Heat shock sensitivity of adult male fertility in the parasitoid wasp Anisopteromalus calandrae (Hymenoptera, Pteromalidae).

In insects, decreased reproduction is a sublethal consequence of high temperatures, with males being more sensitive to this in many species. In hymenoptera, arrhenotokous parthenogenesis means that female offspring are produced using sperm and are thus diploid, while males are haploid. Consequently, sperm stocks in males and females (after copulation) are a key regulator of the sex ratio. Anisopteromalus calandrae is a parasitoid wasp in which males can suffer from subfertility due to a drastic decrease in sperm count after exposure to high temperatures during a critical early pupal stage. However, in this species spermatogenesis continues during adulthood, therefore the heat sensitivity of adult males remains to be studied. Laboratory studies were conducted on virgin and previously mated young adult males under control (30 °C) and heat shock (10 min at 48 °C) conditions to exhaust their initial sperm stock. After heat shock, in both virgin and already mated males, the individual sperm potential was half that of controls. Both groups continuously produced sperm, but sperm stock of heat shocked males' never reached that of the controls. Heat shock reduced survival at 10 days only in previously experienced males but had no impact on the mating ability in competition for a female compared to controls. Despite a reduced sperm count, heat shocked males had fully fertile spermatozoa. Such a physiological response to heat shock in a species with continuous sperm production could be of major interest for both wild populations in a context of temperature variations and parasitoid wasps introduced for agronomical purposes.

Evaluation of Providencia rettgeri pathogenicity against laboratory Mediterranean fruit fly strain (Ceratitis capitata).

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), is often referred to as the most severe agricultural pest. Its biological control is mainly through the Sterile Insect Technique (SIT). Colonization, mass-rearing conditions and the irradiation process impact the competitiveness of sterile males and disrupt symbiotic associations by favoring some bacterial species and suppressing others. Levels of Providencia species have been shown to fluctuate considerably in the gut of the medfly laboratory strain Vienna 8 under irradiation, increasing by up to 22%. This study aimed to determine the pathogenicity of Providencia rettgeri isolated from the gut of laboratory Vienna 8 medfly strains by examining the effects of 1) two different treatment doses on egg-hatching and development and 2) two infection methodologies (ingestion and injection) of male and female adults according to their mating status. Treatment of eggs with P. rettgeri (2%) significantly decreased the mean egg to pupae recovery rate. Our data showed significant high mortality in flies with both injection and ingestion after 24 hours without any effect of sex. Microbial counts demonstrated that the bacteria could proliferate and replicate in adult flies. There was a significant sex-dependent effect after infection, with mortality decreasing significantly for males more than females. Providencia rettgeri can be considered as a potential pathogen of C. capitata. Mating protected males and females against infection by P. rettgeri by triggering an immune response leading to double the levels of Cecropin being secreted compared to infected virgin adults, thus reducing the virulence of the bacteria.

The spatio-temporal partitioning of sperm by males of the prospermatogenic parasitoid Nasonia vitripennis is in line with its gregarious lifestyle.

Male fitness depends on the number of lifetime progeny of their mates and could be constrained by the chance of finding a mate, lifespan and temporal patterns of sperm production and allocation. Here, we used the parasitic wasp Nasonia vitripennis with a two-week lifespan and a gregarious lifestyle, to analyze how the reproductive system is organized to allocate spermatozoa over consecutive matings. Results show that spermatogenesis is synchronized and completed one day before emergence so that males emerge with a full sperm complement. We also found a regulation of spermatozoa transfer between testis and seminal vesicles that allows males to partition small ejaculates over multiple matings. Overall, this study shows that for N. vitripennis, male fertilization potential is determined (1) at the pupal stage, when spermatogenesis takes place to generate a complete life-long stock, (2) on emergence, when transport of spermatozoa from testes to seminal vesicles is initiated and (3) in adulthood, during which spermatozoa are partitioned over successive copulations. Such life history-traits are consistent with the gregarious lifestyle of N. vitripennis.

A combined approach to heat stress effect on male fertility in Nasonia vitripennis: from the physiological consequences on spermatogenesis to the reproductive adjustment of females mated with stressed males.

