Vector Biology JournalISSN: 2473-4810

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Research Article, Vector Biol J Vol: 2 Issue: 1

The Effects of Age at First Mating and Release Ratios on the Mating Competitiveness of Gamma Sterilised Aedes albopictus Males under Semi Field Conditions

David Damiens1,2*, Pauline Olga Tjeck3, Cyrille Lebon1,2, Gilbert Le Goff1,2 and Louis Clement Gouagna1,2
1Institut de Recherche pour le Développement (IRD), UMR IRD 224-CNRS 5290-Université de Montpellier: Maladies Infectieuses et Vecteurs – Ecologie-Génétique, Evolution et Contrôle (MIVEGEC), Montpellier, France
2IRD Réunion / GIP CYROI (Recherche Santé Bio-innovation), Sainte Clotilde, Reunion Island- France
3Laboratoire Biodiversité et Ecosystèmes Tropicaux, Faculté des sciences, Université de La Réunion, Saint-Denis - Reunion Island, France
Corresponding author : David Damiens
IRD Réunion / GIP CYROI, 2 Rue Maxime Rivière, Sainte-Clotilde, 97491Sainte Clotilde, Reunion Island-France
Tel: + 02 62 93 88 13
E-mail: david.dam[email protected]
Received: March 23, 2016 Accepted: April 13, 2016 Published: April 20, 2016
Citation: Damiens D, Tjeck PO, Lebon C, Le Goff, Gouagna LC (2016) The Effects of Age at First Mating and Release Ratios on the Mating Competitiveness of Gamma-Sterilised Aedes albopictus Males under Semi Field Conditions. Vector Biol J 1:1. doi:10.4172/2473-4810.1000107




The success of the Sterile Insect Technique for areawide vector control relies crucially on the ability of sterile males to outcompete their wild counterparts for mating with wild females. Competitiveness tests are commonly performed for assessing quality sterile males intended for field release programs, but there are few reports on the optimal age and ratio for sterile Aedes albopictus male release in a SIT program.


Here, a series of mating competitiveness experiments with different age (1, 3, and 5 d post-emergence) and ratios (1:1, 5:1, and 10:1) of sterile to wild males were carried out in a walk-in field cage under semi-field conditions to examine variations in the mating competitiveness of irradiated adult Ae. albopictus males.


Pupae from laboratory-reared Ae. albopictus were irradiated at 35 Gy and introduced in field cages at different ages post emergence and at different ratios with a cohort of wild males and females. The mating competitiveness of sterile versus fertile males, and sterility induction into wild females measured through the egg hatch were observed.


The age of sterile males Ae. albopictus significantly affected their competitiveness when confronted with wild males of the same age. Competitiveness of sterile males was significantly lower after emergence (1-day old) than when they were older (3-5 days old). Presumably, 3-days old sterile males may be considered as the optimal age for sterile male releases. At this optimal age, induced sterility rates increased as the proportion of irradiated males relative to the wild male increased.


Our study points to the fact that sugar feeding prior to field release, could be administered for 3 days post-emergence in order to enhance the performance of sterile males. Consistent with early investigations under competitive situations differing in sterile male-to-wild male ratio, the level of induced sterility in the wild females increased as the proportion of irradiated males relative to the wild male increased.

