Background
The fast spread of resistance to insecticides observed in the main malaria vectors,
Anopheles gambiae sensu stricto, Anopheles arabiensis and
Anopheles funestus [
1,
2] suggests that the effectiveness of mass distribution of insecticide-treated nets (ITNs) and large-scale indoor residual spraying (IRS) in reducing the incidence of malaria in endemic countries [
3,
4] will reach a plateau in the foreseeable future. There is an urgent need for development of not only new chemical compounds, but also of novel and alternative vector control approaches to complement pesticide-based strategies. This urgency explains the renewed interest in vector control using sterile male releases [
5] and the rapid expansion of research focused on the release of genetically manipulated mosquitoes unable to transmit malaria [
6]. Implicit to these approaches is the necessity to produce large numbers of sexually competitive male mosquitoes from colonized strains in order to target wild vector populations [
7,
8]. The current knowledge base for mosquito mass-rearing techniques has been accumulated over a number of sterile-male mosquito release programmes attempted during the 1970s [
5,
7,
9]. Although some of those programmes significantly impacted the targeted vector populations, results were generally too poor to warrant their continuation and expansion [
5]. These projects generated valuable data about the relative mating competitiveness of laboratory-reared sterile males compared to wild males and putative negative effects of the chemical or radioactivity sterilization steps involved in producing sterile males [
5]. They were, however, generally unable to identify the exact genetic and environmental processes associated with colonization and laboratory rearing that negatively affected the reproductive phenotype of mass-produces males [
5,
8,
9].
Colonized strains that are well adapted to the laboratory are able to mate and lay eggs reliably and predictably in the laboratory setting and as such are the starting point of all release control programmes. In the process of establishing a new laboratory colony, the mosquito population undergoes at least one, and possibly several, selective sweeps and genetic bottlenecks as only a fraction of wild captured individuals survive and reproduce in their new environment and the resulting newly colonized strain progressively adapts to the insectary rearing conditions. Therefore, notwithstanding the potential direct negative fitness effects of sterilization or transgenesis [
10,
11], the genetic changes associated with colonization have the potential to affect the competitiveness and fitness of a candidate release strain [
7,
8,
12]. As an example, the colonization of a wild population of
An. gambiae s.s. resulted in six-fold decrease in microsatellite allelic richness and two-fold decrease in heterozygosity over a period of two years [
13]. Similar patterns have been reported from comparisons of isozyme allelic richness in field population
versus laboratory strains of
Aedes aegypti and
Aedes formosus [
14]. Most of the strains used preferentially for genetic engineering of
An. gambiae have been bred in the laboratory for over 25 years (G3, KIL, etc.) [
15] and are most likely to be considerably inbred. Inbreeding is thought to negatively affect fitness by increasing the frequency of homozygotes at the expense of hererozygotes [
16]. Negative effects can occur either through the accumulation of deleterious recessive alleles leading to unfit homozygotes - the partial dominance hypothesis, or through the loss of favourable heterozygotes - the overdominance hypothesis [
17,
18].
The broad causal relationship between inbreeding, decreased phenotypic quality and fitness is well documented from animal breeding studies [
19]. In addition, the availability of neutral molecular markers in an increasingly large number of organisms has resulted in a recent flurry of heterozygosity-fitness correlation (HFC) studies reporting correlations between estimates of genetic diversity and fitness components in a variety of wild and captive populations [
20]. Currently, none of these studies focus on mosquitoes. However there are some reports of negative effects of inbreeding on the reproductive success of
An. gambiae laboratory populations (e g, [
21]). Moreover, the loss of viability associated with severe inbreeding in attempts to isolate morphological genetic mutants and isogenic lines in Aedine and Anopheline mosquitoes is well documented [
14,
22].
