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.     

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.     

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.     

Copulation, the dynamics of sperm transfer and female refractoriness in the leafhopper Balclutha incisa (Hemiptera: Cicadellidae: Deltocephalinae)

Abstract.  Mature sperm of the leafhopper Balclutha incisa (Matsumara) (Cicadellidae: Auchenorrhyncha: Hemiptera) are stored as a series of sperm bundles within seminal vesicles prior to ejaculation. During transfer, sperm are pumped from the vesicles into the ejaculatory duct to the complex aedeagus. Sperm transfer is marked by a c . 30-fold expansion of the spermatheca to accommodate both sperm and seminal fluid. Sperm number increases exponentially with male age, reaching a maximum of 700 000 after 14 days, while the number of sperm available on days 2–5 is between 70 000 and 100 000. During mating, maximum sperm transfer occurs after 7 min and mating is complete after about 10 min. Ejaculate size, defined by both sperm and associated accessory gland fluid, is influenced by male mating status and the interval since the previous mating. There is a positive correlation between duration of copulation and both ejaculate and the time to subsequent mating. Sperm are more likely to be retained in the testes during mating by males of 2–5 days post-emergence than older males. The number of sperm received by the female can be manipulated experimentally by mating males once (medium ejaculate) or twice (small ejaculate) immediately after their first mating. Females that receive small ejaculates from sperm-depleted males have a far shorter refractory period than females receiving medium to large ejaculates. Both ejaculate size and the time after males have mated influence the female post-mating refractory period as measured by the female's responsiveness to male sexual signalling.     

Effect of irradiation on mating performance and mating ability in the West Indian sweetpotato weevil, Euscepes postfasciatus

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 not only damages the reproductive cells but the somatic cells as well. The mating behavior of irradiated males may be altered over time due to the depressed metabolic activity brought about by sterilization. In this study, we evaluated the mating behavior (copulation behavior, mating performance, and ability of sperm transfer) of irradiated males in Euscepes postfasciatus (Fairmaire) (Coleoptera: Curculionidae) for 16 days after irradiation in the laboratory. The mating performance of males irradiated with a 150 Gy dose, as currently used in the SIT program in Okinawa prefecture for E. postfasciatus, decreased compared to that of control after day 7. As a result, we considered that irradiation had no major effect on male mating behavior for approximately 1 week after irradiation.     

Disparity in sexual behaviour between wild and mass‐reared Mexican fruit flies

The sterile insect technique (SIT) is currently used to control Mexican fruit fly Anastrepha ludens Loew (Diptera: Tephritidae). However, mass‐rearing can alter the quality of released males. If males that are mass‐reared have behaviours different from those of their wild counterparts, then this may diminish the effectiveness of SIT. Questions remain as to whether wild females may be able to detect the male condition before, during and/or after copulation with a mass‐reared male. In the present study, copula duration, female remating, female fecundity and fertility of both mass‐reared and wild A. ludens are evaluated. Marked differences are found between mass‐reared and wild females. Specifically, mating latency is longer and copula duration is shorter for wild females compared with mass‐reared females. Importantly, there are no significant differences in mating latency, copula duration or remating probability between wild females paired with either mass‐reared or wild males. All mass‐reared females remate, whereas only approximately half of the wild females remate after first mating with either a wild or mass‐reared male. Fecundity of wild females mated to either wild or mass‐reared males is approximately one‐third lower than that of mass‐reared females, confirming that mass‐reared females may have been selected for high fecundity and are adapted to laboratory conditions. Fertility of females that mate with a wild male for only 10 min is not significantly different from that achieved via a full‐length copulation. By contrast, females mating with mass‐reared males need copulation durations of at least 40 min to achieve fertility comparable with that achieved via a full‐length copulation. The findings of the present study have important implications for A. ludens controlled through SIT and broaden our understanding on the copulatory and post‐copulatory behaviours between wild females and mass‐reared males.     

Reproductive biology of the predator Macrolophus caliginosus: Effect of age on sexual maturation and mating

Macrolophus caliginosus is a polyphagous mirid bug native to the Mediterranean area where it is widely used as a biological control agent, both in protected and open-field vegetable crops. Its reproductive biology remains largely unknown and a better understanding of it will improve mass rearing and release strategies. In the present work, we studied the development of reproductive organs in females and males and how age conditioned receptiveness to mating in both sexes.Females with mature eggs in their ovarioles appear 3–4 days after adult molt with a similar pattern of ovary maturation in virgin females as in females that were kept with males of the same age. Newly molted males were found to already have active sperm in their seminal vesicles but their accessory glands did not completely develop until 3 days later. Receptivity for copulation paralleled ovarian and accessory gland maturation.     

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.     

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.     