Keywords: Chikungunya; Dengue; Sterile insect technique; SIT; Irradiation


Chikungunya; Dengue; Sterile insect technique; SIT; Irradiation


Aedes albopictus (Skuse) (Diptera: Culicidae), a proven competent vector for several arboviruses including Dengue and Chikungunya viruses, has expanded its distribution area worldwide and now exceeds the limits of its natural tropical range. This Aedes mosquito (Stegomyia) species is considered as the most abundant mosquito species in the Islands of the South west Indian Ocean. It is an introduced invasive species [1] that has almost replaced the previously established Ae. aegypti L. in natural or urban habitats in Reunion Island [2]. Aedes albopictus has been incriminated as the main vector in the recent Chikungunya epidemic outbreaks that occurred in the islands of the Southwest Indian Ocean from 2005 to 2007 [3-5].
Beside vector surveillance, current strategy for controlling Ae. albopictus in Reunion Island involves the classical approach, which strictly enforces breeding site elimination through community participation, and the use of insecticide treatments in targeted areas by vector control authorities. During the dengue (in 2004) and chikungunya epidemics (in 2005/2006), both larvicidal (including temephos and fenithrothion), and adulticidal treatment (e.g. permethrin), were used from 2004 until the beginning of 2006 and then replaced respectively by Bti (Bacillus thuringiensis var. israelensis toxins) and deltamethrin until 2007 [6]. But as in other countries, there are concerns over the use of broad-spectrum insecticides, including the potential selection for insecticide resistance that could affect their efficacy [7-9], in addition to the risk of environmental contamination with possible consequences on non-target organisms [10-12]. Due to the limitations of the current control strategies, there is a renewed interest in alternative or complementary solutions for an efficient and eco-friendly integrated control of Ae. albopictus. Following the Chikungunya outbreak in Reunion Island, considerable attention has been given to the possibility of its prevention by the Sterile Insect Technique (SIT). The SIT is based on inundative and repeated releases of sterile males that induce sterility in the wild population, and consequently suppress the target pest species [13,14], while reducing the use of insecticides. Suitable applications of SIT rely on the use of competitive sterile males that are able to locate and compete for mates in the wild and to transfer their sterile sperm. The fitness of sterile males is thus a key target for SIT programs [15,16] but this is a complex process reflecting the diverse physiological demands of the mass reared males.
To reach the high numbers of sterile males needed for release, technical and material conditions for mass rearing are required [17-21]. Every step of mass production, from colonization to release through the irradiation, has the potential to modify the biology of the sterile insects as well as their ability to survive in the field, and compete with wild males for mating with wild females [22-25]. In Ae. albopictus, colonization increases inbreeding depression [26] and sterilization by irradiation may strongly impact male’s fitness traits such as longevity, flight performance or mating behaviour proportionate to irradiation dose [27]. To prevent such damages while maintaining the competitive mating ability of irradiated versus wild-type males, an established dose of 35 Gy has been chosen to sterilize Ae. albopictus males [28,29].
Various tests have been developed to estimate the life history traits of produced sterile males that influence their efficiency in the field [24]. One of the most classically used is the competitiveness tests. For Ae. albopictus, such tests have been performed in small lab cages or in semi field cages [29-31]. Bellini et al. [31] and Madakacherry et al. [30] showed that irradiated Ae. albopictus males were as competitive as wild males, while "Oliva", et al. [29] showed significant decrease of competitiveness in the presence of wild counterparts. One of the major differences between those experiments was the age of males at the time of release with females. In studies reported by Bellini et al. [31] and Madakacherry et al. [30] the males were 3 days old as opposed to 1 day and 5 days old for males used in another related study by Oliva et al. [29]. In insects, male mating ability, quantity and quality of sperm produced vary with age [32-35]. The first purpose of the study reported herein was to determine the optimal age of sterile males intended for release in an SIT program in Reunion Island. Variation in mating competitiveness of sterile Ae. albopictus males aged 1 day, 3 days and 5 days old post-emergence, when confronted with wild males of the same age was examined.
Classically, releases of sterile males should usually be performed with ‘‘over flooding’’ ratios in order to compensate the reduced competitiveness of irradiated males or to increase the efficiency of the SIT technique by increasing the chances of insemination of wild females by sterile males [36-38]. Previous results from laboratory studies on irradiated male Ae. albopictus released at a ratio of 1:1:5 (wild females: wild males : sterile males), have shown a reduction of 50% of the fertility in wild females [29], while "Madakacherry", et al. [30] observed 80% of reduction. Following the first set of experiments which was designed to determine the optimal age for release, another study aimed to separately evaluate the effect of three treatments differing in sterile male-to-wild male ratio (i.e. 1:1, 5:1 and 10:1) on the female fertility under semi field conditions. This experiment was done for confirmatory purpose and to provide direct information that can serve as a guide in the determination of optimum release strategy in future field testing in Reunion Island where the SIT is currently being developed as a component of area-wide campaigns to suppress Ae. albopictus.

