Conceptual framework and rationale

The sterile insect technique (SIT) has been shown to be an effective and sustainable genetic approach to control populations of selected major pest insects, when part of area-wide integrated pest management (AW-IPM) programmes. The technique introduces genetic sterility in females of the target population in the field following their mating with released sterile males. This process results in population reduction or elimination via embryo lethality caused by dominant lethal mutations induced in sperm of the released males. In the past, several field trials have been carried out for mosquitoes with varying degrees of success. New technology and experience gained with other species of insect pests has encouraged a reassessment of the use of the sterility principle as part of integrated control of malaria vectors. Significant technical and logistic hurdles will need to be overcome to develop the technology and make it effective to suppress selected vector populations, and its application will probably be limited to specific ecological situations. Using sterile males to control mosquito vector populations can only be effective as part of an AW-IPM programme. The area-wide concept entails the targeting of the total mosquito population within a defined area. It requires, therefore, a thorough understanding of the target pest population biology especially as regards mating behaviour, population dynamics, dispersal and level of reproductive isolation. The key challenges for success are: 1) devising methods to monitor vector populations and measuring competitiveness of sterile males in the field, 2) designing mass rearing, sterilization and release strategies that maintain competitiveness of the sterile male mosquitoes, 3) developing methods to separate sexes in order to release only male mosquitoes and 4) adapting suppression measures and release rates to take into account the high reproductive rate of mosquitoes. Finally, success in area-wide implementation in the field can only be achieved if close attention is paid to political, socio-economic and environmental sensitivities and an efficient management organization is established taking into account the interests of all potential stakeholders of an AW-IPM programme.     

Colonisation and mass rearing: learning from others

Mosquitoes, just as other insects produced for the sterile insect technique (SIT), are subjected to several unnatural processes including laboratory colonisation and large-scale factory production. After these processes, sterile male mosquitoes must perform the natural task of locating and mating with wild females. Therefore, the colonisation and production processes must preserve characters necessary for these functions. Fortunately, in contrast to natural selection which favours a suite of characteristics that improve overall fitness, colonisation and production practices for SIT strive to maximize only the few qualities that are necessary to effectively control populations.However, there is considerable uncertainty about some of the appropriate characteristics due to the lack of data. Development of biological products for other applications suggest that it is possible to identify and modify competitiveness characteristics in order to produce competitive mass produced sterile mosquitoes. This goal has been pursued - and sometimes achieved - by mosquito colonisation, production, and studies that have linked these characteristics to field performance. Parallels are drawn to studies in other insect SIT programmes and aquaculture which serve as vital technical reference points for mass-production of mosquitoes, most of whose development occurs - and characteristics of which are determined - in an aquatic environment. Poorly understood areas that require further study are numerous: diet, mass handling and genetic and physiological factors that influence mating competitiveness. Compromises in such traits due to demands to increase numbers or reduce costs, should be carefully considered in light of the desired field performance.     

Male lures and male mating competitiveness in a genetic sexing strain of the melon fly (Diptera: Tephritidae)

The Sterile Insect Technique (SIT) has been used to suppress or eradicate populations of the melon fly, Zeugodacus cucurbitae (Coquillett), a pest of various fruits and vegetables throughout Asia, Africa, and South Pacific Islands. The success of the SIT depends largely on the ability of released, sterile males to compete successfully with wild males for copulations with wild females. Unfortunately, both the sterilization process (via gamma irradiation) and artificial selection under mass-rearing conditions generally reduce the biological competence, including the mating competitiveness, of the released males. However, previous data on Z. cucurbitae show that irradiation has minimal adverse effects and prolonged mass-rearing may or may not result in decreased mating ability. The present study first confirms that neither irradiation nor mass-rearing had deleterious effects on male mating ability of males from a pupal-color, genetic sexing strain (termed T1). Then, the influence of male lures (cue-lure and its natural analogue raspberry ketone) on the mating success of sterile T1 males was measured as a potential pre-release procedure to improve the effectiveness of SIT. Feeding on cue-lure enhanced male mating success, but the effect was short-lived (1 d but not 3 d post-feeding). Feeding on raspberry ketone boosted mating success over a longer time, i.e., at least 5 d but not 10 d post-feeding. However, mixing raspberry ketone into the adult diet had no detectable effect on male mating performance. The implications of these results for SIT against Z. cucurbitae are discussed.     

