Email updates

Keep up to date with the latest news and content from Malaria Journal and BioMed Central.

Open Access Research

Comparison of male reproductive success in malaria-refractory and susceptible strains of Anopheles gambiae

Maarten J Voordouw123*, Jacob C Koella2 and Hilary Hurd3

Author Affiliations

1 Department of Biology, University of Victoria, PO Box 3020, Station CSC, Victoria, BC, V8W 3N5, Canada

2 Division of Biology, Imperial College of London, Silwood Park Campus, Buckhurst Road, Ascot, Berkshire, SL5 7PY, UK

3 Centre for Applied Entomology and Parasitology, School of Life Sciences, Keele University, Staffordshire, ST5 5BG, UK

For all author emails, please log on.

Malaria Journal 2008, 7:103  doi:10.1186/1475-2875-7-103


The electronic version of this article is the complete one and can be found online at: http://www.malariajournal.com/content/7/1/103


Received:13 February 2008
Accepted:5 June 2008
Published:5 June 2008

© 2008 Voordouw et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

In female mosquitoes that transmit malaria, the benefits of being refractory to the Plasmodium parasite are balanced by the immunity costs in the absence of infection. Male mosquitoes, however, gain no advantage from being refractory to blood-transmitted parasites, so that any costs associated with an enhanced immune system in the males limit the evolution of female refractoriness and has practical implications for the release of transgenic males.

Methods

Aspects of the male cost of carrying Plasmodium-refractory genes were estimated by comparing the males' immune response and reproductive success among strains of Anopheles gambiae that had been selected for refractoriness or extreme susceptibility to the rodent malaria parasite, Plasmodium yoelii nigeriensis. The refractory males had a stronger melanization response than males from the susceptible line. Four traits were used as correlates of a male's reproductive success: the proportion of females that were inseminated by a fixed number of males in a cage within a fixed time frame, the proportion of females with motile sperm in their spermathecae, the proportion of ovipositing females, and the mean number of eggs per batch.

Results

Although there were significant differences among groups of males in sperm motility and oviposition success, these differences in male reproductive success were not associated with the refractory or susceptible male genotypes. Contrary to expectation, females mated to early emerging refractory males laid significantly more eggs per batch than females mated to later emerging susceptible males. Sperm motility and oviposition success were strongly correlated suggesting that variation in sperm motility influences female oviposition and ultimately male reproductive success.

Conclusion

An increased melanization response in male A. gambiae does not diminish male reproductive success under the experimental protocol used in this study. That refractory males induced ovipositing females to lay more eggs than susceptible males is an interesting result for any strategy considering the release of transgenic males. That sperm motility influences female oviposition is also important for the release of transgenic males.

Background

Parasitic infections exert strong selection on the immune systems of their hosts. However, evolution and maintenance of a host's immune system are thought to be costly via negative effects on other life history traits [1]. Examples of costs incurred by insects include larvae of Drosophila melanogaster selected for parasitoid resistance that are less competitive than unselected controls [2] and Aedes aegypti mosquitoes selected for early pupation that have a weaker immune response to foreign objects (Sephadex beads) than mosquitoes selected for late pupation [3]. Although immune costs have not always been attributed to precise resistance mechanisms and some mechanisms may not be costly, costs of immune responses that have been identified are a possible explanation for the maintenance of genetic variation in host refractoriness [1,4].

Species of Plasmodium that cause malaria in man and other vertebrate hosts impose fitness costs on their mosquito vectors because they reduce fecundity [5-7] and, in some cases, lifespan [8,9]. It is, therefore, expected that anti-Plasmodium defense mechanisms have evolved and indeed Plasmodium density is reduced by orders of magnitude in many vectors [10-12]. That laboratory colonies of mosquitoes respond readily to artificial selection for refractoriness to Plasmodium [13,14] demonstrates that there is genetic variation for this trait. This has been confirmed with recent observations of considerable genetic variation in refractoriness in natural populations [15-17]. Life history costs of refractoriness to Plasmodium have been measured in two experiments. Ae. aegypti mosquitoes selected for refractoriness to Plasmodium gallinaceum had shorter longevity, smaller body sizes and laid fewer eggs than the susceptible population [18]. Anopheles gambiae selected for refractoriness to Plasmodium yoelii nigeriensis produced fewer offspring when fed on an uninfected blood meal than susceptible mosquitoes after nine generations of selection. However, this difference was observed in only one of the three replicate selection experiments and the effect was not observed after 12 generations of selection [14]. Hence there is some evidence suggesting that in the absence of infection, increased immunity to Plasmodium may be costly for female mosquitoes.

While the costs of having refractory genes may be balanced by the benefits in the female, there is no such balancing selection in the male. Male mosquitoes do not blood feed and therefore never encounter malaria parasites. From the perspective of a male mosquito, genes that protect females from Plasmodium and all the other parasites (e.g. filarial worms) that come with her blood-feeding habit are not beneficial unless these genes protect males against other pathogens. Genes that have a selective advantage in one sex but not the other are called sexually antagonistic genes and have been demonstrated in Drosophila [19]. To date, no one has investigated whether male mosquitoes carrying anti-Plasmodium genes experience a fitness cost. Such costs would be important for the release of transgenic mosquitoes carrying anti-Plasmodium genes, which will most likely involve releasing males [20].

Plasmodium-refractoriness in A. gambiae is likely to be associated with more than one resistance mechanism, including parasite lysis [21] and melanization [13,14]. Two independent selection experiments found that refractory A. gambiae females evolved a mechanism where they deposit melanin on the surface of Plasmodium ookinetes [13,14]. These melanized ookinetes failed to develop to the oocyst stage. More recently it was found that melanization occurs after ookinetes were killed by another mechanism [22]. Regardless of whether it is a primary cytotoxic immune response to Plasmodium [23], the melanization response is an attractive candidate to test for sexually antagonistic immune genes. It can be assayed in both male and female mosquitoes by inoculating them with negatively charged CM C-25 Sephadex beads [24]. Anopheles gambiae males from a Plasmodium-refractory line were shown to have a stronger melanization response towards Sephadex beads than males from a susceptible line [24]. A selection experiment in Ae. aegypti found that evolving a stronger melanization response to Sephadex beads is costly with respect to development time [3]. Hence the melanization response is associated with Plasmodium-refractoriness in females, can be assayed in males and is costly, but does it influence male reproductive success in A. gambiae?

