A reliable morphological method to assess the age of male Anopheles gambiae
© Huho et al;licensee BioMed Central Ltd. 2006
Received: 20 April 2006
Accepted: 27 July 2006
Published: 27 July 2006
Release of genetically-modified (GM) or sterile male mosquitoes for malaria control is hampered by inability to assess the age and mating history of free-living male Anopheles.
Age and mating-related changes in the reproductive system of male Anopheles gambiae were quantified and used to fit predictive statistical models. These models, based on numbers of spermatocysts, relative size of sperm reservoir and presence/absence of a clear area around the accessory gland, were evaluated using an independent sample of mosquitoes whose status was blinded during the experiment.
The number of spermatocysts in male testes decreased with age, and the relative size of their sperm reservoir increased. The presence of a clear area around accessory glands was also linked to age and mating status. A quantitative model was able to categorize males from the blind trial into age groups of young (≤ 4 days) and old (> 4 days) with an overall efficiency of 89%. Using the parameters of this model, a simple table was compiled that can be used to predict male age. In contrast, mating history could not be reliably assessed as virgins could not be distinguished from mated males.
Simple assessment of a few morphological traits which are easily collected in the field allows accurate age-grading of male An. gambiae. This simple, yet robust, model enables evaluation of demographic patterns and mortality in wild and released males in populations targeted by GM or sterile male-based control programmes.
Vector control is one of the few proven ways to reduce malaria transmission [1–7], but the effectiveness of this approach, however, is threatened by the emergence of resistance by mosquitoes to insecticides [8–10]. This phenomenon, combined with the increasing resistance of Plasmodium to chemotherapy [11–15], could substantially exacerbate disease prevalence, morbidity and mortality in Africa. To mitigate the consequences of resistance, new vector control interventions for reducing the malaria burden are urgently needed. One potential new tool is the genetic manipulation (GM) of Anopheles mosquito populations [16, 17], whereby genes that prevent mosquitoes from being infected by malaria are identified and introduced into wild vector populations . Ethical considerations dictate that only male mosquitoes should be used to carry these refractory genes into wild populations [19, 20], as releasing females could increase biting nuisance and transmission of other Anopheles transmitted pathogens, including malaria if the refractory genes are not 100% efficacious.
Given the reliance of GM malaria control strategies on male Anopheles, the need to understand the factors that regulate their reproductive fitness, including their mating competitiveness and survival, is considerable. At present, most knowledge of male Anopheles survival under natural conditions comes from mark-recapture studies [21–25]. Although useful, the dispersal and low recapture of males [23–25] makes it difficult to obtain robust survival estimates from this method. A simpler alternative would be to identify traits that could be used to age-grade and assess the mating status of males on first capture, a feat which is possible with their female counterparts [26, 27]. So far, there has been only two attempts to develop a morphological technique for identifying the age and mating history (mated or virgin) of male mosquitoes [28, 29]. These methods were developed for the Asian malaria vectors Anopheles culicifacies and Anopheles stephensi over twenty years ago, in pioneering work by Mahmood and Reisen [28, 29]. Although generally a successful technique for evaluating age in these species, the method is not widely applied and has never been evaluated for African malaria vectors in the Anopheles gambiae species complex.
Here for the first time, an adaptation of the age and mating status determination method developed for Asian Anopheles [28, 29] to the African malaria vector, An. gambiae s.s. is presented, and its precision in predicting age and mating status of males of unknown background evaluated. The study aimed to test whether male morphological features that are easily observable under field conditions could be used to give robust estimates of male age and mating history. If successful, this methodology could be used to provide baseline measures of male An. gambiae fitness in the wild, based on which the relative performance of GM and/or sterile laboratory-reared mosquitoes could be monitored and compared after release.