Sterile insect technique and Mediterranean fruit fly (Diptera: Tephritidae): assessing the utility of aromatherapy in a Hawaiian coffee field

The sterile insect technique (SIT) is widely used in integrated programs against tephritid fruit fly pests, particularly the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Unfortunately, the mass-rearing procedures inherent to the SIT often lead to a reduction in the mating ability of the released males. One potential solution involves the prerelease exposure of males to particular attractants. In particular, exposure of male Mediterranean fruit flies to ginger, Zingiber officinale Roscoe, root oil (GRO) has been shown to increase mating success in laboratory and field cage trials. Here, we describe a field experiment that compares the level of egg sterility observed in two Hawaiian coffee, Coffea arabica L., plots, with GRO-exposed, sterile males released in one (treated) plot and nonexposed, sterile males released in the other (control) plot. Once per week in both plots over a 13-wk period, sterile males were released, trap captures were scored to estimate relative abundance of sterile and wild males, and coffee berries were collected and dissected in the laboratory to estimate the incidence of unhatched (sterile) eggs. Data on wild fly abundance and the natural rate of egg hatch also were collected in a remote area that received no sterile males. Despite that sterile:wild male ratios were significantly lower in the treated plot than in the control plot, the incidence of sterile eggs was significantly higher in the treated plot than in the control plot. Correspondingly, significantly higher values of Fried's competitiveness index (C) were found, on average, for treated than control sterile males. This study is the first to identify an association between the GRO "status" of sterile males and the incidence of egg sterility in the field and suggests that prerelease, GRO exposure may represent a simple and inexpensive means to increase the effectiveness of Mediterranean fruit fly SIT programs.     

Aromatherapy on a large scale: exposing entire adult holding rooms to ginger root oil increases the mating competitiveness of sterile males of the Mediterranean fruit fly in field cage trials

The sterile insect technique (SIT) is widely used in integrated programs against fruit fly pests, particularly the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Unfortunately, the mass-rearing procedures inherent to the SIT often lead to a reduction in male mating competitiveness. One potential solution involves the pre-release exposure of males to specific attractants. In particular, male exposure to ginger root oil [Zingiber officinale Roscoe (Zingiberaceae); hereafter GRO] has been shown to increase mating success dramatically in field cage trials. Initial studies exposed small groups of males (25 individuals), but more recent work has demonstrated that GRO exposure involving standard storage boxes (containing ≈ 36 000 males) also results in enhanced mating performance. The objective of the present study was to determine whether aromatization of entire trailers, holding ≈ 14 million sterile males from a genetic sexing [temperature sensitive lethal (tsl)] strain, increases male mating success. Independent of the total dose, spatial distribution, or type of dispenser used, sterile males exposed to GRO for a 24-h period displayed greater mating success than non-exposed males in mating cage trials (in which tsl males competed against males from a standard, bisexual strain for females from this same standard strain). Averaged over all experiments, tsl males exposed to GRO obtained 54% of all matings compared to 38% for non-exposed tsl males, an increase of 42%. The implications of these findings for SIT programs against C. capitata are discussed.     

Laboratory adaptation reduces female mating resistance in the sweet potato weevil

Selection for genetic adaptation might occur whenever an animal colony is maintained in the laboratory. The laboratory adaptation of behavior such as foraging, dispersal ability, and mating competitiveness often causes difficulties in the maintenance of biological control agents and other beneficial organisms used in procedures such as the sterile insect technique (SIT). Sweet potato weevil, Cylas formicarius (Summers) (Coleoptera: Brentidae), is an important pest in sub‐tropical and tropical regions. An eradication program targeting C. formicarius using SIT was initiated in Japan with weevils being mass‐reared for 95 generations to obtain sufficient sterile males. The mass‐reared strain of C. formicarius exhibits weaker female resistance to male mating attempts compared with the wild strain. This could affect the success of SIT programs because mating persistence of mass‐reared males might be expected to decrease in response to weak female resistance. We show that high success of sperm transfer to mass‐reared females was due to weak female resistance to male mating attempts. However, the mating behavior of mass‐reared males did not change. In C. formicarius, the trait of male persistence to mate was not correlated with the female resistance traits. Our results suggest that mass‐rearing conditions do not have negative effects on the mating ability of the sterile males of this species, and thus that the current mass‐rearing procedures are suitable for production of sterile males for the weevil eradication program.     