Like most anopheline mosquitoes, A. gambiae mates in male-biased swarms that vary in size from twenty to thousands of individuals [25,26]. In the field, females are rapidly mated and leave the swarm following insemination [27]. Polyandry or female multiple mating is therefore rare in the field [< 3%; reviewed in [28]] suggesting that post-copulatory sperm competition is not important [29]. Females store the sperm in their single spermatheca and are capable of laying up to nine batches of eggs in the laboratory with an average of ~100 eggs per batch [30]. In contrast, males return to the swarm after mating [31,32] and laboratory experiments have demonstrated considerable variation in male reproductive success [33,34]. Male reproductive success can be partitioned into its constituent components: insemination success, oviposition success, and hatching success. This partitioning is useful because different mechanisms influence the different components. For example, insemination success (measured as the proportion of females with sperm in their spermatheca) increases with the size of the swarm [35], the number of nights the sexes are kept together [36,37], the male to female sex ratio [36,37], male body size [32,38], and male age [37,39]. Oviposition success (measured as the proportion of ovipositing females) depends upon the transfer of male accessory gland fluids (MAGS) [[40,41]; but see, [42]] and on nervous signals in the female indicating that her spermathecae is filled with sperm [42,43]. In Anopheles stephensi and Ae. aegypti, hatching success (measured as the proportion of eggs that hatch) decreases over successive batches suggesting either depletion or death of viable sperm in the female's spermatheca [30,44]. Hatching success in Plasmodium-refractory lines of A. gambiae was lower than that in susceptible lines whereas there was no difference in insemination success between the two lines [14]. This observation suggests that Plasmodium-refractory mechanisms may interact in different ways with the different components of male reproductive success.

A range of laboratory mating conditions was recently determined for A. gambiae (minimal swarm size of 10 males, 24 hour mating period, 2:1 sex ratio) that enabled the detection of genetic differences in male reproductive success among families of full-sib males [45]. The success of this study motivated the experimental design in the present study. To test for a cost of refractoriness in males, male reproductive success was compared between lines of A. gambiae that were refractory or susceptible to the rodent malaria parasite, P. y. nigeriensis. Two matched pairs of refractory and susceptible lines of A. gambiae were used: Keele black refractory (BR) versus Keele black susceptible (BS) and Keele red refractory (RR) versus Keele red susceptible (RS). These lines had originally been selected from the same outbred Keele strain [14]. It was recently confirmed that BR and RR are still refractory and that BS and RS are still susceptible to P. y. nigeriensis, three years after the original selection regime. To test whether artificial selection for Plasmodium-refractoriness in females resulted in correlated evolution of the immune response in males, the melanization response was assayed in males from the BR, BS, RR and RS lines. For each of these four lines, male reproductive success was assayed by mating males to females from the Keele population. Male reproductive success was measured in four different ways: the proportion of inseminated females, the proportion of females with motile sperm in their spermathecae 14 days after mating, the proportion of ovipositing females, and the mean number of eggs per batch.

There were significant differences in sperm motility and oviposition success among the 16 groups of males but not between refractory and susceptible males. Females mated to black refractory males laid significantly more eggs per batch than those mated to black susceptible males. In this study, a quarter of all surviving females (57/230) that had taken two blood meals and carried sperm in their spermatheca did not oviposit. These females were almost four times less likely to carry motile sperm in their spermathecae than females that had laid eggs at least once. This suggests that sperm motility influences female oviposition and ultimately male reproductive success.

Methods

General methods

A. gambiae Keele, refractory (R) and highly susceptible (S) strains of mosquitoes were kept in insectaries maintained at a temperature of 27°C, relative humidity of ~70% and a 12:12 light:dark cycle. Adult mosquitoes were kept in 20 cm cubic mesh cages and were fed ad libitum on a solution containing 10% glucose, 0.05% para-aminobenzoic acid (PABA), 0.28% streptomycin/penicillin (Sigma-Aldrich, Poole, UK) and distilled water. Larvae were reared in batches of 50 in small plastic containers (10 × 7 × 5 cm3) containing 200 ml of distilled water. Larvae were fed 0.03, 0.04, 0.08, 0.16, 0.32 mg of ground Tetramin™ per individual on days 1, 2, 3, 4, 5 and 0.60 mg every day thereafter. For the male reproductive success assays, adults were mated in 30 cm cubic cages made of wood and plastic mesh (hereafter referred to as mating cages).

Establishment of 16 sub-lines to separate parental and genetic effects

For each of the four lines – black refractory (BR), black susceptible (BS), red refractory (RR) and red susceptible (RS) – four sub-lines were established as follows (Figure 1). Eight batches of 50 larvae were reared for each of the four lines. Each batch of 50 larvae had been obtained from a single P. y. nigeriensis-infected female for which the number of oocysts that developed following an infective feed was known [see methods in [14]]. Susceptible females contained hundreds of oocysts whereas refractory females contained none. At pupation, ~50 pupae from each batch were evenly distributed among the four sub-lines for a total of ~100 pupae per sub-line. Hence, within a line, the genetic composition of the four sub-lines was the same. For each of the 16 sub-lines, the pupae were placed in mesh cages (20 cm cubic) and the cages were placed in an alternating sequence (BR, BS, RR, RS, etc) on a single shelf in the insectaria. The purpose of establishing the sub-lines was to separate genetic effects from the parental environment.

thumbnailFigure 1. Experimental design. For each of the four lines of Anopheles gambiae – black refractory (BR), black susceptible (BS), red refractory (RR) and red susceptible (RS) – four sub-lines were established with the same genetic background (only shown for the BR line) by dividing ~400 pupae from 8 first generation (G1) females among four replicate cages (A, B, C and D). The adults in the second generation (G2) produced the males (sires) used in the assay of male reproductive success. The G2 adults from the Keele population produced the females (dams) used in the assay of male reproductive success. All third generation (G3) larvae were reared individually in the wells of 24-well tissue culture plates. In the third generation (G3), a cage of 28 males was obtained for each of the 16 sub-lines (only shown for cage A of the BR line). Each cage of males was mated at 3 and 5 days of age to two different groups of 15 virgin Keele females (aged 1 and 3 days, respectively). Males and females were allowed to mate for 20 hours for each mating day. The females were blood fed two days post-mating (pm). Ten blood fed females were haphazardly selected and allocated to individual oviposition cups 3 days pm. All females were given a second blood meal (8 days pm), were monitored for oviposition (3 – 13 days pm) and were dissected for their spermathecae (14 days pm).