Material and Method

Production and irradiation of Ae albopictus males
The Ae albopictus strain used in the present study was collected as eggs in January 2009 at Saint Benoit (La Réunion). The colony is routinely maintained in a climate-controlled insectary (T: 27 ± 2°C, RH: 75 ± 2%, light: 12L: 12D). The females are routinely fed with 2.5 ml of sheep blood through a Parafilm® membrane on the Hemotek® system [39] (Discovery Workshops, Accrington, Lancashire, United Kingdom). Eggs from the two generations F25 and F26 were used for the tests. Egg hatching took place in water with dehydrated rabbit food (hay pellet, Compagnie des Grains du Capricorne, Le Port, Reunion Island) left overnight in the rearing water during 24h. Larvae were collected and reared at a density of approximately 500 larvae (L1) per tray (30×40 cm) containing 1 litre of water. They were fed with dry pellets composed of 50% of rabbit-food and 50% of fish-food (Sera Koi Food, Sera, Heinsberg, Germany). When pupae appeared, they were collected for the experiment purpose. Virgin mosquitoes were obtained by separating females and males as pupae based the genitalia morphology as observed under optical microscope (Leica MZ6). After sex separation, female pupae and a portion of the male pupae were allowed to emerge into separate adult cages without further treatment (fertile group). To produce sterile males, separate batches of pupae (aged between 24 and 30 h) from the same larval cohort were randomly retrieved and sterilized with 35 Gy of ionizing radiation (2.35 Gy min-1 for 15 min) from a Cs-137 source (Gammacell IBL 437, Cis Bio International, Germany) at the Blood bank located at the Bellepierre hospital, St Denis de La Réunion. While the dose required to induce 100% sterility in Ae. albopictus males is about 60 Gy [28], other evaluations of the dose-sterility response in Ae albopictus males indicated that the 35 Gy sterilizing dose used in our study represents a better trade-off between high sterility (approx. 95-97%), and fitness [30,31,40]. At emergence, random batches of 100 sterile males each were maintained in separate cages with continuous access to a 10% (w/v) sucrose solution for 1, 3, and 5 days following emergence to allow ageing, the day of emergence being considered as day 0 of an experimental trial. During the ageing process neither sugar solution nor water was limiting in any treatment.
Production of wild males and females
In addition to the sterile males produced from the laboratory stock, wild (non-sterile) males and females used in the competitiveness test were derived from eggs collected from different field sites (Sainte- Suzanne, Bras-Panon, Saint-Benoît, Sainte-Clotilde CIRAD campus) with oviposition traps (ovitraps). Ovitrap consisted of a 1 liter black plastic jar filled with 650 ml of water collected from a river were placed at ground level in a shaded location. The oviposition substrate was made of a brown germination paper (ovistrip) clipped on a 15 x 10 cm stiff PVC sheet, which was placed inside the ovitrap pot. After 2-3 days, the ovistrips were collected and brought to the laboratory, where each was rinsed with tap water and placed in the dark inside plastic tray covered by a Plexiglas for two days allowing gentle drying and egg maturation. From the third day, the Plexiglas cover was removed to allow faster drying and on day 5, the dried egg paper was placed for 72h in 250 ml of tap water (taken one day earlier) with powdered Rabbit food. Larvae from the hatched eggs were then reared as described above and pupae were collected, sexed by their genitalia and the males and females were placed in separate cages awaiting the experiments. Unless otherwise specified, wild males and females that had emerged from the same field cohorts were used in each semifield experiment. They were maintained in separate laboratory cages (30×30×30 cm3) with continuous access to a 10 % (w/v) sucrose solution for 1, 3 and 5 days as described below.
Effect of sterile male age on competitiveness in semi-field cages