In recent years, several studies have shown a decline in reproductive success in males in both humans and wildlife. Research on male fertility has largely focused on vertebrates, although invertebrates constitute the vast majority of terrestrial biodiversity. The reduction of their reproductive capacities due to environmental stresses can have strong negative ecological impacts, and also dramatic consequences on world food production if it affects the reproductive success of biological control agents, such as parasitic wasps used to control crop pests. Here Nasonia vitripennis, a parasitic wasp of various fly species, was studied to test the effects of 24h-heat stress applied during the first pupal stage on male fertility. Results showed that only primary spermatocytes were present at the first pupal stage in all cysts of the testes. Heat stress caused a delay in spermatogenesis during development and a significant decrease in sperm stock at emergence. Females mated with these heat-stressed males showed a reduce sperm count stored in their spermatheca. Females did not appear to distinguish heat-stressed from control males and did not remate more frequently to compensate for the lack of sperm transferred. As a result, females mated with heat-stressed males produced a suboptimal lifetime offspring sex ratio compared to those mated with control males. This could further impact the population dynamics of this species. N. vitripennis appears to be an interesting biological model to study the mechanisms of subfertility and its consequence on female reproductive strategies and provides new research perspectives in both invertebrates and vertebrates.

Fitness improvement of mass-reared sterile males of Ceratitis capitata (Vienna 8 strain) (Diptera: Tephritidae) after gut enrichment with probiotics.

Successful mass rearing is crucial for sterile insect technique programs. It has been shown that the sterilizing process using gammaradiation results in damage to midgut tissue, cellular organelles, and gut microbiota of flies. This can be responsible for the inferiority of sterile males compared with wild males. A bacteria-enhanced diet could contribute to the improvement of the fly's fitness. We investigated ways of increasing the competitiveness of mass-reared Mediterranean fruit fly, Ceratitis capitata (Wiedemann) sterile males. We tested the hypothesis that the addition of beneficial bacteria to the larvae's diet would lead to a significant increase in their levels in the gut of the sterile adults and consequently improve their size and fitness. As expected, enriching the diet of mass-rearing Vienna-8 strain larvae with beneficial bacteria (Klebsiella pneumonia, Enterobacter spp., and Citrobacter freundii) resulted in increase in the number of Enterobacteriacae communities inhabiting the male's gut and a subsequent significant increase in the size of males and other morphometric traits and enhanced sexual performance of males at emergence.

Heat stress affects male reproduction in a parasitoid wasp.

In insects, reproductive success and survival are affected by temperature. Reproduction is more sensitive than other physiological traits. While the effects of heat stress on females are well known, the effects on males are less clear. Hymenopteran parasitoids are valuable for studying the consequences of heat stress on male reproduction. In these species, through arrhenotoquous parthenogenesis, the sex ratio of the offspring is directly dependent on the sperm stock acquired by females during copulation. In the lab, heat temperature treatments (32-44°C) were applied for 3 days in the pupal stage of Anisopteromalus calandrae males, and development was completed at 30°C. Three different effects were observed depending on the temperature: mortality above 42°C, sterility of emerging males at 40°C, and sub-fertility at 38°C. This sub-fertility is characterized by a dramatic decrease in male sperm supplies, of up to 7% compared to control males. In the course of ageing, the sperm stock of sub-fertile males increases but never reaches the level of control males. Survival was significantly higher in control (30°C) males than those treated at 38°C. Male mating ability was similar whatever the treatment (control and 38°C), but females mated with 38°C-treated males stored 100 times less sperm on average than those mated with control males. The offspring sex ratio of females mated with 38°C-treated males was strongly male biased. The physiological mechanisms are as yet unknown. The relationship between temperature, sperm stock and sex ratio should be taken into account in the management of parasitoids for integrated pest management.

[Parasitic wasps, a model for studying male subfertility]. / Les hyménoptères parasitoïdes: des modèles pour l'étude de l'hypofertilité mâle.