Obtaining refractory and susceptible males and Keele females

For the assay of male reproductive success males from each of the 16 sub-lines were mated to females from the Keele population. To manage the workload, the mothers of the BR and BS males were blood fed one week before the mothers of the RR and RS males (thereby creating a black and red block). Each sub-line was allowed to blood feed for 10 minutes on the forearms of MJV. Three days later, mothers laid eggs in communal oviposition cups and the larvae hatched the following day. For each sub-line, 96 larvae were reared individually in the wells of 24-well tissue culture plates to minimize variation in adult emergence and body size. Larvae were reared on the standard Tetramin™ diet (see General Methods). Due to a dilution error in preparing the Tetramin™ solution, larvae in the black block obtained half the daily food rations on days 5 to 7 as the larvae in the red block. Two days after blood feeding the mothers of the males, the mothers of the females from the Keele population were blood fed. For each block, 768 Keele larvae were reared in the same way as the males.

Mating assay

For each sub-line, 28 males were mated twice, at 3 and 5 days of age, to different groups of 15 Keele females, aged 1 and 3 days, respectively. The purpose of mating the males to two different groups of females was to determine the repeatability of the mating assay. For each sub-line, the pupae were sexed and ~35 male pupae (range = 31 to 43) were transferred to a separate mating cage over a two-day period. The mating cages (i.e. sub-lines) were scanned every 12 hours to estimate the mean male age at emergence. Two days later the Keele pupae were sexed. For each sub-line, two groups of 15 female pupae were transferred to two separate 700 ml polystyrene cups. All the Keele females emerged on the same night. For each block, the 16 groups of virgin females were randomly assigned to one of the 16 combinations of the 8 mating cages and the 2 mating days. Before adding the females, the number of males in each mating cage was standardized to 28. The first group of females (1 day old) was added to the mating cages at 19:00 and was removed the following day at 15:00. This process was repeated two days later with the second group of females (3 days old). Hence each cage of males had 20 hours to inseminate both groups of females with one day of rest between mating days. All the males were frozen after removing the second group of females. For each of the 16 male cages, the mean wing length was estimated from a sample of 10 males. Wing length was measured as the distance between the allula and the distal fringe using a compound microscope (50× magnification) and an ocular micrometer.

Oviposition success and sperm motility phenotype

For each sub-line, the two groups of females were kept in separate 700 ml Polystyrene cups. Two nights after the mating assay (i.e. when the first and second group of females were 3 and 5 days old, respectively), the females were blood fed on the arms of MJV for 10 minutes. Females in the red block were starved for 12 hours before taking their first blood meal, but females in the black block were not. The day after the blood meal, ten blood-fed females were haphazardly selected from each cup and were transferred to individual oviposition cups. For the next five days, the oviposition cups were checked every day to record whether the female had laid eggs or not. For each female, the number of laid eggs was counted. Each of the 320 females was given a second opportunity to blood feed five days after their first blood meal. Females were not starved prior to the second blood meal. Each female was fed for 10 minutes on the left or right forearm of MJV and was subsequently transferred to a fresh oviposition cup. After monitoring oviposition for another five days, all females were checked for insemination 14 days after mating. Females were sacrificed by placing them in 70% ethanol for 20 seconds. Females were dissected for their spermathecae in phosphate-buffered solution (PBS). The spermathecae was placed on a slide in 15 μl of PBS and was gently cracked open with fine needles before covering it with an 18 mm cover glass. The spermatheca was immediately checked for the presence of sperm using a light microscope (100× magnification). For inseminated spermathecae, each sperm bundle was observed for 30 seconds to check for the presence of motile sperm. A sperm bundle was defined as motile if at least one motile sperm was observed; it was defined as non-motile if no motile sperm were observed.

Male melanization phenotype

To test whether refractory males had a more efficient immune response than susceptible males, the melanization phenotype was assayed in male mosquitoes from the BR, BS, RR, and RS lines. For each of the four lines, 200 larvae were reared (see general methods), the pupae were sexed, and the males were blocked by their age of emergence (9, 10 and 11 days after hatching). Each male mosquito was inoculated with one CM C-25 Sephadex bead 24 (± 6) hours after emergence. The beads were hydrated in PBS containing 0.001% methyl green to facilitate visibility. The males were anaesthetized by chilling them on ice for 2 minutes. A single bead and ~0.1 μl of PBS solution was inoculated into the thoracic cavity of the male with a micro-capillary tube pulled into a very fine tip (φ = 40 μm). Inoculated males were placed into 50 ml Falcon tubes that were laid on their sides and that contained moist filter paper and a sugar food source. Males that emerged on days 9, 10 and 11 were assessed for their condition (dead, capable of walking, capable of flying) and then immediately frozen 48, 24 and 12 hours after inoculation, respectively. The following day the thoraxes were dissected in 0.01% methyl green PBS, the bead was searched for up to 10 minutes, and the % melanin cover was estimated for the beads that were found.

Statistical methods

Male reproductive success

There are three binomial measures of male reproductive success: (1) insemination success = the proportion of inseminated females, (2) sperm motility = the proportion of females that had motile sperm in their spermathecae 14 days after mating, and (3) oviposition success = the proportion of females that laid at least one clutch of eggs. There is one normally distributed measure of male reproductive success, the mean number of eggs per batch. Batch refers to the event where a female lays eggs during the five days following a blood meal. The mean number of eggs per batch therefore excludes those events where females did not lay eggs following a blood meal. A generalized linear model (GLM) with a binomial error function is an efficient way to model proportion data. Hence the glm() function in R was used to model the three binomial measures of male reproductive success. For the mean number of eggs per batch, the linear model function in R was used.

