Combining the Sterile Insect Technique with the Incompatible Insect Technique: I-Impact of Wolbachia Infection on the Fitness of Triple- and Double-Infected Strains of Aedes albopictus

The mosquito species Aedes albopictus is a major vector of the human diseases dengue and chikungunya. Due to the lack of efficient and sustainable methods to control this mosquito species, there is an increasing interest in developing and applying the sterile insect technique (SIT) and the incompatible insect technique (IIT), separately or in combination, as population suppression approaches. Ae. albopictus is naturally double-infected with two Wolbachia strains, wAlbA and wAlbB. A new triple Wolbachia-infected strain (i.e., a strain infected with wAlbA, wAlbB, and wPip), known as HC and expressing strong cytoplasmic incompatibility (CI) in appropriate matings, was recently developed. In the present study, we compared several fitness traits of three Ae. albopictus strains (triple-infected, double-infected and uninfected), all of which were of the same genetic background (“Guangzhou City, China”) and were reared under the same conditions. Investigation of egg-hatching rate, survival of pupae and adults, sex ratio, duration of larval stages (development time from L₁ to pupation), time to emergence (development time from L₁ to adult emergence), wing length, female fecundity and adult longevity indicated that the presence of Wolbachia had only a minimal effect on host fitness. Based on this evidence, the HC strain is currently under consideration for mass rearing and application in a combined SIT-IIT strategy to control natural populations of Ae. albopictus in mainland China.     

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.     

The Effects of a Tsetse DNA Virus Infection on the Functions of the Male Accessory Reproductive Gland in the Host Fly Glossina morsitans centralis (Diptera; Glossinidae)

Freshly deposited third instar Glossina morsitans centralis larvae were infected with the tsetse DNA virus by microinjection, and at emergence adult males were separated from the females and fed on rabbit blood every second day for 8 days. A control group treated with sterile saline were handled similarly. They were dissected, and comparative observations made on the appearance and size of the accessory reproductive glands (ARG) in infected and control males. Regularly fed 8-day-old males from infected and control groups were mated to 2-day-old normal females obtained from the insectay. After separation from copula, the females were dissected and the uteri examined for the presence and quality of the spermatophore. The spermathecae were also examined for insemination. ARG tissues from the control and virus infected regularly fed 8-day-old male flies were fixed and processed for electron microscopic studies. The ARGs from control flies were found to be milky in appearance, whereas those from virus-infected flies were transparent in most parts. The ARGs from virus-infected males were significantly smaller in diameters (F = 42.26, p < 0.0001) and shorter (F = 200.4, p < 0.0001) than those of the controls. Most of the virus-infected males failed to form a complete spermatophore, whereas almost all the controls formed complete spermatophore as observed in the uteri of the female mates (Χ² = 111.661, p < 0.0001). The infected males that formed partial spermatophores and those that did not form any at all failed to inseminate their female mates. Histological studies of the ARGs revealed some lesions in the epithelial cells characterized by degeneration of cytoplasmic organelles and detachment of the muscle layer from the basal plasma membrane. However, no virus particles were observed in the affected cells. Received: 18 November 1998 / Accepted: 10 December 1998     

No Evidence for the Effect of MHC on Male Mating Success in the Brown Bear

Mate choice is thought to contribute to the maintenance of the spectacularly high polymorphism of the Major Histocompatibility Complex (MHC) genes, along with balancing selection from parasites, but the relative contribution of the former mechanism is debated. Here, we investigated the association between male MHC genotype and mating success in the brown bear. We analysed fragments of sequences coding for the peptide-binding region of the highly polymorphic MHC class I and class II DRB genes, while controlling for genome-wide effects using a panel of 18 microsatellite markers. Male mating success did not depend on the number of alleles shared with the female or amino-acid distance between potential mates at either locus. Furthermore, we found no indication of female mating preferences for MHC similarity being contingent on the number of alleles the females carried. Finally, we found no significant association between the number of MHC alleles a male carried and his mating success. Thus, our results provided no support for the role of mate choice in shaping MHC polymorphism in the brown bear.     

