Bacterially enriched diet improves sexual performance of sterile male Mediterranean fruit flies

The Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), (medfly) is a polyphagous and cosmopolitan agricultural pest, targeted in many areas for control by the Sterile Insect Technique (SIT). Our objective in the present study was to test the hypothesis that a bacterially enriched diet provided to sterile males will improve their sexual performance in competitive settings that emulate natural conditions. Specifically we determined how feeding on diets enriched with Klebsiella oxytoca affected the ability of sterile males to compete for wild females against wild males, their ability to inhibit female receptivity, and their survival. We found that enriching the sterile male diet with K.oxytoca significantly improved mating competitiveness in the laboratory and in field cages. In addition, bacterially enriched sterile males inhibited female receptivity more efficiently than sugar fed males and survived longer duration of starvation. We conclude that inoculating mass reared sterile flies with bacteria prior to their release is a valid approach to improve the efficacy of SIT.     

Factors affecting Anastrepha fraterculus female receptivity modulation by accessory gland products

In the context of the sterile insect technique (SIT), mass-rearing and male irradiation are imperative. Post-teneral treatments such as the addition of protein in adult's male diet and male hormonal treatment are used to improve sexual performance and to accelerate sexual maturation. In this work we investigated the effect of male accessory glands products (AGPs) on female receptivity of the South American fruit fly Anastrepha fraterculus (Wiedemann), and the effect of strain rearing history, male irradiation, male diet and hormonal treatment on AGPs. Injections of aqueous extracts of male accessory glands into the abdomen of females reduced their receptivity. The AGPs from laboratory males were more effective in inhibiting female receptivity, compared to AGPs from wild males, irrespective of females' origin. The AGPs from fertile males were more effective than AGPs from sterile males. The AGPs from protein-fed males were more effective than AGPs from sugar-fed males. Finally, the AGPs of males treated with juvenile hormone were less effective in inhibiting female receptivity than AGPs of untreated males. We conclude that inhibition of sexual receptivity of A. fraterculus mated females is mediated by products in male accessory gland's and the way that these products act vary widely according to the effect of extrinsic factors. We discuss the results in the perspective of the SIT application for A. fraterculus.     

Transgenic sexing system for Ceratitis capitata (Diptera: Tephritidae) based on female-specific embryonic lethality

Fruit fly pest species have been successfully controlled and managed via the Sterile Insect Technique (SIT), a control strategy that uses infertile matings of sterile males to wild females to reduce pest populations. Biological efficiency in the field is higher if only sterile males are released in SIT programs and production costs are also reduced. Sexing strains developed in the Mediterranean fruit fly Ceratitis capitata (medfly) through classical genetics are immensely beneficial to medfly SIT programs but exhibit reduced fertility and fitness. Moreover, transfer of such classical genetic systems to other tephritid species is difficult. Transgenic approaches can overcome this limitation of classical genetic sexing strains (GSSs), but had resulted so far in transgenic sexing strains (TSSs) with dominant lethality at late larval and pupal stages. Here we present a transgene-based female-specific lethality system for early embryonic sexing in medfly. The system utilizes the sex-specifically spliced transformer intron to restrict ectopic mRNA translation of the pro-apoptotic gene hid^Ala5 to females only. The expression of this lethal effector gene is driven by a tetracycline-repressible transactivator gene tTA that is under the control of promoters/enhancers of early-acting cellularization genes. Despite observed position effects on the sex-specific splicing, we could effectively establish this early-acting transgenic sexing system in the medfly C. capitata. After satisfactory performance in large scale tests, TSSs based on this system will offer cost-effective sexing once introduced into SIT programs. Moreover, this approach is straight forward to be developed also for other insect pest and vector species.     