Mean age of emergence and wing length for males

For the 16 male cages, the mean age at emergence and the mean wing length were normally distributed. These two male traits were modeled as a two-way ANOVA with block (black vs. red), male genotype (refractory vs. susceptible) and their interaction.

Covariance in male reproductive success between mating days and effects of male/female age

The 16 cages of males were mated to two different groups of virgin females. Positive covariance between groups of females mated to the same male cage is expected if some cages of males are consistently better at mating than others. For the proportion data, GLM was used to test for male cage effects. For the mean number of eggs per batch, ANOVA was used to test for male cage effects. Separate analyses were conducted for the black and red blocks to ensure that differences between blocks were not causing the male cage effects. The analyses also tested for male/female age effects. Males were mated at 3 and 5 days of age to females, aged 1 and 3 days, respectively. Therefore, the effects of male and female age cannot be separated.

Male reproductive success in refractory vs. susceptible genotypes

For each of the four measures of male reproductive success, the data from the two groups of females were aggregated thereby obtaining a single mean for each of the 16 male cages. These means were approximately normally distributed. Male reproductive success was modeled as a two-way ANOVA with block, male genotype, and their interaction. A retrospective power analysis was conducted to determine this study's ability to detect significant differences in oviposition success between the refractory and susceptible males [see Additional file 1].

Additional file 1. Power analysis. Post-hoc power analysis to determine the level of replication necessary to detect significant differences in oviposition success between refractory and susceptible males.

Format: DOC Size: 54KB Download file

This file can be viewed with: Microsoft Word ViewerOpen Data

Correlations between the six male traits

The correlations between the six male traits were tested using Pearson's correlation. To control for block effects, the male traits were standardized to z-scores (mean = 0, standard deviation = 1) within each block.

Male melanization phenotype

Three-way ANOVA was used to model % melanin cover as a function of line (black vs. red), male genotype (refractory vs. susceptible), and time to melanize a bead (12, 24, 48 hours).

Results

Summary of results

Of the 320 females that were assigned to individual oviposition cups, 305 survived to the end of the experiment of which 63% (192/305) laid eggs at least once. Of the 275 females that survived and took two blood meals, females that laid a first clutch were eleven times more likely to lay a second clutch (0.93 = 156/167) than females that did not (0.08 = 9/108; χ2 = 194.28, df = 1, p < 0.0001). Spermathecae were scored for the presence of motile or non-motile sperm for 303 females of which 84% were inseminated (254/303) and 49% (148/303) had at least one motile sperm 14 days after mating.

Mean age of emergence and wing length for males

The males in the black block emerged 4.1% later than those in the red block (Table 1). The mean wing length of the males in the black block was 6.8% smaller than that of the red block (Table 1). These significant differences in male emergence and male wing length between blocks (Table 2) were most likely caused by the fact that the larvae in the black block received half as much food on days 5 to 7 as those in the red block. The effect of male genotype on the mean age of male emergence depended on block (Table 2). Black refractory males emerged 3.2% earlier than black susceptible males (Table 1) and this difference was statistically significant. Red refractory males emerged 2.1% later than the red susceptible males (Table 1), but this difference was not significant. There was no significant effect of male genotype or the male genotype*block interaction on mean male wing length (Table 2).

Table 1. Mean male traits for the refractory and susceptible genotypes

Table 2. Male reproductive success in refractory versus susceptible genotypes

Covariance in male reproductive success between mating days and effects of male/female age

The proportion of inseminated females did not differ among male cages in either the black or the red block (Table 3; Figure 2). In both the black and red block, there was a significant effect of male cage on sperm motility and on oviposition success (Table 3; Figure 2). There was no significant effect of male cage on the mean number of eggs per batch (Figure 2) in either the black (F6, 6 = 3.40, p = 0.081) or red block (F7, 7 = 0.39, p = 0.88). The three binomial measures of male reproductive success increased with male/female age: insemination success (0.77 vs. 0.90; Table 3), sperm motility (0.40 vs. 0.57; Table 3), oviposition success (0.50 vs. 0.75; Table 3), but the mean number of eggs per batch (99 vs. 92 eggs; F1,14 = 2.29, p = 0.066) did not.

Table 3. Covariance in male reproductive success between mating days

thumbnailFigure 2. The correlation in male reproductive success between two days of mating in Anopheles gambiae. The correlation between the two groups of females, aged 1 and 3 days, mated to the same cage of males at 3 and 5 days, respectively for four male fitness traits (a) the proportion of inseminated females (r = 0.33, df = 14, p = 0.206), (b) the proportion of females with motile sperm in their spermathecae 14 days after mating (r = 0.58, df = 14, p = 0.019), (c) the proportion of females that oviposited at least once (r = 0.74, df = 14, p = 0.001), and (d) the mean number of eggs per batch (r = 0.47, df = 13, p = 0.078). Females that were mated to BR, BS, RR and RS males are represented by filled circles, filled triangles, open circles and open triangles, respectively. Shown are the lines of best fit from the linear regressions.

Male reproductive success in refractory vs. susceptible genotypes

There was no difference between refractory and susceptible males in insemination, sperm motility or oviposition success (Table 2; Figure 3). There was no effect of block on insemination success or sperm motility (Table 2). Oviposition success in the red block was 1.6 times higher than that in the black block (Table 1). This significant difference in oviposition between blocks (Table 2) was most likely caused by the fact that females mated to red males were starved for 12 hours prior to their first blood meal whereas females mated to black males were not. The red females were therefore more motivated to blood feed than the black females. The results did not change when the mean male age of emergence and mean male wing length were included as covariates.

thumbnailFigure 3. Reproductive success of Anopheles gambiae males from Plasmodium-refractory and Plasmodium-susceptible lines. The effect of male genotype (Plasmodium-refractory vs. Plasmodium-susceptible) and block (black vs. red) on (a) the proportion of inseminated females, (b) the proportion of females with motile sperm in their spermathecae 14 days after mating, (c) the proportion of females that oviposited at least once, and (d) the mean number of eggs per batch. Shown are the medians (bold line), the 25th and 75th percentile (edges of the box) and the minimum and maximum values (whiskers). Each box plot is based on 4 cages.