A novel molecular approach to assess mating success of sterile Ceratitis capitata (Diptera: Tephritidae) males in sterile insect technique programs

Areawide sterile insect technique (SIT) programs against Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), are increasingly implemented worldwide. A key issue in SIT is to assess mating success of released sterile males, which could be currently estimated by egg hatchability and by stored sperm head measurements. We report here on a novel molecular approach that would allow detecting the presence of Mediterranean fruit fly sterile male sperm in the female spermathecae under field conditions, as a precise marker to assess mating performance. The simplicity (only two polymerase chain reactions) and reliability of this method, jointly with the capability to detect Vienna sperm in wild Mediterranean fruit fly maintained in monitoring traps for 7 d under field conditions, suggest that it could be an efficient tool when coupled with areawide SIT programs.     

Sexual competitiveness of a transgenic sexing strain of the Mediterranean fruit fly, Ceratitis capitata

The sterile insect technique (SIT), when used for the control of the Mediterranean fruit fly (medfly), Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), generally relies on the release of sterile flies of only the male sex. Male selection is achieved through the use of a genetic sexing strain (GSS) in which females are killed by heat treatment in the generation prior to release. Transgenic sexing strains (TSS) have been developed that perform the same function of female-lethality, this time by withholding tetracycline (or related compounds) from the larval diet. The use of TSS may allow for certain problems associated with conventional GSS, such as strain instability and reduced productivity in mass-rearing, to be avoided. The performance, and principally the sexual competitiveness, of released male flies is important for the success of an SIT control programme. This study describes field cage experiments in which the competitiveness of males from a TSS (OX3376B) was compared with that of a conventional GSS (VIENNA-8) and two wild-type strains (TOLIMAN and ARG). When competing for female mates with wild-type males, OX3376B male performance was acceptable. When OX3376B males competed directly for mates with VIENNA-8 males, VIENNA-8 slightly outperformed the TSS males. Parallel tests, in which wild-type males competed with either OX3376B or VIENNA-8 males, showed that males from both sexing strains were highly competitive with wild-type males. These results suggest that OX3376B in particular, and TSS in general, show sufficiently good mating competitiveness to merit further research into their suitability for eventual use in SIT programmes.     

Spatial patterns of sterile Mediterranean fruit fly dispersal

The success of the sterile insect technique (SIT) for the control of the Mediterranean fruit fly or medfly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), depends largely on the ability of sterile flies to spread in the target area and compete with the wild males for wild females. Our objectives in the present study were three‐fold: (1) to evaluate the dispersal ability of sterile male medflies and compare their spatial dispersion patterns with that of wild males, (2) to evaluate how different release methods affect subsequent spatial dispersal, and (3) to determine whether manipulating the pre‐release diet of sterile males affects their dispersal. To achieve these objectives, we conducted three experiments in the field where we quantified and analyzed the spatial and temporal dispersal patterns of sterile medflies and the dispersion of resident wild males. Overall, ca. 5% of the released sterile flies were recaptured 100 m from the release point, and ca. 2% were recaptured 200 m from the release point. The released flies rarely survived longer than 5–7 days. We repeatedly found that the spatial dispersion patterns of sterile males significantly correlated with those of wild males. Release methods strongly affected subsequent fly dispersal in the field as significantly more flies were recaptured following a scattered release vs. a central one. Finally, we show that enriching sterile fly pre‐release diet with protein did not affect subsequent dispersal in the field. We conclude that sterile males are able to match the dispersion patterns of wild males, an outcome that is highly important for SIT success. Large releases from central points distant from each other may leave many areas uncovered. Accordingly, scattered releases, repeated twice a week, will provide better coverage of all available aggregations sites. The spatial performance of protein‐fed males suggests that pre‐release diet amendments may be used without detriment as a sexual stimulant in SIT programs.     