Effect of O. porcinus Tick Salivary Gland Extract on the African Swine Fever Virus Infection in Domestic Pig

African swine fever is a haemorrhagic disease in pig production that can have disastrous financial consequences for farming. No vaccines are currently available and animal slaughtering or area zoning to restrict risk-related movements are the only effective measures to prevent the spread of the disease. Ornithodoros soft ticks are known to transmit the African swine fever virus (ASFV) to pigs in farms, following the natural epidemiologic cycle of the virus. Tick saliva has been shown to modulate the host physiological and immunological responses during feeding on skin, thus affecting viral infection. To better understand the interaction between soft tick, ASFV and pig at the bite location and the possible influence of tick saliva on pig infection by ASFV, salivary gland extract (SGE) of Ornithodoros porcinus, co-inoculated or not with ASFV, was used for intradermal auricular inoculation. Our results showed that, after the virus triggered the disease, pigs inoculated with virus and SGE presented greater hyperthermia than pigs inoculated with virus alone. The density of Langerhans cells was modulated at the tick bite or inoculation site, either through recruitment by ASFV or inhibition by SGE. Additionally, SGE and virus induced macrophage recruitment each. This effect was enhanced when they were co-inoculated. Finally, the co-inoculation of SGE and virus delayed the early local spread of virus to the first lymph node on the inoculation side. This study has shown that the effect of SGE was powerful enough to be quantified in pig both on the systemic and local immune response. We believe this model should be developed with infected tick and could improve knowledge of both tick vector competence and tick saliva immunomodulation.     

Effects of Male age and Size on Mating Success in the Bumblebee Bombus terrestris

In social insects, the reproductive strategies adopted by colonies emerge as a complex property of individual behaviours, but as yet we are often unable to fully explain them in evolutionary terms. In bumblebees, colonies adopt either a short-lived strategy specializing in male production, or a longer-lived strategy in which mainly new queens are produced, but this results in males emerging long before mates are available; this strategy can only easily be explained if older males are at a significant reproductive advantage. Here we examine how age and morphological characters influence mating success of male bumblebees. In two separate experiments in which single virgin males and females were confined together, we found that young males and heavy males mated more swiftly and copulated for less time compared to old males or lighter males, respectively. However, in competitive situations age proved to be unimportant and the only factors to influence mating success were the lengths of the fore and hind tibiae, with strong directional selection for long leg length. Fore and hind legs are both used in courtship, so both traits are associated with plausible mechanisms under selection. It has previously been argued that, in times of food stress, bumblebee colonies should produce males as male size is less likely to be strongly correlated with fitness than female size. Our results suggest that this may not be so, since aspects of male size directly impact on their mating success. Our results leave unexplained the emergence of males many days before new queens.     

Starvation and the Mating Success of Wild Male Mediterranean Fruit Flies (Diptera: Tephritidae)

Recent evidence suggests that the nutritional state of male Mediterranean fruit flies, Ceratitis capitata (Wied.) (medfly), is an important influence on various components of their reproductive biology, including mating success. The objective of the present study was to examine experimentally the effect of temporary starvation on the mating success of wild male C. capitata. Males were maintained on protein–sugar or sugar-only diets, and for each diet we compared the mating success of continuously fed males versus males starved for 18 or 24 h immediately before testing. In trials conducted on field-caged, host trees, males starved for 24 h obtained only about half as many matings as fed males for both diets. However, when the starvation period was 18 h, starved males reared on the protein–sugar diet mated significantly less frequently than fed males, whereas starved males reared on sugar mated as often as fed males. Measurements of male pheromone calling and female attraction revealed that reduced mating success likely reflected the decreased signaling activity of starved males.     