Females mated to red males laid 14.3% more eggs per batch than females mated to black males (Table 1; Figure 3). This significant difference in eggs per batch between blocks (Table 2) was most likely caused by the differences in blood feeding mentioned above. The effect of male genotype on the mean number of eggs laid per batch depended on block (Table 2). In the black block, the mean number of eggs per batch of the females mated to the refractory males was 17.5% higher than that of the susceptible males (Table 1; Figure 3) and this difference was statistically significant. In the red block there was no difference in the mean number of eggs per batch between females mated to refractory and susceptible males (Table 1; Figure 3). When the mean male age of emergence was included as a covariate, the male genotype*block interaction term and the main effect of male genotype were no longer significant.

Correlations between the six male traits

After correcting for block effects, insemination success was not correlated with either sperm motility or oviposition success (Table 4). In contrast, sperm motility and oviposition success were significantly correlated (Table 4). There was also a significant correlation between mean male wing length and the mean number of eggs per batch (Table 4).

Table 4. The correlation matrix for the six male traits

Male melanization phenotype

For 116 inoculated males, beads were recovered from 109 individuals, of which 81 were able to fly. Across the black and red lines and the three melanization times, the mean % melanin cover in the refractory males (94 ± 2.3%, n = 54) was significantly higher than that in the susceptible males (84 ± 3.4%, n = 55, F1,105 = 6.70, p = 0.011; Figure 4). There was also a significant effect of melanization time; males given 12, 24 and 48 hours to melanize a bead covered 79 ± 4.5% (n = 41), 92 ± 2.9% (n = 32), and 97 ± 1.9% (n = 36) of the bead, respectively (F2, 105 = 12.73, p < 0.001; Figure 4), but there was no difference between the red and black blocks (F1,105 = 0.05, p = 0.82). Excluding males unable to fly following inoculation did not affect the results.

thumbnailFigure 4. The effect of Plasmodium-susceptibility and refractoriness on melanization response of Anopheles gambiae males. The % melanin cover of a bead increases with the time since the bead was inoculated and the melanization response is more efficient for refractory than susceptible males. Shown are the means and standard errors.

Discussion

No support was found for the main hypothesis of this study, namely that males from populations that are refractory to Plasmodium parasites bear constitutive immunity costs and therefore have lower reproductive success than males from susceptible populations. There was no difference between refractory and susceptible males in insemination success, sperm motility, and oviposition success (Table 2, Figure 3). In the black block, contrary to expectation, females mated to refractory males laid significantly more eggs per batch than females mated to susceptible males (Figure 3). This effect of male genotype on the mean number of eggs per batch could not be separated from the earlier emergence of the black refractory males (Table 1). However, the mean male age of emergence was not correlated with the mean number of eggs per batch across the 16 male cages (Table 4). This observation suggests that the difference in the mean number of eggs per batch between the refractory and susceptible males in the black block was due to their genotype rather than to differences in the mean male age of emergence, which were small (3.2%; Table 1). Regardless of causation, females mated to early emerging refractory males laid significantly more eggs than females mated to later emerging susceptible males.

One critique of this study is that the refractory and susceptible males were not put in direct competition with each other. Such an experiment would ideally require the use of genetic markers that are currently not available for these refractory and susceptible strains. Hence the possibility that refractory costs occur when the two male genotypes compete for a common pool of females cannot be excluded. One of the strengths of this study is that independent replicate cages were established for the generation prior to the male fitness assay. Hence parental effects [recently demonstrated in A. stephensi by [46]] were not confounded with the male genotype. Another strength was that, by allowing the males to mate on two separate days, it was shown that this study was sensitive enough to detect significant differences in reproductive success among males. Because the number of males and females in each cage and the amount of time that the sexes were allowed to mate were controlled, these differences in reproductive success among males probably reflect variation in the larval environment, genetics and parental effects. When this variance among males was included into the power analysis [see Additional file 1], it was found that the experiment's power to detect small differences in the proportion of ovipositing females between refractory and susceptible males was low and that replication would have to be quadrupled to be able to detect moderate differences. Previous studies in A. gambiae [14] and Ae. aegypti [18] detected some female fecundity costs of being refractory to Plasmodium, suggesting an explanation for the maintenance of genetic variation for this trait in the field. The present study suggests that refractory genes are neutral with respect to male fitness in a laboratory setting and that males are unlikely to affect the evolution of Plasmodium-refractoriness in female mosquitoes. However, biotic factors and environmental stresses encountered in the field could affect male fitness in ways that might alter these conclusions.

Although a cost of refractoriness in males was not detected, refractory males had a more efficient melanization response than susceptible ones. Paskewitz and Riehle [24] also showed that the refractory strain of Collins et al [13] had a much stronger melanization response to C-25 Sephadex beads than the susceptible one and that the difference between strains was more pronounced for males than females. Using a time course experiment they showed that the % melanization cover stabilized after 24 hours, which was not the case for our males (Figure 4). In this experiment, variation in the age of emergence (9, 10, 11 days) was confounded with the time given to melanize a bead (48, 24 and 12 hours, respectively), hence the possibility that developmental rates influenced the melanization response, as shown in Ae. aegypti [3], cannot be excluded. The important conclusion, however, is that selection of Plasmodium-refractoriness or -susceptibility in female mosquitoes resulted in a correlated response in the male immune system.

Most studies on anopheline male reproductive success measure insemination success (i.e. the proportion of inseminated females) and assume that this trait is a good estimate of male reproductive success [34,37,39]. This study emphasizes the importance of partitioning male reproductive success into its constituent components such as insemination success, oviposition success, and the number of eggs per batch [e.g. [45]]. Female oviposition is generally conditional on the female having been inseminated. However, the present study shows that there is some independence between these two components. Twenty-five percent of inseminated, blood-fed females in this study did not oviposit. Males differed in their sperm motility (Figure 2b) and oviposition success (Figure 2c) despite having similar insemination success (Figure 2a). Sperm motility and female oviposition were significantly correlated (Table 4). These two observations suggest that the quality or quantity of the male ejaculate influenced female oviposition. Using the G3 strain of A. gambia, it was previously shown that males vary genetically in their ability to induce females to oviposit [45]. The present study suggests a mechanistic link between sperm motility and oviposition.