Mating experiments were conducted under semi-field conditions set at the CIRAD (Centre International de Recherche Agronomique pour le Development) campus in Sainte-Clotilde (20°90’55’’S, 55°49’78’’ E), La Réunion. The experiment took place 2 months in March and April 2013. Five identical walk-in field cages (180×150×150 cm3) were used at the same time so all treatments could be done simultaneous for each replicate. In the competitiveness tests, the release ratio was, in each case, 100:100:100 (numbers of sterile males: wild males: wild females). In addition, control cages have been set up in following ratios: 0:100:100 (fertile control) and 100:0:100 (sterile control). Three age categories have been tested: 1 day-old, 3 days-old and 5 days-old post-emergence. For each selected age at release, three replicates were performed with different batches of virgin mosquitoes. Sterile and wild males were kept with females in the cages for 6 consecutive days from the time of release. Resting places for mosquitoes were provided in the form of a wooden table on top of which a 10% (w/v) sucrose solution was placed for the duration of the experiment. This experimental setup was exposed to ambient environmental conditions at 28ºC ± 5ºC, 80-90% RH. Caged rabbit was provided for female blood-feeding during the first 3 days, after which, the rabbit was removed and three oviposition cups were placed for 3 days consecutively. Subsequently, eggs laid on paper were collected and counted, while adult mosquitoes were collected with a vacuum aspirator and killed by etherisation. Egg counting was performed manually when less than 600 eggs were present on the paper, and for greater number of eggs, counts were done automatically using scanning and counting image J [41]. All eggs were allowed to mature for 2 days under laboratory conditions and then placed in a tray containing tap water with small amount of food (rabbit food) to allow hatching and number of L1 were counted the 3 days following the egg paper immersion.
Effect of different ratios of sterile to wild males on population sterilization process under semi-field conditions
The effect of three different ratios of wild to sterile males on the female fertility was evaluated under the same semi-field conditions as described above. At the start of each experiment, a fixed “optimal” age of males was chosen according to the results obtained in the experiment described above. Different ratios of sterile and wild males of the same age were released into 5 semi-field cages where they were allowed to compete for mating with virgin females of the same age for six consecutive days. Three ratios (sterile males number: wild males number: wild females number) have been tested simultaneously in different field-cages: 100:100:100, 500:100:100 and 1000:100:100. In addition, two other ratios of 0:100:100 (fertile control) and 100:0:100 (sterile control) were included in each experimental trial. Three replicate trials were performed for each release ratio, usually at weekly intervals, using random batched of newly emerged mosquitoes. The five field cages used were randomly assigned to different ratios and the position of each of the sterile/wild male ratio was changed after each replicate trial.
Parameters recorded and statistical analysis
For each treatment and control group in each experiment, the average number of eggs laid per female was established. Subsequently, the observed egg hatching rate was calculated by dividing the number of 1st instars larvae (corresponding to number of hatched eggs) by the total number of eggs laid. The Fried competitiveness index, ‘C’ [42] was calculated using hatch rates from the fertile control (Hn), sterile control (Hs) and the treatment groups (Ho) as follows: where N is the number of ‘normal’ males (wild), S is the number of sterile males.
The analysis of variance (ANOVA) with Tukey’s honestly significant difference post hoc tests and Student’s t-test were used to test differences in hatching rates and competitiveness indices between experimental groups and among replicates. Prior to statistical analyses observed values of egg hatchability were arcsin transformed to increase their fit to the normal distribution. All statistical analyses were carried out using Microsoft Excel 2007 for Windows and Minitab version 2000.
Ethical considerations
The permission for the use of rabbits under semi-field conditions was obtained from the institutional ethical committee after demonstrating that all measures were taken to avoid unnecessary suffering and severe damage to the animals. Animal care was in accordance with the principles and guidelines of the French Ministry of Environment. The corresponding protocol was approved by the Institutional Ethical committed for Animal Care and Experimental Use, GIP CYROI, Sainte Clotilde, Reunion – France.