No Evidence for Significant Effect of Body Size and Age on Male Mating Success in the Spot-legged Treefrog

In anurans, body size and age of individuals generally affect male mating success. To test whether body size and age have effects on male mating success in the foam-nesting treefrog Polypedates megacephalus, a species widely distributed in China, we analyzed differences in body size and age between mated and unmated males for three populations using a Generalized Linear Mixed Model(GLMM). The results showed that mated males did not exhibit larger body size and older age than unmated males, suggesting that large and/or old male individuals did not have greater mating success than small and/or young males. Moreover, we also found a non-significant size-assortative mating pattern for all populations. Our findings suggest that body size and age of the foam-nesting treefrog do not affect male mating success.     

The Male Accessory Gland Substances of Lucilia illustris (I): Its Effect on Mating Receptivity

ABSTRACT In order to verify the physiological functions of male accessory gland (MAG) substances of the blowfly, Lucilia illustris Meigen, the growth and changes in metabolites of male accessory gland with aging, and the effect of male accessory gland extract on mating receptivity of L. illustris were examined. There was not a significant difference in length of MAG with aging, but the width of MAG was grown widely from 0 to 5-days old apparently. Changes in glycogen, total protein and lipid of male accessory gland substances showed same patterns but total protein content was higher than the others. Therefore, male nutrition affected the production of male accessory proteins. During the mating fed unmated male transferred larger amount of protein than that of starved male. When male accessory gland homogenates injected, mating receptivity of gravid female was greatly inhibited comparing to control, suggesting that the accessory gland substances of L. illustris alters female mating receptivity after mating.     

Reproductive Strategies of Aedes albopictus (Diptera: Culicidae) and Implications for the Sterile Insect Technique

Male insects are expected to optimize their reproductive strategy according to the availability of sperm or other ejaculatory materials, and to the availability and reproductive status of females. Here, we investigated the reproductive strategy and sperm management of male and virgin female Aedes albopictus, a mosquito vector of chikungunya and dengue viruses. The dynamics of semen transfer to the female bursa inseminalis and spermathecae were observed. Double-mating experiments were conducted to study the effect of time lapsed or an oviposition event between two copulations on the likelihood of a female double-insemination and the use of sperm for egg fertilization; untreated fertile males and radio-sterilised males were used for this purpose. Multiple inseminations and therefore the possibility of sperm competition were limited to matings closely spaced in time. When two males consecutively mated the same female within a 40 min interval, in ca. 15% of the cases did both males sire progeny. When the intervals between the copulations were longer, all progeny over several gonotrophic cycles were offspring of the first male. The mating behavior of males was examined during a rapid sequence of copulations. Male Ae. albopictus were parceling sperm allocation over several matings; however they would also attempt to copulate with females irrespective of the available sperm supply or accessory gland secretion material. During each mating, they transferred large quantities of sperm that was not stored for egg fertilization, and they attempted to copulate with mated females with a low probability of transferring their genes to the next generation. The outcomes of this study provided in addition some essential insights with respect to the sterile insect technique (SIT) as a vector control method.     

Condition-Dependent Mating Success in Male Fruit Flies: Ingestion of a Pheromone Precursor Compensates for a Low-Quality Diet

Previous studies revealed that males of the oriental fruit fly, Bactrocera dorsalis, require protein in the adult diet to obtain matings and that ingestion of methyl eugenol, which acts as a pheromone precursor, increases male attractiveness and mating competitiveness. The goal of this study was to investigate the interaction between diet quality and methyl eugenol consumption in affecting the mating frequency of B. dorsalis males. In one set of experiments, mature males were deprived of protein for 1, 3, or 7 days and were either given or denied access to methyl eugenol (ME). These males competed against control males (continuously protein-fed, no feeding on ME) for copulations in field cages. Without ME, males held without protein for 3 or 7 days obtained significantly fewer matings than control males. With ME, however, males held for even 7 days without protein achieved higher mating success than control males. In a second set of experiments, mature males were held without protein for 7 days and then given a protein-rich diet for 1, 3, or 7 days before testing and were either given or denied access to ME. Without ME, males were competitively inferior to control males when tested 1 or 3 days after resumption of protein feeding and equivalent to control males only after 7 days of protein feeding. With ME, however, males obtained significantly more matings than control males when tested 3 or 7 days after resumed protein feeding and had similar mating success as control males after 1 day of access to the protein-rich diet. Results show that mating success in this species is condition-dependent, with both nutritional state and ME consumption influencing male mating success. Under the test conditions, feeding on ME counteracted a low quality diet and enhanced male mating success.     