Improving male mating competitiveness and survival in the field for medfly,Ceratitis capitata (Diptera: Tephritidae) SIT programs

The success of the sterile insect technique (SIT) depends critically upon mating between released sterilized males and wild females. In Hawaii, improvements in the efficiency of sterile males were attempted on two separate fronts – mating enhancement and survival improvement. In the former, two methods have been investigated – selective breeding and aromatherapy. In the latter, flies which survived in field cages for several days were selected and bred to produce progeny with enhanced survival ability compared to control flies. Regarding mating selection, standard laboratory-reared males that successfully mated with wild females in field cages were allowed to breed. F1 offspring were inbred, then the selection procedure was repeated for four additional cycles. In the aromatherapy procedure, laboratory-reared males were exposed to ginger root oil for several hours 1 day prior to testing in field cages. Compared to controls, the selected flies improved the mating competitiveness of male flies ca. 3-fold, irradiation reduced this increase to ca. 2.5-fold. Exposing the selected, hybrid strain raised the fitness of the lab males to ca. 9-fold that of wild males. In the ongoing survival selection study, we have obtained lines in which the selected males survived ca. 2-fold better than laboratory control males over several days in an outdoor field cage, with food and water provided. The goal is to combine the traits of higher survival and mating ability into a single strain for SIT release.     

Multiple paternity in a natural population of a wild tobacco fly, Bactrocera cacuminata (Diptera: Tephritidae), assessed by microsatellite DNA markers

Mating frequency has important implications for patterns of sexual selection and sexual conflict and hence for issues such as speciation and the maintenance of genetic diversity. Knowledge of natural mating patterns can also lead to more effective control of pest tephritid species, in which suppression programmes, such as the sterile insect technique (SIT) are employed. Multiple mating by females may compromise the success of SIT. We investigated the level of polyandry and sperm utilization in a Brisbane field population of the tropical fruit fly, Bactrocera cacuminata (Hering), using seven polymorphic microsatellite loci. The offspring of 22 wild-caught gravid females were genotyped to determine the number of males siring each brood and paternity skew, using the programs gerud and scare. Our data showed that 22.7% of females produced offspring sired by at least two males. The mean number of mates per female was 1.72. Paternal contributions of double-sired broods were skewed with the most successful male having sired between 76.9% and 87.5% of the offspring. These results have implications for SIT, because the level of remating we have identified would indicate that wild females could mate with one or more resident fertile males.     

Adaptation of the BG-Sentinel trap to capture male and female Aedes albopictus mosquitoes

In recent years, the remarkable spread of Aedes albopictus (Skuse) (Diptera: Culicidae) throughout the world has drawn attention to this hitherto poorly studied species, particularly after its role in outbreaks of chikungunya fever in the western Indian Ocean and in Italy. Variants of sterile insect technique (SIT), including the release of transgenic males with a dominant lethal gene (RIDL), have been proposed in the search for new and innovative methods of control. Knowledge of male dispersal, mating behaviour and longevity will be critical to the success of this approach. We present an effective and practical method for trapping both male and female Ae. albopictus using a mouse-baited BG-Sentinel trap.     

Age-dependent variation in mating success of sterile male Mediterranean fruit flies (Diptera: Tephritidae): implications for sterile insect technique

The sterile insect technique (SIT) is widely used in integrated programs against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Unfortunately, the mass-rearing procedures inherent to the SIT often lead to a reduction in the mating ability of the released males. To counter this deficiency, SIT programs rely upon the production and release of large numbers of sterile males to achieve high overflooding (sterile:wild male) ratios. To ensure a high release volume, emergence facilities release adult males at a young age (2 d old in some cases). The primary objective of this study was to describe age-dependent variation in the mating propensity and competitiveness of sterile males of C. capitata. Males that were 2 or 3 d old had lower mating propensity than males that were > or =4 d old, and 3-d-old males had lower mating competitiveness than males that were > or =4 d old. Given these results, we measured the effect of a longer holding period on male mortality in storage boxes. With delayed food placement, males held in storage boxes for 4 d after emergence showed no higher mortality than males held for only 2 d (the standard interval). Using large field enclosures, we compared the levels of egg sterility attained via releases of 2- versus 4-d-old sterile males at two overflooding ratios (5:1 and 100:1). At the lower ratio, the proportion of unhatched eggs observed for trials involving 2-d-old sterile males was not, on average, significantly higher than that observed for matings between wild flies (33 versus 25%, respectively), whereas the level of egg sterility observed for releases of 4 d old sterile males was 62%. At the 100:1 overflooding ratio, the proportion of unhatched eggs associated with the 2-d-old sterile males was 58%, a level not significantly different from that induced by 4-d-old sterile males at the 5:1 ratio and significantly lower than the level (79%) observed for 4-d-old sterile males at 100:1 overflooding ratio. The implications of these results for SIT are discussed.     

