The sterilizing effect of pyriproxyfen on the malaria vector Anopheles gambiae: physiological impact on ovaries development

Pyrethroid insecticides are the most widely used compounds in public health because of their high effectiveness and strong excito-repellent effect on insects, as well as low mammalian toxicity [1-3]. Unfortunately resistance to this insecticide class has become widespread in malaria vectors in several malaria endemic countries and that may challenge in the future the success of malaria control programmes [4-7]. Clearly alternative compounds that can complement pyrethroids on long-lasting insecticidal nets (LLINs) are urgently needed. This approach can provide an opportunity to preserve the protectiveness of LLINs through the excito-repellent properties of the pyrethroids while enhancing toxicity through a non-pyrethroid alternative [8,9]. Pyriproxyfen (PPF), an insect juvenile hormone mimic, is recommended by the World Health Organization (WHO) [10,11] for larval control but may also have an impact on life expectancy, fecundity and fertility of adult mosquitoes through tarsal contact. PPF has an extremely low toxicity to humans and shows no cross resistance to other classes of insecticides used in public health [11]. PPF is an insect growth regulator used primarily to inhibit metamorphosis of mosquito larvae and prevent emergence of adults from pupae [12,13], hence its use for mosquito control has been limited to larval stages [14-16]. However, studies have shown that PPF can affect the development and production of eggs (fecundity) and reduces their hatching (fertility) [17,18]. Earlier studies on Aedes mosquitoes have shown that exposure to PPF can reduce the reproductive capacity of adults [19-21] depending on dosage and time of exposure in relation to the blood meal [19]. A complete sterilization of females was observed with Anopheles gambiae exposed to PPF-treated netting [22] and Anopheles arabiensis exposed to PPF in bottle bioassays [23]. More recently, a mixture of PPF and pyrethroids on bed nets achieved a strong sterilizing effect on pyrethroid-resistant An. gambiae s.s. in experimental hut trials [9].

Whilst most studies have focused on evaluating the raw outcomes of PPF on female mosquito fecundity and fertility, none has ever looked at the specific changes operated on the ovaries. Notably, no study has investigated to the best of current knowledge the ability of PPF to interfere with the development of ovaries in mosquitoes (but see Brabant and Dobson [24]). The current study explores this gap and assesses for the first time the physiological impact of PPF on An. gambiae Kisumu female ovaries when exposed to PPF treated nets at different times after blood meals in the laboratory.

The following hypotheses were tested:

In all cases fecundity was measured as the total number of eggs/total number of females that contributed to the oviposition. Similarly fertility was calculated by dividing the total number of larvae/total number of females that contributed to oviposition. The reduction of fecundity and fertility was compared between the group exposed to PPF-treated net and the control group using non-parametric Mann–Whitney tests.

The effect of PPF on fecundity and fertility has been investigated in several insects but the mechanisms by which PPF interferes with female reproductive outcomes has been overlooked specifically in mosquitoes. The main goals in the present study were to describe the physiological impact of PPF on ovarian development and explore the sterilizing effects of PPF at various times before blood meals.