Many studies have reported an increase in male reproductive disorders due to the environment. Despite their ecological importance invertebrates have been poorly studied; they are yet affected in the same way as vertebrates by changes in the environment. Different stresses made on parasitic wasps are able to induce a male subfertility. These insects are easy to rear in the laboratory, used to control insect pests, and have other benefits presented in this article. Parasitic wasps are good models for understanding the mechanisms inducing subfertility and may provide new areas for research in vertebrates.

Improvement of egg hatch of Ceratitis capitata (Diptera: Tephritidae) for enhanced output.

Embryonated eggs of Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) genetic sexing strain (GSS), VIENNA 8 were treated with low concentrations of five disinfectants-formaldehyde, iodine, sodium hypochlorite, peracetic acid, and quaternary ammonium--for decontamination and egg hatch improvement. The newly laid eggs were successfully treated with formaldehyde at 100 ppm for 1 min with 74.2% hatching and with quaternary ammonium at 150 ppm for 1 and 2 min with 70.4 and 69% hatching, respectively. Increased formaldehyde concentration may have affected the embryos, because it resulted in a decrease in the hatching percentage. However, egg viability was not impaired and hatch was not affected by quaternary ammonium treatment compared with controls and eggs treated with other disinfectants. Quaternary ammonium shows promise for decontaminating eggs and improving egg hatch.

Modifications of the chemical profile of hosts after parasitism allow parasitoid females to assess the time elapsed since the first attack.

In solitary parasitoids, only one adult can emerge from a given host. In some of these species, when several eggs are laid on the same host, supernumerary individuals are eliminated by lethal larval fights. In the solitary parasitoid Anisopteromalus calandrae, the probability of a second larva winning the fight depends on the time elapsed since the first oviposition. The older the first egg is at the moment a second egg is laid, the less chance the second egg has of winning the competition. As a consequence, females of this species lay their eggs preferentially on recently parasitized hosts rather than on hosts parasitized by an egg about to hatch. Anisopteromalus calandrae females parasitize bruchid larvae located in cowpea seeds. In a series of choice test experiments using an artificial seed system, we demonstrated that the cue that allows parasitoid females to differentiate between hosts parasitized for different lengths of time comes from the host and not from the artificial seed or the previously laid egg. This cue is perceived at short range, indicating that the chemicals involved are probably partly volatile. Interestingly, although parasitism stops host development, cuticular profiles continue to evolve, but in a different way from those of unparasitized hosts. This difference in the host's cuticular profile after parasitism, therefore, probably underlies the parasitoid female's discrimination.

Could hosts considered as low quality for egg-laying be considered as high quality for host-feeding?

When parasitoid females encounter a host, they can either lay an egg and thus invest in current reproduction or feed on the host and thus invest in future reproduction. However, hosts could have different values according to their parasitized status. Whereas already parasitized hosts represent poor quality for egg-laying, they could have a high nutritive value for feeding. Moreover, the optimal strategy adopted generally depends on the females' physiological state. In this study, the impact of the females' physiological state on their reproductive strategies was investigated in the solitary parasitoid Anisopteromalus calandrae. We analysed how their age and diet influenced (i) the use of hosts (feeding vs. oviposition) and (ii) host selection (previously parasitized vs. unparasitized). Our results show that both age and diet influence the reproductive strategy of A. calandrae females: old females fed with the poorer diet laid fewer eggs and made more host-feeding than others. Females also showed a preference for already parasitized hosts for feeding. This strategy cannot be explained by the nutritive value of haemolymph, as parasitized hosts carry fewer lipids. However, as parasitized hosts are also paralyzed, it could be less costly to feed on them than on unparasitized hosts.

Sperm stock and mating of males in a parasitoid wasp.