Effect of sterile male age on competiveness in semi-field cages
The average numbers of egg produced by females after mating are presented in Figure 1. Regardless of the age at release, there was no evidence of significantly statistical difference in the average numbers of egg produced by females between the treatments and the controls (F4,10=2.94, P=0.07, Figure 1). The mean egg hatching rates significantly differed between treatments and controls (F4,10=63.5, P<0.001). However, due to a relatively high variability of the observed hatch rates in the 5 days old treatment, there was no statistical evidence of significantly variation between eggs hatching rates according to the age of males. Overall, there was no significant effect of age of male on the competitiveness index (CI=0.24 ± 0.08, 0.51 ± 0.11 and 0.56 ± 0.39 for sterile males aged 1, 3 and 5 days old, respectively). The lack of age dependent regulation of sterile mating competitiveness probably occurred as a result of the high variability in the results obtained in the 5 days old treatment. If analyzed without 5days old results, the competitiveness index of 1 day old sterile males and 3 days old are significantly different (T test, t=11.56, df=5, P=0.027).
Figure 1: Variation in the mean of egg numbers and hatch rates (± SE) for egg batches laid by females in field cages containing either irradiated Ae. albopictus males only (Control sterile), wild males only (Control fertile), and an even ratio of sterile to wild males of three different ages classes (1, 3 and 5 days old). The number of mosquitoes used for each experiment (sterile males: wild males: wild females) was 100:100:100 in the treatment, 0:100:100 in the control fertile group, and 100:0:100 in the control sterile one × 3 replicates. Different letters indicate significant difference in Tukey’s posthoc tests following ANOVA test for eggs hatch rate.
Effect of different ratios of sterile to wild males on population sterilisation process under semi-field conditions
Although there was an apparent variability in the number of eggs produced among treatment groups and controls, the differences in the average number of eggs recorded were unrelated to variations in the sterile: wild male ratio (F4,10=2.03, P=0.16, Figure 2). The mean egg hatching rates were significantly different according to the treatment (F4,10=160.17, P<0.001) and compared to controls (Figure 2). As expected, there was generally a marked decline in the observed egg hatching rates between the 1:1:1 and 5:1:1 and 10:1:1 ratios’, confirming that the level of sterility induced among females is influenced by the ratio of sterile males. Surprisingly, the treatment ratio of 1000:100:100 showed high variability in the egg hatching rate, precluding significant differences with the ratio of 500:100:100, but results obtained in these treatment were significantly different from the two controls (fertile and sterile). In parallel, full induced sterility was not attained during the experiments, even with the highest sterile to wild-type ratio 10:1, though the latter ratio yielded significantly higher level of sterility compared to the sterile control group. The induced sterility rates were 33.6 ± 5.2, 49.7 ± 1.6 and 61.7 ± 11.0% for the ratio of 100:100:100, ratio 500:100:100, ratio 1000:100:100 respectively. Competitiveness index significantly varied according to the release ratio (F2,6=15.15, P<0.005), with 0.51 ± 0.11, 0.20 ± 0.02 and 0.18 ± 0.09 recorded for the ratio of 100:100:100, 500:100:100, 1000:100:100, respectively.
Figure 2: Variation in the mean of egg numbers and hatch rates (± SE) for egg batches laid by females in field cages containing either three different ratio of sterile to wild Ae. albopictus males (sterile males number: wild males number: wild females number) 100:100:100, 500:100:100 and 1000:100:100 , irradiated males only (ratio 0:100:100, Control sterile) and wild males only (ratio 100:0:100, Control fertile). Different letters indicate significant difference in Tukey’s posthoc tests following ANOVA test for eggs hatch rate.


