While mean oocyst densities estimated earlier in the infection were able to capture the non-linear trends in sporozoite recovery, the continued migration of sporozoites to the salivary glands also resulted in higher sporozoite yields over 17–29 days post-bloodmeal. The effect of mean oocyst densities and time were independent of parasite strain, and the number of salivary glands pooled, further suggesting that the effects described here should be broadly applicable. Taken together, we describe a framework where mean oocyst densities estimated earlier in the infection could be used to approximate yields a priori, with the potential for yields to be maximized by leveraging the relationship between sporozoite yields and time. For example, at all mean oocyst densities, our model suggests that compared to 17 days post-bloodmeal, yields may be 1.3-, 1.7-, and 2.2-fold higher at 21, 25, and 29 days post-bloodmeal, respectively (Fig. 3). While yields will be dependent on mean oocyst densities, in general, by accounting for the effect of time, our results offer several benefits such as dissecting fewer mosquitoes, increasing the efficiency of otherwise cumbersome technical procedures, and providing greater reliability in the results of the downstream assays.
The range of sporozoites reported here generally corroborates previous findings from individual or pooled mosquitoes infected with P. berghei [17–19, 21, 33–38, 56–60]. The quadratic effect of mean oocyst densities on sporozoite yields increased with mean oocyst densities in a non-linear manner, wherein the initial increase in yields until ~ 50 oocysts were followed by a decline. In general, this non-linearity has been noted for sporozoites produced by both rodent and human Plasmodium species; current evidence suggests sporozoite densities may be dependent on complex interactions between nutritional availability, vector mortality, and immune responses to the oocysts and/or sporozoites [22, 61–63]. For P. berghei, the decline in sporozoite densities was noted at mean oocyst densities ≥ 50 per midgut [19, 21], with at least one study suggesting vector mortality during sporozoite egress as the most likely explanation for the reduced recovery . A major benefit of this analysis is that we can now leverage our knowledge of the relationship between sporozoite yields and time post-infection to reduce the risk of losing sporozoites at high mean oocyst densities (e.g., > 50) [19, 21], while also reducing dissection effort by boosting yields at lower oocyst densities. Although the model was unable to detect any evidence of a density-dependent delay (lack of clear association between mean oocyst densities and time), the clear effect of time suggested that the continued invasion of salivary glands should still be beneficial to sporozoite yields. Taken together, the effects of oocyst densities and time carry two implications for maximizing sporozoite yields. First, the non-linear effect of mean oocyst densities suggests that as oocyst densities increase (> 50), more mosquitoes would need to be dissected to obtain yields comparable to groups with lower densities. Second, although yields may increase linearly over time for all oocyst densities, groups with mean oocyst densities over 50 may still yield more sporozoites than groups with oocyst densities under 50, even if, for instance, sporozoite collection was delayed to 29 days post-bloodmeal (Fig. 3).
In addition to testing the potential for yields to decline with increasing mean oocyst densities (≥ 50), we also tested whether yields would decline over time by including a quadratic (“humped”) effect of days post-bloodmeal on sporozoite yields (supplementary tables 2 and 3). While the model was unable to identify a time point when yields started declining, simulations based on the model fit suggested that irrespective of mean oocyst density, yields became increasingly uncertain starting ~ 25 days post-bloodmeal (Fig. 1b and Fig. 2). Whether this was due to a lack of data, nutritional resources, heightened immune responses, a sign of deteriorating host health and increased mortality, or sporozoite senescence [19–21], is not possible to ascertain here. As such, until more data is available, we suggest sampling mosquitoes at ~ 25 days post-bloodmeal, but no later than 29 days when sporozoite infectivity has been suggested to decline . Based on our results, the model predicts 1.7-fold higher yields at 25 days (vs 17 days), irrespective of mean oocyst densities.
Together, mean oocyst densities and time were able to account for just under 50% of the overall variation in sporozoite yields from our dataset (e.g., Table 1). Allowing the contributions of the two predictors to vary randomly among groups significantly improved the model’s account of the total variation (R2 > 70%, Table 1), indicating the presence of other confounders specific to each group contributed significantly to the variation around the predicted yields (Fig. 1 and Fig. 3). For instance, of the groups where more information was available, one potential source of variation was a group of PbANKA-infected mosquitoes, which, despite only carrying a mean of 10.1 oocysts/midgut provided unexpectedly high sporozoite yields over time. However, only seven of the 17 individuals dissected (41%) showed evidence of having fed on the mouse (presence of eggs in ovaries during oocyst quantifications, mean feeding rates for all groups ± se = 72.4% ± 2.7), suggesting that < 50% of the group could contribute to the sporozoite pool (albeit with the caveat that estimation of feeding rates may be masked by eggs being reabsorbed by the mother [64–66]). These seven blood-fed individuals were carrying a mean of 25 oocysts per midgut, which was higher than the 10.1 calculated earlier, but more likely to explain the higher sporozoite yields observed for the group. In contrast, a group of PbGFP-LUCCON-infected mosquitoes with a mean oocyst density of 46.2 should have yielded, according to the model, significantly more sporozoites than the 5,250 recovered from 15 salivary glands (or 350 per mosquito) at 17 days post-bloodmeal. To determine why the yields were low, we quantified oocysts in the midguts of these 15 individuals at the time of sporozoite collections (i.e., 17 days) and found that they were carrying 17 mean oocysts per midgut, which was lower than the 46.2 mean oocysts measured earlier from the same group. Although we are uncertain whether this was due to excess mortality in the cage or overestimation of mean oocyst densities earlier, the low oocyst densities were the likely reason for the low yields observed. While the models identified these groups as sources of variation, in general, they were consistent with the conclusion that yields were driven by mean oocyst densities and time.
While the instances above highlight how ‘accurate’ estimation of mean oocyst densities is also critical to predicting sporozoite yields over time, despite the consistency, the effect of time on sporozoite yields was less clear (Table 1). Future experiments with sporozoites collected over a more balanced sampling schedule, and a fixed number of individuals could address some of the study limitations and offer clearer insight into whether rates of sporozoite migration do indeed vary with oocyst density. As oocyst densities increase, sporozoite migration over time may be the primary reason for increased yields (Fig. 1a), however, a clear estimation of its contribution may be confounded by pooling sporozoites from individuals with increasingly heterogenous oocyst densities (shaded areas, Supplementary Fig. 2). Further, this heterogeneity between individuals in their contribution to the sporozoite pool could explain why adjusting for differences in the number of salivary glands did not identify a clearer effect of time (Supplementary table 3). Thus, estimating the relationship between oocyst density and sporozoite yields from pooled salivary glands likely only provides qualitative insight into a process that may vary quantitatively among individuals. Considering this, we suggest that isolating sporozoites from all mosquitoes at a single day post-bloodmeal (e.g., 25–26 days), instead of at fixed intervals, may be a more efficient use of mosquitoes and time, while still achieving maximal sporozoite yields.
There are two additional caveats to our study and analysis. First, although the main effects of mean oocyst densities and time were common to both parasite strains, our objective was not to compare strains. Thus, we cannot disregard the possibility that strains may differ in overall sporozoite output . Second, differences between mosquito colonies due to genetic (e.g., An. stephensi “Indian” vs “SDA–500”) [67, 68] and/or epigenetic effects (for instance, larval culture conditions and its carry-over effect on adults) , may manifest as differences in parasite infection, replication, and sporozoite yields. While these differences suggest that more careful consideration may be necessary before settling on specific (or range of) parameters, the fundamental nature of the relationship between oocyst densities, time, and sporozoite yields should be consistent.