Assessing mating performance of male Glossina pallidipes (Diptera: Glossinidae) using a walk-in field cage

To monitor the quality of male tsetse for use in the sterile insect technique (SIT), a field cage test was developed and evaluated. Mating competitiveness was tested with male Glossina pallidipes Austen that emerged from pupae stored for different periods at 15 degrees C. Control males emerged from pupae stored at 23-24 degrees C and emerged at 26.5 degrees C. Each sample of test males was divided into two groups with one group being irradiated at 120 Gy; the other group was not irradiated. More than 70% of the maximum possible number of mating pairs occurred in all tests. Males emerged from pupae kept at low temperature and then irradiated formed a greater proportion of mating pairs than the controls. Males emerged from pupae kept at 15 degrees C generally started mating more quickly than the standard colony males although there was no significant difference. Insemination rates were above 99%. Pooled data indicated that mean spermathecal values for females mated with irradiated males were significantly lower than for control males. The duration of copulation varied significantly between treatment groups and was significantly longer for irradiated male flies; there was no correlation between duration of copulation and mean spermathecal value.     

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.     

Female presence enhances sexual performance of sterile Anastrepha ludens males of the Tapachula-7 GSS strain

The sterile insect technique (SIT), used for the control of many tephritid fly pests, is based on the rearing and release of large numbers of sexually competitive sterile insects into a wild population. In the interest of reducing expenses and increasing SIT effectiveness, genetic sexing strains (GSS) have been developed. These strains allow the production and release of only males. The objective of our study was to assess the effects of pre-release adult exposure to methoprene and to females on the mating propensity and mating competitiveness of GSS sterile males of Anastrepha ludens (Loew) (Diptera: Tephritidae). GSS sterile males were kept on a protein-sugar (protein-fed) or a protein-sugar-methoprene diet and were exposed to different proportions of females for the normal pre-release period of 5 days. Using laboratory and field-cage bioassays, we examined the influence of methoprene and female presence on the mating success of sterile males of 3–9 days old, in competition for wild females with untreated males and with wild males. Methoprene and female exposure had no significant effects on male mating success in the laboratory, whereas age had a positive relationship with the number of copulations observed. However, in field-cage bioassays, males exposed to females obtained a higher number of copulations than unexposed control males. Possible implications of these findings for programs that use GSS and especially for the campaign against Mexican fruit flies are discussed.     

The mating-induced refractoriness of Lucilia cuprina females: manipulating the male contribution

Abstract Three manipulations of the male contribution to copulation were used to investigate what determines the switch-off of female receptivity that follows mating in Lucilia cuprina. Multiple matings by males led to a reduction in their effectiveness at switching-off females: the first twelve matings caused more than 50% of females to be switched-off 1 day after mating, while the first four matings caused more than 50% of females to be switched-off for 7 days after mating. Males mated up to twenty-three times and on average ten times during 10 h of continuous access to virgin females. The number of sperm transferred declined logarithmically through a series of matings as did the quantity of material transferred from the male accessory gland (measured as radiolabeled material) through the first six matings. Overnight isolation of multiply-mated males led to considerable recovery of their ability to switch-off females, and to limited renewal of their ability to transfer sperm. Castrated males transferred similar quantities of accessory gland material to females as did sham-operated males during their first five matings and in their first three matings switched-off 95% of females for 1 day but only 48% for 7 days. When normal mating pairs were separated at increasing intervals after coupling, an increasing proportion of females were switched-off at 1 or 7 days afterward. 7 days after 2 min matings no females were switched-off though 40% of the number of sperm transferred in a full mating had been transferred by this time. The proposed explanation for these data is that both the initial switch-off and its duration are determined by the quantity of a receptivity-inhibiting substance that normally enters the female haemo-lymph after being injected by the male into the wall of the bursa copulatrix. It is proposed that when castrated males mate, an absence of sperm results in most accessory gland secretion entering the empty spermathecae (rather than the wall of the bursa as usually occurs) and hence being unable to reach the haemolymph and exert its influence. The effective dose of the receptivity-inhibiting substance is measured on a logarithmic scale.     

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.     

Anopheles culicifacies: effects of age on the male reproductive system and mating ability of virgin adult mosquitoes

Abstract. Under controlled laboratory conditions of 28–30^oC and 16:8 L:D photoperiod, an attempt was made to develop an age-grading technique for Anopheles culicifacies males. Mating activity was maximal when females were 5–12 days old and males were 5–7 days old. The numbers of total and mature spermatocysts declined significantly with age, and the proportion of the testes occupied by the sperm reservoir increased as virgin males grew older. Mating resulted in the loss of spermatozoa and accessory gland substance from the reproductive system. Loss of mating ability of older virgin males seemed to be age-related, because the reproductive system contained ample supplies of accessory gland substance and spermatozoa. Morphological changes of the reproductive system, due to mating and age, were used to infer the age and reproductive history of unknown males in a laboratory evaluation.     