The results of the present study provided further evidence that tarsal contact with PPF-treated nets sterilized adult females of An. gambiae under laboratory conditions. Importantly, bioassays analysis showed that PPF impaired the development of eggs in the ovaries of An. gambiae, one of the most important malaria vectors in many part of sub-Saharan Africa. Exposure to PPF affected the rate at which ovarian follicles develop from the previtellogenic resting stage to maturity. Females of Anopheles require blood meals to mature their eggs. Females emerge from the larval stage with their primary follicles at stage I and need a blood meal to complete follicle development to stage V. In the present study, three minutes exposure of female An. gambiae to PPF-treated nets halted follicle maturation as they never reached stage V. Christophers [25] divided the course of ovarian development of Anopheles into five stages (I to V), based on the appearance of follicles under the microscope; it is now possible to relate Christophers stages to some of the physiological events of oogenesis. Several external factors influence development toward these stages in mosquitoes including nutrition [26]. Large well nourished females are able to develop their primary follicles to the previtellogenic resting stage without blood-feeding, therefore at the expense of reserves accumulated during larval life. Most wild An. gambiae need two blood meals to develop their first batch of eggs [26]. In females of Aedes aegypti that take a small blood meal, the development of follicles arrests in stage III but resumes development after a further blood meal [27,28]. In the present study, female An. gambiae were given full blood meals but still could not lay eggs after being in contact with PPF-treated nets. Juvenile hormone has important functions in the previtellogenic phase, stimulating development of stage I follicles to the previtellogenic stage and inducing the fat body to become competent to synthetize vitellogenin. PPF, a juvenile hormone analogue, may not initiate these functions and that might disrupt these hormonal routes leading to a failure of egg development. Further juvenile hormone level in the mosquito blood stream consistently drops after blood meal and that initiates the secretion of ecdysone by the ovary. Once released, the ecdysone directs the development of eggs ([24] and references therein). It is suspected that in the present case, PPF, the analogue juvenile hormone, was still highly present in PPF exposed females blood stream, inhibiting the release of ecdysone. Methoprene, another mimic juvenile hormone, was shown in a recent study to significantly reduce the length of ovary and the size of oocyte in Ae. aegypti females [24]. In the current study, a fine scale observation of the egg development showed that after each cycle of oviposition, follicles in the ovarioles of females not exposed to PPF underwent successive developmental stages known as Christopher developmental stages. Follicles were all in the same developmental stage (Christopher stage II) and deposit of yolk could be seen in the ovum at the base of the follicles. In contrary in females exposed to PPF-treated nets, the follicles were very slow to develop and never reached the complete maturation stage and showed different developmental stages. Some were still in the early developmental stage (Christopher stage II) with a round structure, while some were elongated and dark, a sign of egg abortion.

That females exposed to PPF-treated nets could not lay eggs after the first blood meal but also on subsequent blood meals, indicates the long, residual activity of this chemical on An. gambiae reproduction. In addition, mosquitoes exposed to PPF and blood-fed five days later could not lay eggs. Mosquitoes that had undergone a first oviposition cycle and were then subsequently exposed to PPF could no longer lay eggs. Most of the few eggs laid in certain cases did not hatch suggesting that either the larvae failed to develop or they died after development has been completed. These results are in apparent contradiction to those of Harris et al. [23] who did not show a long residual activity of PPF. It should be noted that while Harris et al. [23] used An. arabiensis in their study, the present study used An. gambiae and the dosage of PPF was ten times higher than that of their study. Moreover the substrate of treatment in the previous study [23] was made of bottle while the present study, PPF was incorporated in bednet. Interestingly Brabant and Dobson [24] showed that methoprene could impair oviposition over two gonotrophic cycles in Ae. aegypti and larval breeding sites treatment with PPF could inhibit larval development up to five months [20].

A short contact time (three minutes) with PPF treated net and the long-residual effect of the chemical on mosquito reproductive output make it a very attractive control tool of malaria vectors. Most malaria vectors have developed resistance to pyrethroids and many other insecticides, threatening the future of vector control. Mixing PPF with pyrethroids on the nets can help control and limit the spread of resistance to pyrethroids in the distribution of malaria vectors. A couple of studies have already shown the potential of such mixtures raising the hope of a viable new vector control tool to help combat insecticide resistance [9,29]. Specifically previous studies with PPF hand-dipped nets demonstrated complete sterilization of laboratory reared pyrethroid susceptible Anopheles gambiae in bioassays. Further to this study, N’gufor et al. [9] have provided strong evidence that pyrethroid resistant An. gambiae were sterilized if they come to contact with bed nets that contain PPF. They concluded that a better reduction in vector abundance can be achieved with community wide use of Olyset Duo compared to LNs treated only with pyrethroids. In microcosms containing breeding sites to simulate the natural ecosystem, Ohba et al. [21] have shown that PPF had a strong sterilizing effect on Aedes albopictus.