Male irradiation affects female remating behavior in Anastrepha serpentina (Diptera: Tephritidae)

Female remating in target pest species can affect the efficacy of control methods such as the Sterile Insect Technique (SIT) but very little is known about the postcopulatory mating behavior of these pests. In this study, we investigated the remating behavior of female Anastrepha serpentina (Diptera: Tephritidae), an oligophagous pest of Sapotaceae. First, we tested how long the sexual refractory period of females lasted after an initial mating. Second, we tested the effect of male and female sterility, female ovipositing opportunities and male density on female propensity to remate. Lastly, we tested if the amount of sperm stored by females was correlated to the likelihood of females to remate. We found that receptivity of mass-reared A. serpentina females had a bimodal response, with up to 16% of mass-reared A. serpentina females remating five days after the initial copulation, decreasing to 2% at 10 and 15 days and increasing to 13% after 20 days. Compared to fertile males, sterile males were less likely to mate and less likely to inhibit females from remating. Copula duration of sterile males was shorter compared to fertile males. Remating females were less likely to mate with a sterile male as a second mate. Sterile females were less likely to mate or remate compared to fertile females. Opportunity to oviposit and male density had no effect on female remating probability. Sperm numbers were not correlated with female likelihood to remate. Information on the post-copulatory behavior of mass-reared A. serpentina will aid fruit fly managers in improving the quality of sterile males. We discuss our results in terms of the differences this species presents in female remating behavior compared to other tephritids.     

Riding the Trojan horse: combating pest insects with their own symbionts

Insects form an extremely large group of animals and bear a consequently large variety of associated microbes. This microbiota includes very specific and obligate symbionts that provide essential functions to the host, and facultative partners that are not necessarily required for survival. The Tephritidae is a large family that includes many fruit pests such as the Mediterranean fruit fly (the medfly, Ceratitis capitata) and the Olive fly (Bactrocera oleae). Community and functional analyses showed that the microbiota of both flies contribute to their diet, and affect host fitness parameters. The analysis of the microbiota's community structure of mass‐reared, sterilized medfly males used in the sterile insect technique revealed a strong reduction in Klebsiella spp. compared with non‐sterile and wild flies. Inoculation of sterile males with this gut population affected female mating behaviour as they preferentially mated with inoculated versus non‐inoculated males. These studies suggest that control can be significantly improved by manipulating symbionts in pest animals.     

Radiation effects on Drosophila suzukii (Diptera: Drosophilidae) reproductive behaviour

Female remating is a widespread behaviour, reported in several insect species. This behaviour can affect the efficiency of sterile insect technique (SIT); however, little is known about the postcopulatory behaviour of some pest species considered as candidates to be controlled by this technique, such as Drosophila suzukii (Matsumura, 1931) (Diptera: Drosophilidae). In this study, we investigated the effects of male and female sterilization on mating and remating behaviour of D. suzukii. First, we tested the occurrence of multiple mating in different combinations between sterile and fertile males and females. Then, we tested the effects of male and female sterility on female propensity to mate and remate. We found an overall low remating rate by D. suzukii females. Male sterility did not influence mating and remating likelihood; however, copula duration of sterile males was shorter compared to fertile males. On the other hand, sterile females were less likely to mate. Our findings encourage further research regarding the use of SIT to control D. suzukii.     

Sexual competitiveness of sterile Ceratitis capitata males exposed to essential oils from non-host plant species native to Argentina

The sterile insect technique (SIT) is used to control fruit fly pests, such as the Mediterranean fruit fly (medfly), Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Enhancing sexual competitiveness of mass-reared sterile males can contribute to making this technique more effective. It has been shown that exposure to volatiles from essential oils (EOs), such as ginger root oil (GRO) and those from host fruits, increases male mating success. We evaluated the effect of EOs from non-host species native to Argentina, Schinus polygama (Cav.) Cabrera (Anacardiaceae) and Baccharis spartioides (Hook. & Arn.) Remy (Asteraceae), on the sexual competitiveness of sterile C. capitata males. In field cage experiments, sterile males exposed to S. polygama EO increased their mating success. In addition, sterile males exposed to this EO achieved more matings on trees than non-exposed males, thus suggesting the former are more prone to locate and defend the pheromone-calling territory. Deprivation of water and/or food significantly reduced males' life span, but exposure to S. polygama EO and GRO did not affect their survival. Schinus polygama EO is composed of mono- and sesquiterpenes with behavioral and electroantennographic responses in medflies, indicating that semiochemicals that boost medfly sexual competitiveness combine in this EO.     