The expected negative effects of inbreeding on laboratory-reared mosquitoes have led to different schemes for reconstituting their genetic diversity prior to mosquito release programmes [
7]. These approaches require crossing and backcrossing laboratory strains with the progeny of field-collected individuals, and are thus not always practical to implement regularly and efficiently [
7]. Critically, these schemes ignore the independent contribution of selection for laboratory conditions, another genetic process that could impact the future mating competitiveness of released individuals. Consequently, such schemes can only be considered as hit-or-miss approaches. In addition, there is currently very little understanding of which reproductive traits are negatively impacted by colonization and of how these changes could potentially translate into decreased mating competitiveness in the field [
8,
23]. Without that knowledge it is virtually impossible to improve on current breeding schemes and laboratory-rearing practices [
7].
Here changes in sperm length, testes size and male accessory gland size of
An. gambiae occurring at different stages of the colonization process were investigated through comparisons of the progeny of field-collected individuals and different laboratory strains aged two to 35+ years. Sperm length has been shown to be very variable in laboratory strains of
An. gambiae [
24] and one study reported that longer sperm were more likely to be stored in the female spermathecae upon mating than shorter ones [
25]. There is also limited evidence that sperm length could correlate with male reproductive success in
An. gambiae [
26]. There are currently no studies focusing on variation in testes and male accessory glands size among laboratory or field anopheline populations. In anophelines, the size of both organs is known to increase with male mosquito age and culminate five to six days after emergence [
27‐
29]. Testes size is expected to correlate with the size of the sperm reservoir, and thus could potentially affect the total number of females that males can inseminate. In addition to transferring sperm, male mosquitoes deposit a mating plug in the female atrium during copulation. The mating plug is produced by the male accessory glands and, once deposited in the female, acts as a physical barrier that decreases the likelihood of females mating with other males [
21,
30]. These plugs also contain an array of sex-peptides that are responsible for inducing a cascade of behavioural changes in females [
30‐
32]. These changes include refractoriness to further mating [
30,
33,
34], host finding, feeding [
35], and the initiation of oogenesis [
36]. Changes in the size of male accessory glands could affect the size and/or number of plugs that males are able to transfer to females, and therefore determine the number of females they can inseminate.
In addition to comparing those reproductive characters in relation to the age of mosquito colonies, these traits were compared in a colony used to produce two genetically-modified (GM) strains. These strains had been genetically-modified using a two-phase transformation system [
37]. The procedure required for genetic transformation leads to two successive genetic bottlenecks that could potentially affect the reproductive phenotype of these and other GM strains created using similar approaches. Finally, we performed crosses between strains and the progeny of field-caught females to create genetically-refreshed outbred strains for comparison with non-refreshed ones. Crosses were also made between old strains to generate heterotic hybrid males. Both types of crosses enabled us to better compare the effects of inbreeding from the effects of selection on the male reproductive phenotype.
This study is the first to describe broad phenotypic changes affecting sperm length, and the size of testes and male accessory glands during the colonization process of laboratory strains of An. gambiae and to shed light on the underlying genetic processes leading to these changes. The results have important implications for ecological studies focusing on mosquito reproductive success in the laboratory, as well as for protocols of mass mosquito rearing that are critical to the success of malaria control strategies relying on mosquito releases.
Discussion
This is the first study to examine evolutionary changes in reproductive traits following colonization and adaptation to the laboratory environment in An. gambiae and to distinguish the effects of different evolutionary forces acting on its reproductive phenotype. Contrasting changes were observed in the length of sperm, and the size of testes and male accessory gland in relation to the age of mosquito colonies. Laboratory mosquitoes generally had increasingly larger testes but shorter sperm and smaller accessory glands than their wild-type counterparts. Sperm length decreased with time of colonization. Comparisons among genetically transformed, genetically refreshed, and in heterotic males supported the idea that this decrease in male sperm length was due to inbreeding. In contrast to that pattern, testes size was found to increase over time and was larger in the long-established KIL and KIS strains, suggesting progressive adaptation to laboratory conditions. Furthermore, testes size did not differ between the KIL strain and the derived transgenic EE and Evida3 lines, suggesting that this change was driven by laboratory selection rather than by the two sequential genetic bottlenecks associated with the two-phase genetic transformation system. In addition testes size was not recovered with strain refreshment or in heterotic males confirming that this change was driven by laboratory selection. Finally, the size of male accessory glands decreased over time following a trajectory opposite to that of testes, albeit at a much faster rate, suggesting that selection for laboratory conditions led to a quick decrease of this organ’s size. Here again, further comparisons of these organs in relation to genetic transformation suggested that accessory gland size did not change in relation to genetic bottlenecking. In addition, accessory gland size was not improved in refreshed strains and in heterotic males thereby supporting the idea that adaptation to laboratory conditions drove these size change rather than inbreeding.