An. gambiae s.s. pupae were obtained from a colony maintained at the Ifakara Health Research & Development Centre (IHRDC). This colony was established in 1996 from individuals collected from Njage village in Kilombero District, Tanzania. In the insectary, pupae were collected and held individually in plastic tubes (4.9 × 2.9 cm) that were covered by netting. Pupae were left overnight for emergence, and the sex of the emerged adults identified visually the following day. Thereafter, adults of the same sex and age were pooled in groups of 50 and held in netted cages (20 × 20 × 15 cm). Mosquitoes were classified as being '0' days old on the day of their emergence. All females were at least two days old before being used in experiments described below. While in cages, mosquitoes were fed on a 10% glucose solution that was administered by placing a soaked cotton wool pad on top of the cage.
Age and mating status determination experiments
Virgin males isolated at emergence were left for periods of 1–20 days. On each day of age, the gonads of at least 10 males were dissected and observations made as described below (following Mahmood & Reisen ), in order to assess how the morphology of their reproductive organs changed through time. In order to test whether morphological traits could predict whether mating had occurred, and whether any observed relationships between male age and morphology were altered by mating, experiments were conducted to provide a sample of males who had copulated before observation. On each day of mating experiments, 50 male An. gambiae of the same age were placed in front of a window prior to dusk (age groups ranged from 1–18 days old). Activity inside the cages was observed to begin approximately 45 minutes before dusk. Once males began to swarm, 20 virgin females (2–4 days old) were introduced into the cage. Two observers monitored activity in the cage with the assistance of a red light. When pairs of mosquitoes in copula were observed, they were siphoned from the cage and transferred together into a separate holding cup. The following morning, both males and females captured in copula were dissected. First, the spermatheca of the female was dissected to confirm whether she had been inseminated . Then the male partner of the inseminated female was dissected, and the morphological features of their reproductive system compared to those of male virgins of the same age. A further sample of males caught in copula were left for a number of days after mating (2–5) before being dissected in order to estimate the duration of any observable morphological changes associated with mating. Males were not observed to mate on the day of their emergence, so these experiments were restricted to males that were 1 day post-emergence or older.
Male dissection and morphological examination
Development of the qualitative model
Specifications of the qualitative model for age-grading, describing the required combination of morphological traits for a male to be estimated as 'young' (≤ 4 days), or 'old' (> 4 days) under this model.
Number of spermatocysts
Percentage of testis occupied by the sperm reservoir
Clear area present
Predicted age (days)
3 – 5
10 – 50%
Yes or no
0 – 2
50 – 100%
Development of a predictive model and statistical analysis
General linear models were performed to investigate the relationship between the known age of males and: (1) the number of spermatocysts in individual testes, and (2) the proportion of the testes occupied by the sperm reservoir. Before analysis, data collected as proportions (% of testes occupied by the sperm reservoir) were logit transformed to improve their fit to a normal distribution. The additional explanatory variable of male mating status (virgin or mated the night before) was included in these models to examine whether it influenced any apparent relationship between male age and spermatocyst number, or sperm reservoir. The initial maximal statistical models for both spermatocyst number and proportional size of the sperm reservoir included the main effects of male age (days), mating status and their interaction (age × mating status). Additionally, logistic regression was used to investigate relationships between the presence of a clear area around the accessory glands and male age and mating history. The presence of a clear area was treated as a binary response variable ('0' if absent, '1' if present), with male age, mating status, and their interaction, being treated as independent explanatory variables. In all analyses, non-significant terms were sequentially eliminated to yield the minimum statistically significant model for each trait.
Description of model fitting procedures used in four quantitative statistical models of male An. gambiae age. In all cases, the outcome variable (y) was male age group, defined as 'young' (≤ 4 days), or 'old' (> 4 days). In each model, the relationship between three explanatory variables and the outcome variables of spermatocyst number (x1), proportional size of the sperm reservoir (x2), and the presence or absence of a clear area (x3) was tested. In all models, x3 was treated as a categorical variable, with the treatment of x1 and x2 varying between models as described below (a-d representing distinct categories).