Remating in Ceratitis capitata sterile males: Implications in sterile insect technique programmes

Sterile insect technique (SIT) is used, among other biological control tools, as a sustainable measure for the management of Ceratitis capitata Wiedemann (Diptera: Tephritidae) in many agricultural regions where this pest can trigger severe economic impacts. The tendency of wild females to remate multiple times has been deeply studied; it has been a common point of controversy when evaluating SIT programmes. Nevertheless, the remating potential of the released sterile males remains unknown. Here, under laboratory conditions, the remating capability of mass-reared sterile males was determined. Wild-type virgin females were offered to sterile males (Vienna-8 strain), which had the opportunity to mate up to four consecutive times. The remating assays were carried out at 24 hr, 48 hr, 4 days and 7 days after the first mating. At the end of each tested time period, males were divided according to their mating response, mated or unmated, and subsequently reused for the next round of mating assays. The frequency of successful remating in each tested time period was obtained. Insemination was confirmed by determining the sperm transfer in mated female spermathecae by quantitative real-time PCR. Our results demonstrate that 73% of the mass-reared sterile males were able to remate 24 hr after the first mating, 55% of which remated again the day after. Close to 25% of the V8 sterile males tended to copulate in all of the four mating opportunities. The qPCR analysis of the spermathecae contents verified an effective transfer of V8 sperm to wild females with every mating; 99% of copulations resulted in sperm transfer. These findings shed light on the remating potential of V8 sterile males, an aspect until now underestimated in many SIT programmes.     

The Sterile Insect Technique for Controlling Populations of Aedes albopictus (Diptera: Culicidae) on Reunion Island: Mating Vigour of Sterilized Males

Reunion Island suffers from high densities of the chikungunya and dengue vector Aedes albopictus. The sterile insect technique (SIT) offers a promising strategy for mosquito-borne diseases prevention and control. For such a strategy to be effective, sterile males need to be competitive enough to fulfil their intended function by reducing wild mosquito populations in natura. We studied the effect of irradiation on sexual maturation and mating success of males, and compared the sexual competitiveness of sterile versus wild males in the presence of wild females in semi-field conditions. For all untreated or sterile males, sexual maturation was completed within 13 to 20 h post-emergence and some males were able to inseminate females when 15 h old. In the absence of competition, untreated and sterile males were able to inseminate the same number of virgin females during 48 h, in small laboratory cages: an average of 93% of females was inseminated no matter the treatment, the age of males, and the sex ratio. Daily mating success of single sterile males followed the same pattern as for untreated ones, although they inseminated significantly fewer females after the ninth day. The competitiveness index of sterile males in semi-field conditions was only 0.14 when they were released at 1-day old, but improved to 0.53 when the release occurred after a 5-day period in laboratory conditions. In SIT simulation experiments, a 5∶1 sterile to wild male ratio allowed a two-fold reduction of the wild population’s fertility. This suggests that sterile males could be sufficiently competitive to mate with wild females within the framework of an SIT component as part of an AW-IPM programme for suppressing a wild population of Ae. albopictus in Reunion Island. It will be of interest to minimise the pre-release period in controlled conditions to ensure a good competitiveness without increasing mass rearing costs.     