Effects of male age on the mating behavior of both sexes in the sorghum plant bug, Stenotus rubrovittatus (Hemiptera: Miridae)

In the sorghum plant bug Stenotus rubrovittatus (Matsumura) (Hemiptera: Miridae), mating behavior consists of male courtship and female mating receptivity. Previous studies have found that female age is correlated with ovary development and that mature females with developed ovaries are more receptive to male courtship. Thus, we examined whether male age affects the mating behavior of both sexes and male accessory gland development. Unmated males 0–9 days after emergence and 3-day-old virgin females receptive to male courtship were studied. Immediately after emergence, only 20 % of the males courted females (n = 25). At 3 days old, 68 % of the males courted females (n = 25), the most active age. In contrast, more than 75 % of the courted females were receptive to the male courtship regardless of male age. These results indicate that only male courtship behavior is affected by male age, although it is not enhanced in proportion to male age. Male accessory glands developed with male age. However, no clear relationship was detected between male courtship behavior and accessory gland development. In S. rubrovittatus, it is difficult to explain male courtship behavior solely from male age and accessory gland development.     

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.     

The control of receptivity and ovulation in the tsetse fly, Glossina morsitans

ABSTRACT. A rapid decline in receptivity of mated female Glossina morsitans morsitans Westwood is shown to depend on both physical and chemical stimuli associated with copulation. Radiolabelling revealed the transfer of substances from the male to the haemolymph of the female during copulation. Implantation of male tissues or their injection as homogenates into virgin females showed the chemical stimulus to come from the male accessory glands. Receptivity decreased in females mated to males with ejaculatory ducts severed or testes removed and also in females which had a glass bead inserted into their uterus and/or the tip of their abdomen covered with wax, suggesting that a physical stimulus inducing refractoriness is provided by distension of the uterus and/or stimulation of their terminalial setae. Exposing virgin females to daily short matings in which no male materials were transferred, confirmed this. Receptivity also declined slowly with age in unexposed virgin females. Transfusion of haemolymph from mated females (up to 11 days old) into virgins did not indicate the existence of a haemolymph-borne ovulation-inducing factor; apparently only physical stimuli from mating are involved in the induction of ovulation, and somehow prime the ovarian tissue so that it responds appropriately later when the egg has matured. Whether the stimulus is transmitted to the ovary neurally or hormonally is unknown.     

Males of the seed bug Togo hemipterus (Heteroptera: Lygaeidae) use accessory gland substances to inhibit remating by females

In species in which females mate repeatedly, males can adopt several strategies to reduce the risk of sperm competition with future males. The refractory period of females significantly increased as the mating duration increased in the seed bug Togo hemipterus (Heteroptera: Lygaeidae). To elucidate the mechanisms by which mated females are inhibited from remating, we investigated the effects of male-derived substances on the inhibition of mating receptivity of virgin females by injecting the substances into their abdomens. The length of time from injection to mating in virgin females was significantly longer for females injected with accessory gland B solution than for those injected with seminal vesicle, accessory gland A, or control solutions. This is the first report showing that heteropteran males inhibit female remating by using substances from an accessory gland. We discuss and consider the adoption and evolution of this strategy by T. hemipterus males by focusing on female genitalia structures, oviposition habit, and paternity and comparing these traits with those of other heteropterids.     

Impact of Salivary Gland Hypertrophy Virus Infection on the Mating Success of Male Glossina pallidipes: Consequences for the Sterile Insect Technique

Many species of tsetse flies are infected by a virus (GpSGHV) that causes salivary gland hypertrophy (SGH). Female Glossina pallidipes (Austen) with SGH symptoms (SGH+) have reduced fecundity and SGH+ male G. pallidipes are unable to inseminate female flies. Consequently, G. pallidipes laboratory colonies with a high prevalence of SGH have been difficult to maintain and have collapsed on several occasions. To assess the potential impact of the release of SGH+ sterile male G. pallidipes on the efficacy of an integrated control programme with a sterile insect technique (SIT) component, we examined the mating efficiency and behaviour of male G. pallidipes in field cages in relation to SGH prevalence. The results showed in a field cage setting a significantly reduced mating frequency of 19% for a male G. pallidipes population with a high prevalence of SGH (83%) compared to 38% for a male population with a low prevalence of SGH (7%). Premating period and mating duration did not vary significantly with SGH status. A high percentage (>80%) of females that had mated with SGH+ males had empty spermathecae. The remating frequency of female G. pallidipes was very low irrespective of the SGH status of the males in the first mating. These results indicate that a high prevalence of SGH+ in G. pallidipes not only affects colony stability and performance but, in view of their reduced mating propensity and competitiveness, releasing SGH+ sterile male G. pallidipes will reduce the efficiency of a sterile male release programme.