The Impact of Hepatitis A Virus Infection on Hepatitis C Virus Infection: A Competitive Exclusion Hypothesis

We address the observation that, in some cases, patients infected with the hepatitis C virus (HCV) are cleared of HCV when super-infected with the hepatitis A virus (HAV). We hypothesise that this phenomenon can be explained by the competitive exclusion principle, including the action of the immune system, and show that the inclusion of the immune system explains both the elimination of one virus and the co-existence of both infections for a certain range of parameters. We discuss the potential clinical implications of our findings.     

Ultrastructural evidence for trans-ovum transmission of the DNA virus of tsetse,Glossina pallidipes (Diptera: Glossinidae)

The occurrence of vertical transmission of the DNA virus of tsetse was studied in virus-infected, femaleGlossina pallidipes with hypertrophied salivary glands (HSG). Ultrastructural examination of tissue components of ovaries of these females revealed virus particles within both germ cell cystocyte clusters and in the follicles, sparsely distributed within nurse cells and in the oocyte cytoplasm. The presence of the virus particles within the ooplasm demonstrates the ovum as a vehicle through which theG. pallidipes virus is disseminated in nature.     

Vibratory Communication in the Jumping Spider Phidippus clarus: Substrate‐borne Courtship Signals are Important for Male Mating Success

Recently, work has shown that multimodal communication is common throughout the animal kingdom but the function of multimodal signals is still poorly understood. Phidippus clarus are jumping spiders in which males produce multimodal (visual and vibrational) signals in both male–male (aggressive) and male–female (courtship) contexts. The P. clarus mating system is complex, with sex ratios and the level of male competition changing over the course of the breeding season. Vibrational signal components have been shown to function in male aggressive contests but their role in courtship has not been investigated. Here, we performed an experiment to test the role of vibrational signaling in courtship by observing mating success for males that were experimentally muted. We show that vibratory courtship signals, and in particular signaling rate, is an important component of mating success and potentially a target of female choice. While the ability to produce vibratory signals significantly increased mating success, some muted males were still able to successfully mate. In these trials, signaling rate also predicted mating success suggesting that redundant signal components may compensate for errors and perturbations in signal transmission or that vibratory signals function to enhance the efficacy of visual signals.     

Combining the Sterile Insect Technique with Wolbachia-Based Approaches: II- A Safer Approach to Aedes albopictus Population Suppression Programmes,Designed to Minimize the Consequences of Inadvertent Female Release

Due to the absence of a perfect method for mosquito sex separation, the combination of the sterile insect technique and the incompatible insect technique is now being considered as a potentially effective method to control Aedes albopictus. In this present study first we examine the minimum pupal irradiation dose required to induce complete sterility in Wolbachia triple-infected (HC), double-infected (GUA) and uninfected (GT) female Ae. albopictus. The HC line is a candidate for Ae. albopictus population suppression programmes, but due to the risk of population replacement which characterizes this triple infected line, the individuals to be released need to be additionally irradiated. After determining the minimum irradiation dose required for complete female sterility, we test whether sterilization is sufficient to prevent invasion of the triple infection from the HC females into double-infected (GUA) populations. Our results indicate that irradiated Ae. albopictus HC, GUA and GT strain females have decreased fecundity and egg hatch rate when irradiated, inversely proportional to the dose, and the complete sterilization of females can be acquired by pupal irradiation with doses above 28 Gy. PCR-based analysis of F₁ and F₂ progeny indicate that the irradiated HC females, cannot spread the new Wolbachia wPip strain into a small cage GUA population, released at a 1:5 ratio. Considering the above results, we conclude that irradiation can be used to reduce the risk of population replacement caused by an unintentional release of Wolbachia triple-infected Ae. albopictus HC strain females during male release for population suppression.     