Genetic elimination of field-cage populations of Mediterranean fruit flies

The Mediterranean fruit fly (medfly, Ceratitis capitata Wiedemann) is a pest of over 300 fruits, vegetables and nuts. The sterile insect technique (SIT) is a control measure used to reduce the reproductive potential of populations through the mass release of sterilized male insects that mate with wild females. However, SIT flies can display poor field performance, due to the effects of mass-rearing and of the irradiation process used for sterilization. The development of female-lethal RIDL (release of insects carrying a dominant lethal) strains for medfly can overcome many of the problems of SIT associated with irradiation. Here, we present life-history characterizations for two medfly RIDL strains, OX3864A and OX3647Q. Our results show (i) full functionality of RIDL, (ii) equivalency of RIDL and wild-type strains for life-history characteristics, and (iii) a high level of sexual competitiveness against both wild-type and wild-derived males. We also present the first proof-of-principle experiment on the use of RIDL to eliminate medfly populations. Weekly releases of OX3864A males into stable populations of wild-type medfly caused a successive decline in numbers, leading to eradication. The results show that genetic control can provide an effective alternative to SIT for the control of pest insects.     

Enhancing mating performance after juvenile hormone treatment in Anastrepha fraterculus: a differential response in males and females acts as a physiological sexing system

Methoprene (a mimic of juvenile hormone) treatment can reduce the time required for sexual maturation in Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae) males under laboratory conditions, supporting its use as a treatment for sterile males within the context of the sterile insect technique (SIT). We evaluated sexual behaviour, mating competitiveness of methoprene-treated males, and female readiness to mate after methoprene-treatment in field cages. The study involved two strains of A. fraterculus from Argentina and Peru, which show several polymorphisms in relation to their sexual behaviour. We also analyzed whether methoprene treatment affected male and/or female behaviour in the same way in these two strains. Methoprene-treated males were equally competitive with untreated mature males, and became sexually competitive 6 days after emergence (3–4 days earlier than untreated males). In contrast, methoprene did not induce sexual maturation in females or, at least, it did not induce a higher rate of mating in 7-day-old females. These results were observed both for the Argentina and the Peru strains. Altogether, our results indicate that methoprene treatment produces sexually competitive males in field cages. In the absence of a genetic sexing system, and when sterile males and females of A. fraterculus are released simultaneously, the fact that females do not respond as do males to the methoprene treatment acts as a physiological sexing effect. Therefore, in the presence of mainly sexually immature sterile females, released sexually mature sterile males would have to disperse in search of wild fertile females, thereby greatly reducing matings among the released sterile insects and thus enhancing sterile insect technique efficiency.     

Regulation of sexual receptivity of female Mediterranean fruit flies: old hypotheses revisited and a new synthesis proposed

The objective of this study was to examine the relative contributions of copula duration and sperm transfer to the inhibition of sexual receptivity of female Mediterranean fruit flies (Ceratitis capitata, Diptera: Tephritidae). Females choosing to remate had significantly fewer sperm in their spermathecae than females who chose not to remate. Duration of a female's first copulation did not affect her subsequent receptivity. Furthermore, on the first day following copulation significantly more females whose first mate was sterile and from a laboratory strain (sterile males transfer fewer sperm than wild males) chose to copulate than did females whose mate was fertile and recently derived from wild stock. Finally, we offer a synthesis of the available information on remating in this species, and suggest that while females are facultatively polyandrous, copula duration, sperm transfer and male accessory gland secretions act in succession to inhibit female receptivity.     

Remating by female Mediterranean fruit flies (Ceratitis capitata, Diptera: Tephritidae): Temporal patterns and modulation by male condition

We determined the temporal pattern of female remating in the Mediterranean fruit fly, Ceratitis capitata, and how mating with sterile males affects remating. In addition, we examined the hypotheses that sterile male nutrition and age affect the subsequent receptivity of their mates. Temporally, female receptivity varied significantly throughout the experimental period. Relatively high levels of remating (14%) on the days following the first copulation were followed by a decline, with a significantly low point (4.1%) 2 weeks after mating. Subsequently, receptivity is gradually restored (18%) 3 and 4 weeks after the initial copulation. When females were first mated to sterile males, significantly higher remating percentages were recorded. The ability of sterile males to inhibit receptivity of both wild and laboratory reared females on the day of first mating was significantly improved when they were fed a nutrient rich diet. Male age at first mating also affected female receptivity: sterile males of intermediate age (11 days old) inhibited female remating significantly more than younger or older flies. Although further studies are needed to determine the relative roles of natural and sexual selection in modulating patterns of female sexual receptivity, the Sterile Insect Technique may be improved by releasing well nourished, older sterile males.     

