respectively)., Conclusions: Our results revealed that both irradiation and colonization alter reproductive traits. However, different developmental stages are not equally affected. It is necessary to consider as many fitness traits as possible to evaluate the efficacy of the sterile insect technique.