Limited understanding of mosquito biology and ecology poses a challenge for the development of effective vector control approaches. Laboratory colonization of target species provides an opportunity to address these knowledge gaps by facilitating detailed investigation of vector biology under controlled conditions where experimental manipulation is possible. Here we characterized the fitness traits of An. funestus s.s during colonization attempts from a wild population in Tanzania to identify the bottlenecks that make this species so difficult to colonize. To our understanding this is the first documentation of fitness constraints during attempted colonization of this species and the first report of attempted colonization of An. funestus from Tanzania.
Consistent with most previous attempts, colonization of this wild An. funestus population proved unsuccessful with no offspring being produced from the F1 generation. Several life history processes and demographic traits were identified as being impaired when FUTAZ were brought into the laboratory. First, there number of eggs laid by wild FUTAZ when brought in the insectary was lower than in the well-establish FUMOZ line, as were hatching rates, larval survival, mating success and adult female/male survival. F1 FUTAZ body sizes were slightly smaller compared to their maternal generation in the wild, but did not differ with the FUMOZ strain indicating this trait is unlikely to predict colonization success. Of all these fitness traits, the primary hurdles to colonization are likely to be the extremely low mating success and larval survival of F1 An. funestus in the laboratory. Until these fitness traits can be improved under laboratory conditions, the colonization of An. funestus is unlikely to be successful and repeatable.
Eggs laid by wild-FUTAZ An. funestus had low proportion of hatching compared to those of FUMOZ, though they were both lower than 50%, indicating many were unviable eggs laid by non-inseminated females. Previous studies investigating the impact of different water sources used for larval rearing in an An. funestus colony (FUMOZ) indicated that their egg hatching rate can exceed 70% [59, 60]; confirming hatch rates in this study were low. It is known that females of other Anopheles species can produce unviable eggs without successful mating, or after mating with sperm-less males . Therefore, poor hatching observed in the nascent strain (FUTAZ) here is likely due to the low mating success of An. funestus in captivity as has been previously documented in other Anopheles species .
The larval development period of F1-FUTAZ (11–14 days) was similar to that reported for An. funestus in other laboratory settings [59, 60, 62], but faster than FUMOZ development period (21–23 days) observed in this study. The duration of larval development in An. funestus (FUTAZ and FUMOZ) observed here were considerably longer than described for An. gambiae complex in the laboratory . For example, life table analyses of An. gambiae indicate larval development period from eclosion to adult emergence of about 11 days at 27oC [63–67]. This long larval development period for F1-FUTAZ results in a long estimated generation time of 30–33 days from eggs to first oviposition; which is higher than estimated for other African Anopheles malaria vectors [59, 68, 69]. Other life table analyses performed on An. funestus colonies estimated a generation time of approximately 33 days in insecticide-resistant (FUMOZ ) and susceptible strains (FANG), . As a consequence of this extended period of larval development, the egg to pupa survival was very low; approximately 6% for F1-FUTAZ and 27% for FUMOZ. Due to this long larval development and associated high larval mortality, very large numbers of eggs would be required generate modest numbers of adults in the laboratory. Therefore, the fitness and reproductive success of these resulting adults would have to be very high to yield a further generation.
Analysis of wild FUTAZ adults and their F1 offspring indicate their fitness is reduced compared to that of a stable An. funestus colony (FUMOZ). Wild-FUTAZ An. funestus s.s. brought into the laboratory laid 16% fewer eggs than the FUMOZ colony, and the F1 generation of FUTAZ produced no viable eggs at all. A previous study measuring the fecundity of F1 An. funestus using Madagascan population reported that this species can lay and average of 56 to 108 eggs per mosquito in captivity , which corroborate with 65 and 76 eggs from wild-FUTAZ and FUMOZ respectively from the current study. The number of eggs here is consistent to that reported in resistant and susceptible An. funestus strains in the laboratory, .
