Streamlining sporozoite isolation from mosquitoes by leveraging the dynamics of migration to the salivary glands

Abstract

Background Sporozoites isolated from the salivary glands of Plasmodium-infected mosquitoes are a prerequisite for several basic and pre-clinical applications. Although salivary glands are pooled to maximize sporozoite recovery, insufficient yields pose logistical and analytical hurdles; thus, predicting yields prior to isolation would be valuable. Preceding oocyst densities in the midgut is an obvious candidate. However, it is unclear whether current understanding of its relationship with sporozoite densities can be used to maximize yields, or whether it can capture the potential density-dependence in rates of sporozoite invasion of the salivary glands. Methods This study presents a retrospective analysis of Anopheles stephensi mosquitoes infected with two strains of the rodent-specific Plasmodium berghei. Mean oocyst densities were estimated in the midguts earlier in the infection (11–15 days post-blood meal), with sporozoites pooled from the salivary glands later in the infection (17–29 days). Generalized linear mixed effects models were used to determine if (1) mean oocyst densities can predict sporozoite yields from pooled salivary glands, (2) whether these densities can capture differences in rates of sporozoite invasion of salivary glands, and (3), if the interaction between oocyst densities and time could be leveraged to boost overall yields. Results The non-linear effect of mean oocyst densities confirmed the role of density-dependent constraints in limiting yields beyond certain oocyst densities. Irrespective of oocyst densities however, the continued invasion of salivary glands by the sporozoites boosted recoveries over time (17–29 days post-blood meal) for either parasite strain. Conclusions Sporozoite invasion of the salivary glands over time can be leveraged to maximize yields for P. berghei. In general, however, invasion of the salivary glands over time is a critical fitness determinant for all Plasmodium species (extrinsic incubation period, EIP). Thus, delaying sporozoite collection could, in principle, substantially reduce dissection effort for any parasite within the genus, with the results also alluding to the potential for changes in sporozoites densities over time to modify infectivity for the next host.

Blood meals from ‘dead-end’ vertebrate hosts enhance transmission potential of malaria-infected mosquitoes

“…Data analysis and modeling was performed in the RStudio IDE (version 2022.07.1, Build 554) running R software (version 4.2), as described in detail previously [25][26][27]. Unless stated otherwise, blood feeding rates and parasite prevalence (EIP) were modeled as the proportion gravid or infected from total dissected respectively, with a binomial distribution in generalized linear mixed effects models (GLMMs).…”

“…One cage was not offered a second blood meal (control, or 'none' herein) while the remaining three experimental cages were offered a second blood meal of bovine, canine or human origin respectively. After the blood meal, 20-25 mosquitoes from each group were sampled at regular intervals, with salivary glands and ovaries (see below) assessed for sporozoite and gravid status respectively, as described previously [25][26][27]; for P. falciparum, mosquitoes were sampled on 10, 12, 14, 16, 18, and 21 days post-infection (or 2, 4, 6, 8, 10 and 13 days post-second blood meal), while P. berghei-infected mosquitoes were assessed at 14, 16 and 18 days post-infection (or 2, 4 and 6 days post-second blood meal).…”

“…The copyright this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.24.521872 doi: bioRxiv preprint one source of blood over another: no clear differences in gravid status between the various species' blood meals would confirm the lack of preference. Note that repeated measurements of gravid status from different individuals over time in the respective groups would also impart more robustness to the results [25].…”

“…On the day of infection (0 days post-infection), 1250 female An. stephensi were infected with P. falciparum NF54 or P. berghei (PbGFP-LUCCON) respectively, as described previously [25][26][27]. At 6 days post-infection, oviposition sites were introduced into the mosquito cages infected with either parasite.…”

“…To stimulate oviposition at 6 days post-infection, mosquitoes were offered open-top polypropylene cups (4.5 inches diameter x 2.5 inches height) containing ~200ml deionized water. Parasite densities and infection status in the mosquitoes were estimated at the stated time points as described before [25][26][27]. Mortality was assessed daily in all the cages.…”