At this point, the factors that influence sperm motility are not clear. Sperm motility decreases with the time taken to dissect a spermatheca (Voordouw, unpublished data). It appears to be robust to the duration of the observation interval and little additional information is gained from observing the sperm bundle for more than 30 seconds (Voordouw, unpublished data). It is possible that other methodological factors such as the composition of the PBS affect sperm motility [47]. However such methodological factors cannot account for the correlation between sperm motility and female oviposition (Table 4). Perhaps the sperm motility phenotype measured in this study captures some measure of sperm quality such as sperm viability or sperm mobility that have been shown to influence male reproductive success in other systems [48]. Alternatively, sperm motility may represent some measure of sperm quantity if the probability of detecting at least one motile depends on the amount of sperm initially transferred. In the fly, Scathophaga stercoraria, the viability of sperm in the female's spermathecae decreases rapidly after mating and was shown to vary significantly among males [49]. In S. stercoraria, males have heritable variation in sperm length [50] leading Bernasconi et al [49] to speculate that sperm morphology influences variation in sperm longevity. Klowden and Chambers [51] recently reported that A. gambiae produces polymorphic sperm and that the female reproductive tract was more likely to contain longer sperm. Long sperm bias in the female spermatheca has also been observed in Drosophila pseudoobscura where males produce equal amounts of short and long sperm but only the latter fertilizes the eggs [52]. Future research will focus on how sperm length and sperm motility influence male reproductive success in A. gambiae.

A previous laboratory study has shown that large A. gambiae males are more likely to acquire mates than small males [38]. In Anopheles freeborni, examination of the accessory glands and testes in field-captured males, suggests that large individuals are more likely to mate than small ones [32]. The mechanism that gives larger anopheline males a mating advantage is not known. Large males may be better at displacing small males from their mates (if such take-overs occur in anopheline mating swarms) or may be better at catching and subduing females. Alternatively, large males may have bigger energy budgets that allow them to swarm for longer periods of time as documented in swarming chironomid midges [53]. In this study, male wing length was not correlated with male insemination or male oviposition success (Table 4). However, male wing length was significantly correlated with the mean number of eggs per batch (Table 4); females mated to larger males laid more eggs per batch. A previous laboratory study has shown that A. gambiae males prefer to mate with large females that produce larger batches of eggs [35]. Size assortative mating may explain why the mean number of eggs per batch was correlated with male wing length while oviposition success was not.

The three binomial measures of male reproductive success increased over the two days of mating. On the first day of mating, the males were 3-days old-virgins and the females were 1-day-old virgins. On the second day of mating, the males were 5-days-old with one day of mating experience and the females were 3-days-old virgins. Previous work on other strains of A. gambiae found that the proportion of inseminated females peaks at seven days of age for both males and females [37], whereas in another study, the proportion of ovipositing females was not affected by the age of the female but was higher for 2-days-old males than 6-days-old males [54]. This study cannot separate male and female age effects or male experience, but this does not influence the comparison of the refractory and susceptible lines as the two mating days were combined.

As mentioned previously, there were significant differences among male cages in oviposition success. These results are similar to a study on the G3 strain of A. gambiae that found significant differences among families of full-sib males in oviposition success [45]. In both studies, there was considerable variation in male reproductive success. In the black block of this study for example, oviposition success of the most successful swarm was three times higher than that of the least successful swarm (81% vs. 26%). In the red block, there was a two-fold difference between the most and least successful swarm (100% vs. 53%). If male oviposition success contains a genetic component [e.g. [45]] there is considerable scope for the evolution of male reproductive success. Given plans for the eventual release of transgenic male mosquitoes, identifying and manipulating the genetic mechanisms underlying this variation in male reproductive success may considerably improve their performance in the field.

Conclusion

In conclusion, Plasmodium-refractory males had a more efficient immune response than their susceptible counterparts, but this did not translate into immune-related fitness costs in the laboratory when the immune system was un-challenged. Black refractory males induced ovipositing females to lay more eggs per batch than black susceptible males. Hence the present study suggests that males do not restrict the evolution of Plasmodium refractoriness in female mosquitoes in the absence of agents that infect males. Male reproductive success was repeatable across two days of mating with respect to oviposition success and sperm motility. These two measures of male fitness were strongly correlated suggesting a mechanistic link. Future research will be directed towards characterizing what male factors influence sperm motility and oviposition success and whether these can be manipulated to improve the reproductive success of transgenic males in the field.

Authors' contributions

MJV conceived the idea, ran the experiment and analyzed the data. MJV, JCK and HH designed the experiment, interpreted the data and wrote the paper.

Acknowledgements

This study was supported by a Natural Sciences and Engineering Research Council (NSERC) of Canada postdoctoral fellowship to MJV (November 2005 – November 2007). Thanks to Ann Underhill and Dr. Pam Taylor for assistance with experiments. Also thanks to Simon Fellous and three anonymous referees whose comments improved the manuscript.

References

  1. Sheldon BC, Verhulst S: Ecological immunology: costly parasite defences and trade-offs in evolutionary ecology.

    Trends Ecol Evol 1996, 11:317-321. Publisher Full Text OpenURL

  2. Kraaijeveld AR, Godfray HCJ: Trade-off between parasitoid resistance and larval competitive ability in Drosophila melanogaster.

    Nature 1997, 389:278-280. PubMed Abstract | Publisher Full Text OpenURL

  3. Koella JC, Boete C: A genetic correlation between age at pupation and melanization immune response of the yellow fever mosquito Aedes aegypti.

    Evolution 2002, 56:1074-1079. PubMed Abstract OpenURL

  4. Stearns SC: The evolution of life-histories. Oxford: Oxford University Press; 1992.

  5. Hogg JC, Cawardine S, Hurd H: The effect of Plasmodium yoelii nigeriensis infection on ovarian protein accumulation by Anopheles stephensi.

    Parasitol Res 1997, 83:374-379. PubMed Abstract | Publisher Full Text OpenURL

  6. Hogg JC, Hurd H: Malaria-induced reduction of fecundity during the first gonotrophic cycle of Anopheles stephensi mosquitoes.