The effects of sterile males and two braconid parasitoids,Fopius arisanus (Sonan) and Diachasmimorpha krausii (Fullaway) (Hymenoptera), on caged populations of Mediterranean fruit flies,Ceratitis capitata (Wied.) (Diptera: Tephritidae) at various sites in Guatemala

Area-wide control of the Mediterranean fruit fly (=medfly), Ceratitis capitata (Wiedemann), typically involves sterile insect technique (=SIT), and at present the “Temperature Sensitive Lethal” (=TSL) strain is commonly mass-reared for such releases. In theory, and with some experimental support, the augmentative addition of parasitoids to sterile releases can suppress pest populations to a greater extent than either technique alone. The efficacies of TSL males, parasitoids, and TSL males and parasitoids were compared in large field cages erected over coffee grown at four locations and three altitudes (relatively high, medium and low for the crop) in Guatemala. Two species of opiine braconid parasitoids, the larval–pupal parasitoid Diachasmimorpha krausii (Fullaway) and the egg-pupal parasitoid Fopius arisanus (Sonan), were released either together or in combination with sterile males into cages along with fertile medflies. Results of this evaluation were assessed by comparing the number of pupae and adult insects that completed development (F1 generation) as a result of the reproduction of a parental generation released into each field cage. The TSL males significantly suppressed F1 fly populations but only in one of four study sites. However, the inclusion of F. arisanus and D. krausii always provided significant suppression and the effect was frequently substantial. In one site there was a significant interaction between the capacity of sterile males and parasitoids to suppress caged fly populations. There was no effect of host-fruit abundance on the numbers of flies recovered, however, there were significant interactions between maximum and minimum temperatures and the effects of sterile males and parasitoids, respectively. The results suggest that mass-reared sterile medflies and biological control agents should be tested for both consistent sexual-quality and their ability to perform in the various environments in which they will be released.     

Optimal life stage for radiation sterilization of Anopheles males and their fitness for release

Mating by male Anopheles mosquitoes (Diptera: Culicidae) was evaluated in the laboratory to assess fitness effects of radio-sterilization applied during different life stages of the malaria vectors An. stephensi Liston and An. gambiae Giles sensu stricto. After reproductive sterilization by gamma-irradiation (120 Gy), equal proportions of sterile and fertile (unirradiated) male adults were released into cages with virgin females and allowed to compete for matings. Radio-sterilization was applied when the males were pupae aged 0-7 h or 24-32 h, or adults aged <24 h or 24-55 h. After being radio-sterilized in the adult stage, males of both species competed effectively with unirradiated males, whereas those sterilized in the pupal stage obtained significantly fewer matings than unirradiated males from the same cohort. There was no evidence of females obtaining multiple inseminations. These findings emphasize the need to radio-sterilize males as adults in order to minimize the fitness cost. Such males may be intended for sterile insect technique population suppression or for trial releases of transgenic anophelines.     

The male ejaculate as inhibitor of female remating in two tephritid flies

The inhibition of female receptivity after copulation is usually related to the quality of the first mating. Males are able to modulate female receptivity through various mechanisms. Among these is the transfer of the ejaculate composed mainly by sperm and accessory gland proteins (AGPs). Here we used the South American fruit fly Anastrepha fraterculus (where AGP injections inhibit female receptivity) and the Mexican fruit fly Anastrepha ludens (where injection of AGPs failed to inhibit receptivity) as study organisms to test which mechanisms are used by males to prevent remating. In both species, neither the act of copulation without ejaculate transfer nor sperm stored inhibited female receptivity. Moreover, using multiply mated sterile and wild males in Mex flies we showed that the number of sperm stored by females varied according to male fertility status and number of previous matings, while female remating did not. We suggest female receptivity in both flies is inhibited by the mechanical and/or physiological effect of the full ejaculate. This finding brings us closer to understanding the mechanisms through which female receptivity can be modulated.     