Impact of the Timing of Male Emergence on Mating Capacity of Males in Trichogramma evanescens Westwood

In species with highly structured population, such as Trichogramma spp., mating occurs mostly at emergence on the patch and early emerging males have an advantage, as they are present when the first females emerge. However, early emergence of male could also enable males to mature sexually before the emergence of females or enhance their capacity to induce higher receptivity in females. We measured time of emergence of male and female Trichogramma evanescens Westwood (Hymenoptera: Trichogrammatidae) from hosts that were parasitized within a 30 min period. We also measured the effect of male age on their capacity to mate, and the ability of inseminated females to produce daughters. To verify if early emerging males induced higher receptivity in females, we observed the females mate choice between males of different ages. Our results indicated that the mean time of emergence between males and females was 29 min for individuals that develop in 9 days and 10 min for individuals that develop in 10 days. The protandry observed in this species could be the result of either a faster development of males or a male first strategy by the ovipositing female. In T. evanescens, protandry does not permit males to mature sexually nor to induce higher receptivity in females. The main advantage of protandry for males thus appears to be early access to females as they emerge.     

The Effect of Abiotic Factors on the Male Mate Searching Behavior and the Mating Success of Aphidius ervi (Hymenoptera: Aphidiidae)

The effect of wind speed and distance from the source on the male response of the aphid parasitoid, Aphidius ervi (Hymenoptera: Aphidiidae), to a pheromone source was studied in a wind tunnel. The number of males taking flight, entering the plume and successfully reaching the source, decreased at wind speeds >50 cm/s. Furthermore, the proportion of those attempting upwind flight that fell to the ground increased with increasing wind speed. In contrast, distance from the source had no significant effect on any of the parameters examined. While male flight behavior was significantly reduced at 70 cm/s, some males walked to the source when there was a bridge connecting the pheromone source and the release platform. This suggests that ambulatory behavior could be a significant component of male mate searching in A. ervi when wind conditions are too strong for upwind flight. The possible effects of variation in atmospheric pressure on male flight behavior to the long distance pheromone, as well as to the short distance one, were also investigated. No significant effects of atmospheric pressure were observed. These findings differ significantly from those previously reported for another aphid parasitoid, A. nigripes, and the reasons for such differences are discussed.     

Exploitation of the Medfly Gut Microbiota for the Enhancement of Sterile Insect Technique: Use of Enterobacter sp. in Larval Diet-Based Probiotic Applications

The Mediterranean fruit fly (medfly), Ceratitis capitata, is a pest of worldwide substantial economic importance, as well as a Tephritidae model for sterile insect technique (SIT) applications. The latter is partially due to the development and utilization of genetic sexing strains (GSS) for this species, such as the Vienna 8 strain, which is currently used in mass rearing facilities worldwide. Improving the performance of such a strain both in mass rearing facilities and in the field could significantly enhance the efficacy of SIT and reduce operational costs. Recent studies have suggested that the manipulation of gut symbionts can have a significant positive effect on the overall fitness of insect strains. We used culture-based approaches to isolate and characterize gut-associated bacterial species of the Vienna 8 strain under mass rearing conditions. We also exploited one of the isolated bacterial species, Enterobacter sp., as dietary supplement (probiotic) to the larval diet, and we assessed its effects on fitness parameters under the standard operating procedures used in SIT operational programs. Probiotic application of Enterobacter sp. resulted in improvement of both pupal and adult productivity, as well as reduced rearing duration, particularly for males, without affecting pupal weight, sex ratio, male mating competitiveness, flight ability and longevity under starvation.