    Med Vet Entomol 1995, 9:176-180. PubMed Abstract | Publisher Full Text OpenURL

  7. Hogg JC, Hurd H: Plasmodium yoelii nigeriensis : The effect of high and low intensity of infection upon the egg production and bloodmeal size of Anopheles stephensi during three gonotrophic cycles.

    Parasitology 1995, 111:555-562. PubMed Abstract OpenURL

  8. Anderson RA, Knols BGJ, Koella JC: Plasmodium falciparum sporozoites increase feeding-associated mortality of their mosquito hosts Anopheles gambiae s.l.

    Parasitology 2000, 120:329-333. PubMed Abstract | Publisher Full Text OpenURL

  9. Ferguson HM, Read AF: Why is the effect of malaria parasites on mosquito survival still unresolved?

    Trends Parasitol 2002, 18:256-261. PubMed Abstract | Publisher Full Text OpenURL

  10. Alavi Y, Arai M, Mendoza J, Tufet-Bayona M, Sinha R, Fowler K, Billker O, Franke-Fayard B, Janse CJ, Waters A, Sinden RE: The dynamics of interactions between Plasmodium and the mosquito: a study of the infectivity of Plasmodium berghei and Plasmodium gallinaceum, and their transmission by Anopheles stephensi, Anopheles gambiae and Aedes aegypti.

    Int J Parasitol 2003, 33:933-943. PubMed Abstract | Publisher Full Text OpenURL

  11. Sinden RE, Alavi Y, Raine JD: Mosquito-malaria interactions: a reappraisal of the concepts of susceptibility and refractoriness.

    Insect Biochem Mol Biol 2004, 34:625-629. PubMed Abstract | Publisher Full Text OpenURL

  12. Vaughan JA, Hensley L, Beier JC: Sporogonic development of Plasmodium yoelii in five anopheline species.

    J Parasitol 1994, 80:674-681. PubMed Abstract | Publisher Full Text OpenURL

  13. Collins FH, Sakai RK, Vernick KD, Paskewitz S, Seeley DC, Miller LH, Collins WE, Campbell CC, Gwadz RW: Genetic Selection of a Plasmodium-Refractory Strain of the Malaria Vector Anopheles gambiae.

    Science 1986, 234:607-610. PubMed Abstract | Publisher Full Text OpenURL

  14. Hurd H, Taylor PJ, Adams D, Underhill A, Eggleston P: Evaluating the costs of mosquito resistance to malaria parasites.

    Evolution 2005, 59:2560-2572. PubMed Abstract | PubMed Central Full Text OpenURL

  15. Niare O, Markianos K, Volz J, Oduol F, Toure A, Bagayoko M, Sangare D, Traore SF, Wang J, Blass C, Dolo G, Bouare M, Kafatos FC, Kruglyak L, Toure YT, Vernick KD: Genetic loci affecting resistance to human malaria parasites in a West African mosquito vector population.

    Science 2002, 298:213-216. PubMed Abstract | Publisher Full Text OpenURL

  16. Riehle MM, Markianos K, Lambrechts L, Xia A, Sharakhov I, Koella JC, Vernick KD: A major genetic locus controlling natural Plasmodium falciparum infection is shared by East and West African Anopheles gambiae.

    Malaria Journal 2007, 6:87. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  17. Riehle MM, Markianos K, Niare O, Xu J, Li J, Toure AM, Podiougou B, Oduol F, Diawara S, Diallo M, Coulibaly B, Ouatara A, Kruglyak L, Traore SF, Vernick KD: Natural malaria infection in Anopheles gambiae is regulated by a single genomic control region.

    Science 2006, 312:577-579. PubMed Abstract | Publisher Full Text OpenURL

  18. Yan G, Severson DW, Christensen BM: Costs and benefits of mosquito refractoriness to malaria parasites: implications for genetic variability of mosquitoes and genetic control of malaria.

    Evolution 1997, 51:441-450. Publisher Full Text OpenURL

  19. Rice WR: Sexually antagonistic genes: experimental evidence.

    Science 1992, 256:1436-1439. PubMed Abstract | Publisher Full Text OpenURL

  20. Alphey L, Beard CB, Billingsley P, Coetzee M, Crisanti A, Curtis C, Eggleston P, Godfray C, Hemingway J, Jacobs-Lorena M, James AA, Kafatos FC, Mukwaya LG, Paton M, Powell JR, Schneider W, Scott TW, Sina B, Sinden R, Sinkins S, Spielman A, Toure A, Collins FH: Malaria control with genetically manipulated insect vectors.

    Science 2002, 298:119-121. PubMed Abstract | Publisher Full Text OpenURL

  21. Vernick KD, Fujioka H, Seeley DC, Tandler B, Aikawa M, Miller LH: Plasmodium gallinaceum : a refractory mechanism of ookinete killing in the mosquito, Anopheles gambiae.

    Exp Parasitol 1995, 80:583-595. PubMed Abstract | Publisher Full Text OpenURL

  22. Blandin S, Shiao SH, Moita LF, Janse CJ, Waters AP, Kafatos FC, Levashina EA: Complement-like protein TEP1 is a determinant of vectorial capacity in the malaria vector Anopheles gambiae.

    Cell 2004, 116:661-670. PubMed Abstract | Publisher Full Text OpenURL

  23. Christensen BM, Li J, Chen CC, Nappi AJ: Melanization immune responses in mosquito vectors.

    Trends Parasitol 2005, 21:192-199. PubMed Abstract | Publisher Full Text OpenURL

  24. Paskewitz S, Riehle M: Response of Plasmodium refractory and susceptible strains of Anopheles gambiae to innoculated Sephadex beads.

    Dev Comp Immunol 1994, 18:369-375. PubMed Abstract | Publisher Full Text OpenURL

  25. Charlwood JD, Pinto J, Sousa CA, Madsen H, Ferreira C, do Rosario VE: The swarming and mating behaviour of Anopheles gambiae s.s. (Diptera : Culicidae) from Sao Tome Island.

    J Vector Ecol 2002, 27:178-183. PubMed Abstract OpenURL

  26. Charlwood JD, Thompson R, Madsen H: Observations on the swarming and mating behaviour of Anopheles funestus from southern Mozambique.

    Malaria Journal 2003., 2 PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  27. Yuval B: Mating systems of blood-feeding flies.