These results are important because they are the first to clearly highlight significant morphological differences between laboratory strains and wild mosquitoes and therefore serve to emphasize the need to validate laboratory findings with semi-field or field studies particularly when focusing on mosquito mating ecology. Although the exact relationship between the size of these male traits and mating success and fecundity was not demonstrated here, there is evidence from previous studies suggesting that changes in sperm, testes and accessory glands may affect male fitness (see below). Thus these changes have the potential to affect their mating competitiveness in the context of sterile or GM mosquito releases. Furthermore, whilst the negative effects of inbreeding on sperm size was counteracted in males from refreshed strains and heterotic males, strain refreshment was not sufficient to restore the wild-type-like testes and accessory gland phenotype. This suggests that producing males with a mating phenotype comparable to that of wild males might require complex breeding and rearing scheme. The possibility of creating heterotic ‘supermales’ with enhanced mating performance from old inbred lines adapted to the laboratory is an exciting development that may be an effective way for producing large numbers of competitive males. This exciting discovery warrants further evaluation.
That sperm length progressively decreases with colonization time, hence inbreeding, suggests that it could be used as a practical biomarker for describing levels of inbreeding in mosquito colonies. This constitutes a substantial improvement over measures of inbreeding relying on molecular markers heterozygosity since correlations between heterozygosity at neutral markers and fitness are notoriously weak [
20,
52]. The unreliability of inbreeding estimates based on molecular markers is further compounded by their sensitivity to demographic events commonly affecting mosquito colonies, such as contaminations with other strains that can occur unbeknown to mosquito colony users. Thus sperm length comparisons between laboratory strains and between these strains and wild individuals from their population of origin provide a simpler way of comparing levels of inbreeding than the comparatively time consuming and expensive molecular approaches.
Currently the exact relationship between sperm length and male mosquito fitness is unknown and further studies are underway to establish that causal link. In anopheline mosquitoes, some studies suggest that larger sperm have a higher likelihood of fertilizing the eggs. A comparative study of
Anopheles quadriannulatus,
Anopheles darlingi and
An. gambiae s.s. revealed high degrees of sperm length polymorphism in males from these four taxa [
24]. It is noteworthy that for
An. gambiae the size reported in that study ranged from 0.026-0.100 mm which is smaller than the range of the most inbred KIL (0.054-0.372 mm) and Kisumu strains (0.020-0.321 mm) [
24]. Interestingly, the same study [
24] and a study of
An. arabiensis [
25] showed that sperm recovered in the female spermathecae were comparatively larger than those measured from testes suggesting that larger sperm have the highest likelihood of fertilizing the eggs than smaller ones [
24,
25]. In the outbred Keele strain, average sperm length was found to be ~0.199 mm (range 0.100-0.250 mm) and negatively genetically correlated with oviposition success [
26]. These results are not necessarily incompatible with the patterns of sperm length in relation to inbreeding reported here and would suggest that the Keele strain with its intermediate sperm length was indeed not strongly inbred at the time of that study [
26]. The same study showed that there was significant intra-specific variation in sperm length among males from the same
Anopheles species as shown here. The exact function of sperm polymorphism in
An. gambiae is currently still unknown and, despite the strong effect of inbreeding observed in this study, it is noteworthy that strains of increasing age retained comparable levels of sperm length variation despite a constant decrease in mean size and a shift towards higher proportions of small sperm. As outlined elsewhere [
26], sperm variation could simply be maintained because of natural variation in the size of female sperm storage organs. Comparative studies in anopheline species [
24] and stalk-eyed flies [
53] suggest that sperm length and the female spermatheca size broadly co-evolve. Within species, experiments in
Drosophila [
54] and dung beetles [
55] showed that the competitiveness of different-size sperm depended on the size of the female sperm storage organs. Taken together, these findings suggest that optimal sperm length could vary with spermatheca size, which strongly correlates with female body size [
56]. Because female size depends on the female larval growth conditions [
51] having polymorphic sperm might allow males to have higher reproductive success across a wide range of female body and spermatheca sizes.