Microsatellite analysis reveals remating by wild Mediterranean fruit fly females,Ceratitis capitata

Accurate estimates of remating in wild female insects are required for an understanding of the causes of variation in remating between individuals, populations and species. Such estimates are also of profound importance for major economic fruit pests such as the Mediterranean fruit fly (Ceratitis capitata). A major method for the suppression of this pest is the sterile insect technique (SIT), which relies on matings between mass-reared, sterilized males and wild females. Remating by wild females will thus impact negatively on the success of SIT. We used microsatellite markers to determine the level of remating in wild (field-collected) Mediterranean fruit fly females from the Greek Island of Chios. We compared the four locus microsatellite genotypes of these females and their offspring. Our data showed 7.1% of wild females remated. Skewed paternity among progeny arrays provided further evidence for double matings. Our lowest estimate of remating was 3.8% and the highest was 21%.     

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.     

Potential for controlling codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) in Argentina using the sterile insect technique and egg parasitoids

Codling moth is the main pest affecting apples and pears worldwide. Most pest control strategies used against this insect have relied on the use of broad‐spectrum insecticides which have led to non‐desirable effects like pesticide resistance, residues in the environment, human health concerns and the reduction of access to international markets. Therefore, alternative pest control strategies that would result in sustainable fruit production systems while taking care of the environment are strongly promoted. The use of the sterile insect technique has proven to be a valuable pest control tactic within area‐wide integrated pest management strategies, and its synergistic effect for Lepidoptera pests when combined with other biological control tactics such as parasitoids has been documented. The purposes of this research were to evaluate the response of an Argentinean codling moth strain to a sub‐sterilizing radiation dose of 100 Gy and to assess the acceptability and suitability of sterile codling moth eggs by the egg parasitoids, Trichogramma cacoeciae (Marchal) and Trichogramma nerudai (Pintureau and Gerding). Irradiated female moths survived better than irradiated male moths and non‐irradiated male and female moths. Also, the fecundity of irradiated female moths was reduced by more than 30% as compared to non‐irradiated ones whereas their fertility was close to zero. The F₁ generation was male biased with a lower fertility (inherited sterility) than the parental generation. Trichogramma cacoeciae and T. nerudai parasitized both fertile and sterile eggs. However, there was a significant reduction in acceptability for sterile eggs. Trichogramma nerudai parasitized more eggs than T. cacoeciae, but egg acceptability for this species was proportionally lower than for T. cacoeciae especially on eggs oviposited by irradiated females. Development to adult of both parasitoids species was not substantially affected by the origin of the eggs and the wasps had acceptable levels of adult emergence, survival and fecundity. These results provided useful information on the potential for controlling the codling moth using egg parasitoids and the sterile insect technique in Argentina.     

Sexual behavior of mutant strains of the medfly Ceratitis capitata (Diptera: Tephritidae)

Males of the mutant strains (blind, vestigal-winged) of the Mediterranean fruit fly (medfly), Ceratits capitata (Wiedmann) showed differences in behavior compared with control (mass-reared) males. Mutant males made fewer mating attempts and achieved fewer matings than control males. Vestigal-winged females copulated less frequently with both mutants. Blind males climbed rather than jumped onto females and copulated in very low numbers compared with control and vestigal males. Blind females copulated normally with control, males and in very low numbers with both types of mutant males.     

Precocious Mexican fruit fly methoprene‐fed males inhibit female receptivity and perform sexually as mature males

The sterile insect technique (SIT) has been used successfully for the control of fruit flies. The efficiency of this technique can be significantly reduced when sterile released insects are exposed to adverse conditions and predators, as a great number of sterile insects die before reaching sexual maturity and thus fail to mate with wild females. Treatments with juvenile hormone (JH) analogues such as methoprene (M) significantly reduce the time to reach sexual maturity by sterile Anastrepha ludens (Loew) (Diptera: Tephritidae) males. In this study, we compared the sexual performance of non‐treated sexually mature males with young males that had been sexually accelerated with M. Furthermore, we compared the ability of M‐fed males in inhibiting female remating compared with sexually mature males. Results showed that at 5 days M‐fed males had lower mating success than mature males; however, 6‐day‐old (0.1%) M‐fed males had the same amount of matings as mature 13‐day‐old males. Young 5‐ to 10‐day‐old M‐fed males also had similar number of matings as mature non‐treated 12‐ to 17‐day‐old males. There were no differences in copula duration between treatments. Moreover, there were no differences between the fertility, fecundity or refractory period of females mated with either young male fed M or normal sexually mature males. These results indicated that young males that were sexually accelerated with M have the same sexual performance as non‐treated sexually mature males. Implications of using M as a pre‐release treatment for A. ludens controlled through SIT are discussed.