Treatment of explanatory variables x1 & x2
No. of categories
Spermatocyst number (x1)
% Size of sperm reservoir (x2)
a = 0–2
b = 3–5
a = < 60%
b = ≥ 60%
a = 0
b = 1–3
c = 4
d = 5
a = ≤ 40%
b = 41 – 59%
c = 60 – 94%
d = ≥ 95%
a = 0
b = 1–3
c = 4
d = 5
a = ≤ 30%
b = 31 – 60%
c = 61 – 90%
d = ≥ 91%
Logistic regression was also applied to examine the association between mating status (treated as a dependent variable) and all three morphological traits. As with age, four different quantitative models were fit to explain variation in mating status (Table 2). Similar to age, all three morphological traits and their interaction were combined in a logistic regression model and used to predict the probability that a male was a virgin or had mated. This model predicted male mating status as a probability ranging between 0 and 1. Males whose mating status was predicted to be less than 0.5 were classified as virgins, and those assigned a value higher or equal to 0.5 were designated as having mated. Unless otherwise stated, error estimates accompanying means represent one standard error. All statistical analyses were performed using SAS version 8.2.
Validation of the statistical model for age and mating status determination
In a blind trial, laboratory-reared males whose age and mating status were known only by one insectary worker were given to another researcher to dissect. The observer recorded the morphological features of these unknown males, and entered them as independent variables into the age and mating status predictive models described above. The accuracy of predictions of both age and mating status were compared to the actual values (as revealed post hoc). The predictions of male age obtained from the statistical model were compared with those obtained from the qualitative 'rule of thumb' (Table 1).
Male reproductive morphology and age and mating status
Using morphology to predict age and mating status
Comparative success of four different statistical models aiming to predict male An. gambiae age group as a function of their reproductive morphology (Table 2). Overall success indicates the percent of a given data set (original or blind trial) whose age-grade was correctly predicted by the model; with age-specific success indicating the proportion of 'young' and 'old' males correctly classified within these groups.
Proportion of variation explained (original data)
Percent of data classified into the correct age groups (%)
Original Data Set
Blind Trial Data Set
(r 2 )
Comparative accuracy with which the qualitative (Table 1) and quantitative (Table 2, breakpoints) models predicted the age-grade of male An. gambiae examined in a blind trial.
Actual age group (days)
Proportion of correct classification
Comparative success of four different statistical models aiming to predict the mating status of male An. gambiae as a function of their reproductive morphology (as described in Table 2). Mating-specific success indicates the proportion of virgin (0) and once-mated males (1) that were correctly classified by each model.
Proportion of variation explained (original data)
Percent of data classified into the correct mating status (%)
Original Data Set
Blind Trial Data Set
(r 2 )
Mating-status specific success
Mating-status specific success
Predictions of male An. gambiae age-grade as a function of their morphology, as obtained from the optimally performing breakpoints model. Text indicates whether males of a particular trait combination would be classified as being ≤ 4 days old (YOUNG), or older (OLD). Numbers in parentheses give the probability of males with a particular trait combination being in the 'OLD' age group, with the model categorizing all males with a probability of lower < 0.5 as being 'YOUNG', and 0.5 ≥ as being 'OLD'. Following this classification guide, the age status of 'YOUNG' and 'OLD' male An. gambiae should be correctly predicted on 81% and 95% of occasions respectively.
Proportion of testis occupied by sperm reservoir
Age class as predicted by the optimal model (BREAKPOINTS)
Clear area absent
Clear area present
Mahmood and Reisen were able to use these same morphological features to age-grade male An culicifacies . Interestingly, the overall success rate of their morphological age-grading method when applied to An. stephensi was identical to that which has been obtained for An. gambiae (89% when applied to blind trial data in both cases, ). This similarity in accuracy of age prediction between this model and that of Mahmood and Reisen suggests these male mosquito reproductive traits are broadly indicative of age; both across Anopheline species and geographic locations. Investigation of the utility of these traits for age-grading other mosquito genera (e.g Aedes and Culicine) would be of great use to evaluate the overall generality of this approach.
In contrast to the success of the age-grading model, attempts to predict male An gambiae mating history based on these morphological traits failed. Both when applied to the original and blind trial data set, the statistical model, failed to identify virgins (Table 5). This bias was substantial, with 82–99% of virgins being incorrectly classified as mated. Although all three selected morphological traits changed with mating, the effect was relatively small compared to those caused by age (Figures 4, 5, 6). Thus without a priori knowledge of male age (precise to the day), it is unlikely that these morphological traits alone can infer whether a male An gambiae has mated or not. Thus additional morphological, physiological and/or behavioural traits that are more tightly linked to mating must be identified in order to reliably assess the mating history of male An gambiae from field samples.