Hymenoptera are haplodiploid insects, consequently sex ratio depends on female's sperm management which itself arises from the reproductive capacity of neighbouring males. To study the influence of ageing on male reproductive potential, laboratory experiments were conducted on Dinarmus basalis (Hymenoptera, Pteromalidae) males, a tropical wasp in which sperm counts are known to constrain sex ratio. Two groups of virgin males were compared: 1-day and 30-days old. Parameters recorded were sperm quantity and viability in seminal vesicles, shape of testis, mating ability in both individual and competitive situations and sperm stored by females after male multiple mating. Older males had twice as much sperm as young males, but their reproductive capacities did not differ. They were able to copulate with 20 successive virgin females in a short period. Sperm stored in spermathecae decreased with female mating order. In competition, old and young males had the same access to females. The difference between old and young males was visible at the level of reproductive tract: young males have functional testis and old males have empty non-functional testis. Spermatozoa are kept viable in male seminal vesicles for long periods. In this species, the reproductive potential of males is not altered by ageing. At the population level this may represent an adaptation for maintaining continuous reserves of sperm at the disposal of females.

Sperm production and mating potential of males after a cold shock on pupae of the parasitoid wasp Dinarmus basalis (Hymenoptera: Pteromalidae).

For ectothermic species, temperature is a key environmental factor influencing several aspects of their physiology and ecology, acting particularly on reproduction. To measure the consequences of a severe thermal stress during development on male reproduction, a cold shock (1h at -18 degrees C) was tested on Dinarmus basalis pupae. D. basalis (Hymenoptera: Pteromalidae) is a parasitoid wasp in which sperm management in both male and female is of prime importance. After a cold shock, developmental success was reduced, with a quarter of cold-shocked males not emerging correctly. The stress effects were estimated at the level of sperm stock in seminal vesicles of males at different ages and on the ability of 2-day-old males to access females in single and multiple mating and in male-male competition. Cold-shocked males had a reduced sperm stock compared to control males and this difference persisted with age. The rate of sperm production was similar in both groups. The consequences of a cold shock on male reproductive ability were perceptible in multiple mating and male-male competition but not in single mating. Cold-shocked males were at a disadvantage, inseminating fewer females and copulating less frequently. Finally, male pupae of D. basalis were able to withstand severe temperature stresses and their reproductive functions were partially preserved.

Effect of host size on male fitness in the parasitoid wasp Dinarmus basalis.

The effect of host size on male fitness was tested in the parasitoid wasp Dinarmus basalis (Hymenoptera, Pteromalidae) using hosts of different fresh weight. Fitness was measured as the sperm stock in seminal vesicles, and the ability to access females in single or competition situations. Both body size and sperm in seminal vesicles increased with host fresh weight. Males from small hosts had a reduced size and sperm stock compared to those from larger hosts. In single situations, males from both small and large hosts had similar reproductive capacities, whereas in multiple mating or competition situations, males from small hosts were at a disadvantage, inseminating fewer females and copulating less frequently. However, females did not appear to choose between males, and no effect on sperm stored in the spermatheca was observed. Being small does not prevent a D. basalis male mating and producing progeny in single situations, although more offspring could be expected from larger males because of their better competitive abilities.

[Reproductive capacity of females Eupelmus vuilleti (Eupelmidae) inseminated by hyperparasitoid males developed upon the primary parasitoid Dinarmus basalis (Pteromalidae)]. / Capacité de reproduction des femelles Eupelmus vuilleti (Eupelmidae), inséminées par des mâles développés en hyperparasitoïdes de Dinarmus basalis (Pteromalidae).

Eupelmus vuilleti is a primary and solitary ectoparasitoid of the larval stages of Bruchids (Callosobruchus maculatus, Bruchidius atrolineatus). In a context of intense competition for healthy hosts, E. vuilleti displays ovicide and larvicide behaviours towards the Pteromalid D. basalis during its development (kleptoparasitism), and in an extreme expression of kleptoparasitism the E. vuilleti females hyperparasitize the final larval stage (L5 stage) of D. basalis. In this study, we compared the variability of reproductive success in males that had developed in the context of hyperparasitism to that in males that had developed on primary hosts. The adaptation capacity of the males when 24 h old was analysed in terms of their weight, of the quantity of spermatozoids stored in the seminal vesicles, of the quality of insemination determined from the quantity of spermatozoids stored in the spermatheca of the females after the first mating, and of the number of daughters produced. Adults of E. vuilleti, the larvae of which had developed as hyperparasitoids, are smaller than those that have developed on primary hosts, but they keep all the abilities required to parasite a population of primary hosts once the competitive pressure is reduced.