Mosquito body size is often interpreted as a proxy of their fitness [70–72]. Here we found that wild FUTAZ were somewhat larger in body size than F1-FUTAZ and FUMOZ. Consistent with the hypothesis of body size being an indicator of fitness, wing length was positively correlated with fecundity in the wild population of An. funestus (FUTAZ). However, the opposite was seen in the stable FUMOZ strain where wing size was negatively associated with fecundity. Thus at least in this one stable laboratory colony, large body size in An. funestus was not a good indicator of reproductive success. Thus caution is required in extrapolating fitness differences based on An. funestus body size, particularly between field and laboratory strains. Although body size fell between wild-FUTAZ and F1-FUTAZ, these mosquitoes were still bigger than the FUMOZ which had the highest fecundity.
The mating success of An. funestus from these populations was extremely low in the laboratory, supporting hypothesis that mating is the key bottleneck for the colonization of this species. Compared to wild-FUTAZ, insemination rates in F1-FUTAZ were extremely low (9.2% vs. 72%) and insufficient to establish a further generation F2-FUTAZ. This poor mating success is likely due eurygamy, the inability of some Anopheles species including An. funestus to initiate natural swarming behavior in flight [73, 74]. Our findings match those of other studies documenting mating as the major obstacle for successful colonization of Anopheles funestus [34, 35, 75]. To overcome this problem, techniques such as forced mating and exposing mosquitoes during sunset to induce swarming have been applied [76, 77]. Other studies have experimented with the use of large cages to stimulate natural mating for Anopheles, and simulate sunset which may be crucial cue for mating [78, 79]. However so far these methods have had little or no success over multiple attempts [32, 34]. In the current study, no F2-FUTAZ offspring were generated because none of the F1-FUTAZ laid viable eggs. Further research on how to induce mating behavior in An. funestus, particularly using more realistic semi-field systems, would be of great value. Such studies must focus on both females and males, to determine if males are unwilling to initiate swarming behavior or not fit enough to do so.
Analysis of adult mosquito survival indicated that the nascent Tanzania colony (F1-FUTAZ) had a reduced lifespan compared to stable An. funestus colony (FUMOZ). However adult survival in both cases was relatively high (32 median days for FUTAZ and 52 days for FUMOZ); with both strains living well beyond the minimum period required to produce eggs and transmit malaria. Our results corroborate another laboratory study conducted on FUMOZ where adult life span ranged from 39 to 64 days ; again much higher than F1-FUTAZ here. The shorter life span of FUTAZ relative to FUMOZ may be a result of the stress from the change of environment, or lack of adaption to laboratory conditions. Nevertheless, this F1-FUTAZ survived much longer compared to another competent vectors of malaria transmission, An. arabiensis and An. gambiae s.s. in the laboratory conditions . Previous experiments on parity shows that the median survival of An. funestus in the wild is much shorter, ranging from 7–10 days in the wild population . Thus poor adult survival relative to the wild cannot explain the failure of colonization here.
A potential limitation of our study is that the unfed An. funestus were used to seed laboratory colonies, requiring us to blood feed them artificially (on chicken blood) to acquire eggs for the next generation. This means the mating status and age of the wild population was uncertain and variable, and that variation in fecundity between strains (e.g. FUTAZ and FUMOZ) may have been impacted by differences in host blood source (human versus chicken). An alternative would have been to collect only visibly blood fed (likely from humans) An. funestus from the wild and use their eggs to generate the F1 generation. This was considered, but given the much lower abundance of blood fed An. funestus inside houses compared to the numbers of unfed females that can be obtained in CDC light traps; we choose the latter approach to ensure sufficiently large samples were obtained for colonization experiments. These wild mosquitoes could not be provided with a human blood meal given their malaria infection status was unknown and they were not adapted to membrane feeding, thus chicken blood was provided. This variation in host blood source could have generated some differences in fitness between strains. However, we consider this to be unlikely given that previous studies indicate that human and chicken blood meals generate similar egg production in other African malaria vector species (An. arabiensis and An. gambiae ). The F1 population, upon which the main fitness indicators were assessed, was fed on human blood.