Before testing quantitative statistical models for age and mating status determination, attempts to identify general 'rules of thumb' that could be followed to directly predict male age without statistical analysis were made. However, the statistical approach was substantially more robust than the qualitative 'rule-of-thumb' model discerned from early observation (89% vs. 60% overall success rate). Qualitatively, the only morphological trait linked to mating history was whether a male had a clear area around his accessory glands or not. It was noticed that male An. gambiae who had mated were more likely to have a clear halo around their accessory gland than those who had not, an observation also shared by Mahmood and Reisen that supports the conclusion that mating depletes accessory gland fluid . Dissection of a cohort of males suggested that although mating prompts the appearance of a clear area, this feature is lost within 3–4 days of mating. This observation is also consistent with Mahmood and Reisen's investigation of An stephensi and An culicifacies , and confirms the notion that this feature is a transient indicator of mating, and highly confounded by age. Thus, no simple means to assess male An. gambiae mating history on the basis of morphology equivalent to that which is available for females was found.
In developing these age-grading and mating status models, much was learned about the basic biology and development of the male An. gambiae reproductive system. Similar to Mahmood and Reisen [28, 29], it was established that the number of spermatocysts in male testes fell as males grew older, and that on average virgin males had slightly fewer spermatocysts than those allowed to mate once (Figure 4). As expected, the decrease in spermatocyst numbers was met by a corresponding increase in proportion of the testes occupied by the sperm reservoir (Figure 5). These changes reflect the rate of reproductive maturation of An. gambiae males as influenced by mating and age, with the sperm reservoir expanding as spermatocysts gradually break down and release spermatozoa into the anterior end of the testis. Working on An. culicifacies, Mahmood and Reisen  found that spermatozoa (and thus size of the sperm reservoir) and accessory gland substances were also depleted with mating. Here it was found that virgin An. gambiae expanded their sperm reservoir faster than mated males; suggesting that mating may deplete spermatozoa in this species also. The depletion of sperm through mating may prompt the testis to produce and mature their spermatocysts, a phenomenon that would explain why mated males of the same age had a slightly higher number of spermatocysts than virgins.
Based on these findings, it is concluded that this quantitative statistical model can serve as an excellent tool for field biologists to age-grade free-living male An. gambiae, and be used to characterize the average survival of males from different populations. Furthermore, this model is simpler, quicker and substantially less costly to apply than some of the intensive laboratory methods currently under development. However, caution should be taken when applying this model, as researchers must be aware that it identifies 'young' males (< 4 days) with slightly lower accuracy than those who are 'old' (> 4 days, 81% vs. 95% success respectively). Thus the model may modestly overestimate the proportion of the male population in the 'old' class; and thus the survival profile of a population. Attempts to distinguish between males who had mated and males who had not on the basis of morphology were largely unsuccessful. Hopefully, other morphological or physiological traits that are more reliably linked to mating history can be identified, and used to track the mating success of genetically modified or sterile males if released into the wild. Field studies are currently underway to evaluate the utility of this age-grading method during routine mosquito surveillance. If successful, it is hoped that vector biologists working on An. gambiae in the wild will adopt this method widely to increase the range of tools at their disposal for understanding and reducing the expansion of this deadly disease vector.
The authors are grateful to Japheth Kihonda, Hassani Ngonyani and Nicolas Kasigudi in the Public Health Entomology Unit at the IHRDC for their technical assistance in conducting these experiments. Additionally we thank the IHRDC, Wageningen University, Swiss Tropical Institute and the University of Dar es Salaam for institutional support. We thank Dr. Tom Smith for his advice on model interpretation. This work was financed by VIDI grant (no. 864.03.004) awarded by the Dutch Scientific Organization (NWO) to BGJ Knols, and a grant by the International Atomic Energy Agency to HMF (URT 13295).
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