The changes observed in testes and accessory gland size in relation to colonization time can be explained by the unique mating conditions associated with insectary rearing. In natural populations males await females in male-dominated swarms thereby creating conditions in which male competition for females is high and reproductive success may largely be driven by female choice [
57]. This type of conditions, which bear analogies with leks, typically leads to very skewed distributions of male reproductive success with males of higher phenotypic quality securing most copula [
57,
58]. The 50:50 sex ratio artificially created by combining distinct cohorts of freshly hatched female and male imagoes in small laboratory cages results in starkly different selection pressures on males. Anopheline males can typically inseminate up to five females per night [
59]. Given the large number of virgin females available to males in crowded cages, the best males cannot possibly secure all mating, hence there may be more mating opportunities for males of lesser phenotypic quality. Male reproductive success may then depend less on the male phenotypic quality and female choice than on the male capacity to inseminate as many females as possible in a short window of time. In other words, laboratory rearing leads to increased sperm competition and larger testes size may be strongly selected for as they enable more frequent mating. The relationship between testes size and sperm competition is well described across a large number of taxa including insects [
60‐
62]. Experimental evolution studies have also shown an increase in testes size in relation to increased sperm competition in
Drosophila [
63] and the bruchid beetle
Callosobruchus maculatus [
62].
Positive selection for testes size could result in negative selection on male accessory gland size if there is a negative genetic correlation between these two traits. Such a negative correlation could exist if, for example, there is a trade-off between sperm and sex-peptide production. However, the decrease in male accessory gland size over time appears to have been quicker than that of testes size. In anophelines the size of accessory gland is highly dependent on male mating status and decreases following mating [
28,
29,
64]. The mating plug produced by the accessory glands is thought to acts as a physical barrier to further mating [
21,
30]. It is noteworthy that
An. gambiae males are thought capable of inseminating up to five females per night but of producing only two full mating plugs [
64]. If cage rearing leads to scramble competition for females the importance of securing as many copula as possible might outweigh that of preventing females from further mating through the physical barrier of a full plug. Alternatively, female fecundity might not depend on a full mating plug under insectary conditions. Plugs contain sex-peptides that are responsible for inducing a number of behavioural changes [
30‐
32] such as refractoriness to further mating [
30,
33,
34], host finding and feeding [
35], and the initiation of oogenesis [
36]. They also contain the vitellogenic steroid hormone 20-hydroxyecdysone that may be an important determinant of female fecundity [
65]. Thus the adaptive reduction in plug size observed in colonized strains could be linked to one or several changes in the female traits that are mediated by plug composition. Detailed analyses of changes in plug composition following colonization would therefore be required in order to delineate which of these is driving the observed changes in accessory gland size.
This study found no correlation between the mean sperm length and the body size of males in any of the strains studied or across all strains. In a study on the Keele strain of
An. gambiae, the two traits were significantly correlated in some but not all datasets [
26]. Here, there was an overall significant linear relationship between body size and testes size across all strains but the relationship did not hold when correcting for strain effects. In contrast, accessory gland size strongly correlated with body size across and within strains.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
RB, NE, FT, MC, SFT planned the experiments. RB, NEE, DP, MT conducted the experiments. RB and FT analysed the data. RB and FT wrote the manuscript. All authors read and approved the final manuscript.