Mass‐rearing conditions do not affect responsiveness to sex pheromone and flight activity in sweetpotato weevils

For ensuring the effectiveness of sterile insect technique (SIT) programmes, maintaining the reproductive competitiveness and dispersal ability of mass‐reared sterile males is essential. Inadvertent selection is an important genetic process that frequently occurs during mass rearing to produce sterile males. We investigated the effect of mass‐rearing conditions on the responsiveness to sex pheromones and spontaneous flight activity of males of the sweetpotato weevil Cylas formicarius (Coleoptera: Brentidae). There were no significant differences in the responsiveness to sex pheromones and spontaneous flight activity between wild and mass‐reared strains. These results indicate that mass‐reared strains of C. formicarius might not cause serious problems for implementing SIT programmes.     

Insect quality control: synchronized sex,mating system,and biological rhythm

The sterile insect technique (SIT) is a method of eradicating insects by releasing mass-reared sterilized males into fields to reduce the hatchability of eggs laid by wild females that have mated with the sterile males. SIT requires mass-production of the target insect, and maintenance of the quality of the mass-reared insects. The most important factor is successful mating between wild females and sterile males because SIT depends on their synchronized copulation. Therefore, understanding the mating systems and fertilization processes of target insects is prerequisite. Insect behavior often has circadian rhythms that are controlled by a biological clock. However, very few studies of relationships between sterile insect quality and circadian rhythm have been performed compared with the amount of research on the mating ability of target insects. The timing of male copulation attempts with receptivity of females is key to successful mating between released males and wild females. Therefore, we should focus on the mechanisms controlling the timing of mating in target insects. On the other hand, in biological control projects, precise timing of the release of natural enemies to attack pest species is required because behavior of pests and control agents are affected by their circadian rhythms. Involving both chronobiologists and applied entomologists might produce novel ideas for sterile insect quality control by synchronized sex between mass-reared and wild flies, and for biological control agent quality by matching timing in activity between predator activity and prey behavior. Control of the biological clocks in sterile insects or biological control agents is required for advanced quality control of rearing insects.     

Effect of irradiation on mating ability in the male sweetpotato weevil (Coleoptera: Curculionidae)

The sterile insect technique (SIT) is widely used for suppressing or eradicating target pest insect populations. The effectiveness of SIT depends on the ability of released sterile males to mate with and inseminate wild females. Irradiation is the effective manner to sterilize mass-reared insects. The negative impacts of this procedure are not limited to damage on reproductive cells. Gamma-radiation damages the epithelial tissue of midgut, which affects the alimentation in insects. Irradiated males alter their mating behavior over time because of the depression of metabolic activity by sterilization. In this study, we evaluated the male mating performance and sexually compatibility of irradiated male Cylas formicarius elegantulus (Summers) (Coleoptera: Curculionidae) with a 200-Gy dose, as currently used in the SIT program in Okinawa Prefecture, throughout 16 d after irradiation in the laboratory. The mating ability of irradiated males did not differ from that of control males for about a week. However, the mating ability of irradiated male drastically decreased thereafter. We consider that irradiated male C. formicarius elegantulus with a 200-Gy dose had no major effect on male mating behavior approximately for a week after irradiation.     

Fractionated irradiation improves the mating performance of the West Indian sweet potato weevil Euscepes postfasciatus

• 1 The sterile insect technique (SIT) is widely used to suppress or eradicate target pest insect populations.
• 2 The effectiveness of SIT depends on the ability of released sterile males to mate with and inseminate wild females. The use of gamma radiation to induce sterility, however, negatively affects both somatic cells as well as reproductive cells. Consequently, mating performance of sterilized individuals decreases drastically over time. The mating propensity of sterilized Euscepes postfasciatus (Fairmaire) males irradiated with a single dose of 150 Gy (the current standard of the Okinawa Prefecture SIT programme) is equal to that of non‐irradiated weevils for the first 6 days.
• 3 Fractionated irradiation, in which a sterilizing dose is delivered over time in a series of smaller irradiations, reduces the damage of irradiation in insects. In the present study, we evaluated the effect of fractionated irradiation on male fertilization ability, longevity and mating propensity of E. postfasciatus for a period of 16 days after irradiation.
• 4 Although fractionated irradiation totalling 150 Gy was found to induce full sterility regardless of the number of individual doses, the mating propensity of male weevils sterilized by fractionated irradiation was maintained for the first 12 days. These results demonstrate that fractionated irradiation can be highly advantageous in programmes aimed at eradication of E. postfasciatus.