    Annu Rev Entomol 2006, 51:413-440. PubMed Abstract | Publisher Full Text OpenURL

  28. Tripet F, Toure YT, Dolo G, Lanzaro GC: Frequency of multiple inseminations in field-collected Anopheles gambiae females revealed by DNA analysis of transferred sperm.

    Am J Trop Med Hyg 2003, 68:1-5. PubMed Abstract | Publisher Full Text OpenURL

  29. Parker GA: Sperm competition and its evolutionary consequences in the insects.

    Biol Rev Camb Philos Soc 1970, 45:525-567. Publisher Full Text OpenURL

  30. Suleman M: Intraspecific variation in the reproductive capacity of Anopheles stephensi (Diptera, Culicidae).

    J Med Entomol 1990, 27:819-828. PubMed Abstract OpenURL

  31. Yuval B, Bouskila A: Temporal dynamics of mating and predation in mosquito swarms.

    Oecologia 1993, 95:65-69. OpenURL

  32. Yuval B, Wekesa JW, Washino RK: Effect of body size on swarming behavior and mating success of male Anopheles freeborni (Diptera: Culicidae).

    J Insect Physiol 1993, 6:333-342. OpenURL

  33. Bock ME, Reisen WK, Milby MM: Lifetime mating pattern of laboratory-adapted Culex tarsalis males.

    Mosq News 1983, 43:350-354. OpenURL

  34. Mahmood F, Reisen WK: Anopheles stephensi (Diptera: Culicidae): changes in male mating competence and reproductive system morphology associated with aging and mating.

    J Med Entomol 1982, 19:573-588. PubMed Abstract OpenURL

  35. Okanda FM, Dao A, Njiru BN, Arija J, Akelo HA, Toure Y, Odulaja A, Beier JC, Githure JI, Yan G, Gouagna LC, Knols BGJ, Killeen GF: Behavioural determinants of gene flow in malaria vector populations: Anopheles gambiae males select large females as mates.

    Malaria Journal 2002, 1:1-7. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  36. Charlwood JD, Jones MDR: Mating behaviour in the mosquito, Anopheles gambiae s.l. I. Close range and contact behaviour.

    Physiol Entomol 1979, 4:111-120. Publisher Full Text OpenURL

  37. Verhoek BA, Takken W: Age effects on the Insemination rate of Anopheles gambiae sl in the laboratory.

    Entomol Exp Appl 1994, 72:167-172. Publisher Full Text OpenURL

  38. Ng'habi KR, John B, Nkwengulila G, Knols BGJ, Killeen GF, Ferguson HM: Effect of larval crowding on mating competitiveness of Anopheles gambiae mosquitoes.

    Malaria Journal 2005, 4:1-9. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  39. Mahmood F, Reisen WK: Anopheles culicifacies : effects of age on the male reproductive system and mating ability of virgin adult mosquitoes.

    Med Vet Entomol 1994, 8:31-37. PubMed Abstract | Publisher Full Text OpenURL

  40. Bryan JH: Results of consecutive matings of female Anopheles gambiae species B with fertile and sterile males.

    Nature 1968, 218:489. PubMed Abstract | Publisher Full Text OpenURL

  41. Bryan JH: Further studies on consecutive matings in the Anopheles gambiae complex.

    Nature 1972, 239:519-520. PubMed Abstract | Publisher Full Text OpenURL

  42. Klowden MJ: Sexual receptivity in Anopheles gambiae mosquitoes: absence of control by male accessory gland substances.

    J Insect Physiol 2001, 47:661-666. PubMed Abstract | Publisher Full Text OpenURL

  43. Klowden MJ: Switchover to the mated state by spermathecal activation in female Anopheles gambiae mosquitoes.

    J Insect Physiol 2006, 52:679-684. PubMed Abstract | Publisher Full Text OpenURL

  44. Marina CF, Ibarra JE, Arredondo-Jimenez JI, Fernandez-Salas I, Liedo P, Williams T: Adverse effects of covert iridovirus infection on life history and demographic parameters of Aedes aegypti.

    Entomol Exp Appl 2003, 106:53-61. Publisher Full Text OpenURL

  45. Voordouw MJ, Koella JC: Genetic variation of male reproductive success in a laboratory population of Anopheles gambiae.

    Malaria Journal 2007, 6:1-11. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  46. Grech K, Maung LA, Read AF: The effect of parental rearing conditions on offspring life history in Anopheles stephensi.

    Malaria Journal 2007, 6:1-9. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  47. Werner M, Simmons LW: Insect sperm motility.

    Biol Rev (Camb) 2008, 83:191-208. PubMed Abstract | Publisher Full Text OpenURL

  48. Snook RR: Sperm in competition: not playing by the numbers.

    Trends Ecol Evol 2004, 20:46-53. PubMed Abstract | Publisher Full Text OpenURL

  49. Bernasconi G, Hellriegel B, Heyland A, Ward PI: Sperm survival in the female reproductive tract in the fly Scathophaga stercoraria (L.).

    J Insect Physiol 2002, 48:197-203. PubMed Abstract | Publisher Full Text OpenURL

  50. Ward PI: Sperm length is heritable and sex-linked in the yellow dung fly (Scathophaga stercoraria).

    Journal of Zoology 2000, 251:349-353. Publisher Full Text OpenURL

  51. Klowden MJ, Chambers GM: Production of polymorphic sperm by anopheline mosquitoes and their fate within the female genital tract.

    J Insect Physiol 2004, 50:1163-1170. PubMed Abstract | Publisher Full Text OpenURL

  52. Snook RR, Markow TA, Karr TL: Functional nonequivalence of sperm in Drosophila pseudoobscura.

    Proc Natl Acad Sci USA 1994, 91:11222-11226. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  53. Neems RM, McLachlan AJ, Chambers R: Body size and lifetime mating success of male midges (Diptera: Chironomidae).

    Anim Behav 1990, 40:648-652. Publisher Full Text OpenURL

  54. Chambers GM, Klowden MJ: Age of Anopheles gambiae Giles male mosquitoes at time of mating influences female oviposition.

    J Vector Ecol 2001, 26:196-201. PubMed Abstract OpenURL