Under semi-field experimental conditions in Reunion Island, gamma irradiated Ae. albopictus males exhibited a CI value that varies according to the age, ranging from 0.24 for 1 day old males to 0.51 and 0.56 for 3 days old and 5 days old, respectively (where a value of 1.0 suggests equal competitiveness). Mating competitiveness was significantly lower when sterile males were confronted with wild males soon after emergence (1-day old) than when they were 3 days old, though holding sterile males under laboratory conditions for 5 days prior to confrontation with wild males did not allow improving the CI as compared with other age classes. This suggests that 3 days old sterile males could be the most competitive compared to the other younger and older ones. These results were consistent with the increase of competitiveness observed by "Oliva", et al. [40] between one day old and five days old males released in semi field cages with wild males. Presumably, such an increase in CI with age could be linked to the variation of mating activity intensity during adult life in most mosquitoes. The peak of mating activity of Anopheles stephensi Liston [43] and An culicifacies [44] takes place between the third and the seventh days. In Anopheles gambiae and Anopheles arabiensis, mating ability of seven-day-old males is higher than the one of males younger than three days old [45]. However, the observed change in competitiveness value with age may not be due to an increase of intrinsic male mating ability, but instead associated with the sugar meals supplied for 2-3 days following emergence, leading to an increase in fitness of sterile males [40]. Indeed, the provision of energetic substances to adults prior to release could offset the reduction of competitiveness caused by radiation and mass rearing process, thereby improving the competitiveness of sterile males [18,46,47]. Bellini et al. [48] thus created a sugar feeding device (associated with the releasing device) on which newly emerged sterile males (released as pupae) could acquire sugar meal prior to disperse into the wild. Such sugar fed released males showed an increase of about 25% of the value of competitiveness index compared to unfed males.
Even if no significant difference in competitiveness has been observed between irradiated males aged 3 days and 5 days old, high variability in the competitiveness of 5 days old sterile males suggests that ageing may also affect the mating competitive ability of sterile males. A loss of sperm cell present in male storage organ with time has already been observed in irradiated An. arabiensis males [49]. Examination of the testes of irradiated virgin males kept for 6 days showed four times less sperm than non-irradiated males of same age. Such phenomena could be suspected in Ae. albopictus. Indeed, Oliva et al. [50] have already shown that the sterile males that copulate with 4-5 successive females until the third day post-emergence were unable to replenish their sperm supply, suggesting that the immature sperm cells may have been impaired by irradiation exposure. Collectively, these findings suggest that despite a significant decline in male fitness following radiation sterilization, holding sterile males under optimal conditions with access to sugar meal ideally for 3 days post-emergence may be influential enough to consider the pre-release period.
Regardless of the age, the sterile Ae. albopictus males used in our conditions were at their best (in ratio 100:100:100) approximately half as competitive as wild-type males, suggesting a significant impact of irradiation and/or mass rearing. Moreover, the Competitiveness Index found in our experiment was comparable to that found by Oliva et al. [29] 0.14 for 1 day old and 0.55 for 5 day old males. In contrast, our results are very different from those found in previous experiments in Italy [31] and in Vienna [30], which showed that irradiated Ae. albopictus males were as competitive as wild males when they were 3 days old. The results concordance with Oliva, et al. [29] could be due to the fact that similar methodologies regarding cage sizes, release ratios and adult densities have been used, while experimental conditions were strongly different from Bellini et al. [31] and Madakacherry et al. [30] set up. However, the same range of Competitiveness Index was found in the latter investigations, although the experimental setups were relatively different. Beside the difference in experimental conditions, another reason that may explain the discrepancy between the previous results and our findings is probably the difference in strains of Ae. albopictus used (Reunion strain for the current study and Oliva et al. [29] and Rimini strain for Bellini et al. [31] and "Madakacherry", et al. [30]). To the best of our knowledge, very few information exist in the literature regarding potential difference in mating competitiveness between different strains in Ae. albopictus. Intraspecific variations in life history traits such as rates of larval growth [51], diapause [52], female longevity and reproduction [53], fitness and hybrid production [54] or competitive interactions with Ae. aegypti [55], has been already documented in Ae. albopictus from distinct geographic origins. Thus, different Ae. albopictus strains from distinct geographic regions likely exhibit different male quality after mass rearing or irradiation treatment. This may have direct implications in the efficiency of the sterile male insect technique as a control strategy across borders.
The efficiency of the sterile male insect technique as a control strategy relies on releases of sterile males in numbers adequate to obtain high over-flooding ratios in the field. In the experiment reported herein, despite a positive relationship between induced sterility and ratio of the wild to sterile males, meaningful difference in induced sterility was visible when sterile males were confined with wild males and females in the ratio of 500:100:100 (5:1:1) compared to the 1000:100:100 (10:1:1). The level of induced sterility at the ratio of 5:1 (49.7%) as observed in the present study is lower than the 81% observed in Rimini (Italy) strain Ae. albopictus [30], but relatively closed to the 53% previously recorded with 1 day old La Réunion strain Ae. albopictus [29]. This again might be related to strain differences in the competitiveness of sterile Ae. albopictus males when confronted with wild rivals at different ratios. Gaining a fuller understanding of strain-specific control of mating competitiveness in irradiated males will be important in the future before establishing any long term sustainable strategy for the suppression of Ae. albopictus by sterile male releases in different field situations.
This situation in Ae. albopictus competitiveness suggests that even a higher sterile-to-wild male ratio would be required to achieve meaningful population suppression in the field situations. Proof-ofprinciple demonstrations of this idea that a ratio much higher than 10:1 may be sometimes needed to achieve a meaningful suppression have been reported in previous SIT programs against other insects. For example, a significant decline in fertility of a wild population of Mediterranean fruit flies (Ceratitis capitata) in Guatemala was obtained when sterile males were released in the field at a ratio of 100:1 sterile male: wild male [37], while ratios of 40:1 were required for the suppression of codling moths (Cydia pomonella) populations in British Colombia, Canada [36]. Taking into consideration the effect of radiation on the life history parameters of Ae. albopictus [31,29] some uncertainty will remain on whether the ratio superior to 10:1 will be sufficient to effectively suppress the population of the naturally occurring Ae. albopictus mosquitoes in diverse field situations. Results from the current and related studies at least motivate further evaluation in small-scale open release trials with increasing ratios of sterile to wild males. However, mathematical models and numerical simulations may help gaining an appreciation on how experimental trials such as that reported here, and real-world baseline ecological data can be brought together to establish the appropriate release strategies for long-term suppression of wild Ae. albopictus populations.
Under our studied conditions, sterile Ae. albopictus males show a decrease of competitiveness with the increase of sterile male ratio and that could explain why high levels of sterility were not obtain when increasing the release ratio from 5:1 to 10:1. Such reduction of CI with the increasing ratio is difficult to interpret. Few papers deal with this ratio effect on male competitiveness, Maiga et al. [56] observed such decrease for Anopheles coluzzii while Yamada et al. [57] does not observe that for An. arabiensis. No real explanation has been proposed. One hypothesis could be that the higher density of male lead to higher disturbance between males during copulations due to the known aggressive behaviour of Ae. albopictus males under laboratory or semi-controlled conditions. In 100:100 ratios, each male probably could mate “quietly” while in 500:100 and 1000:100 ratios, disturbances and fights probability increase in which normal male could be the winner against the irradadiated male. Indeed, as observed in other some insect species, somatic damages link to the irradiation could have affect locomotor activity [58], flight ability [59] or male vigour [60] and could be detrimental during the fights.


As observed in numerous studies that have been set to develop or assess the efficacy of Sterile Insect Technique (SIT), irradiation inevitably impacts on the competitiveness of sterile males. Consistent with previous studies, we found that the age of the sterile Ae. albopictus males at release interacted with other physiological factors (such as access to sugar meal after sterilization) to affect their mating competitiveness. In our study, 3 days post-emergence was found optimum and thus may be considered in the future application of SIT against Ae. albopictus. "Oliva", et al [29] suggested the release of 5 days old male in their paper but high variability of mating ability observed in the current study suggests an agedependant increasing effect of irradiation process. Considering this requirement, our results further indicate that effective field release strategies against Ae. albopictus may require sterile-to-wild male ratios greater than 10:1. Keeping this in view, an inundative sterile male release may be very effective in reducing the density of the target vector population if used as a component of areawide integrated vector management, depending on technical constraints and specifics field situations. Indeed some other ecofriendly techniques could be used to enhance the efficiency of the SIT as the association with Wolbachia-induced phenotypes, such as cytoplasmic incompatibility and pathogen interference [61,62], or behavioural-based control programs as “boosted SIT” (Autodissemination of a juvenile hormone analogue the pyriproxyfen by sterile males [63]) and mosquitocidal botanicals [64]. Such integrated vector management could lead to a limitation of the high numbers of sterile males to release to a threshold economically viable and need to be tested in further experiments.


We are thankful to the technicians at the vector control Division of the Regional Health Agency for their assistance during field mosquito sampling. This study was part of the “SIT feasibility programme” in Reunion Island, jointly supported by the French Ministry of Health and the European Regional Development Fund (ERDF) under the 2007-2013 Operational Convergence Program.

Authors’ Contributions

Conceived and designed the experiments: LCG, DD. Performed the experiments: OD, GLG and CL. Analyzed the data: DD and LCG. Wrote the paper: DD and LCG. All authors read and edited the manuscript and approved the final version.

Competing Interest

The authors declare no competing interests.



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