Bernard Pintureau

Wolbachia endosymbionts responsible for various alterations of sexuality in arthropods

Rickettsia-like maternally inherited bacteria have been shown to be involved in a variety of alterations of arthropod sexuality, such as female-biased sex ratios, parthenogenesis, and sterility of crosses either between infected males and uninfected females or between infected individuals (cytoplasmic incompatibility). We have characterized several of these microorganisms through partial sequences of the small (16S) and large (23S) subunit ribosomal DNA. All the symbionts identified, which include several cytoplasmic incompatibility microorganisms, several endosymbionts of terrestrial isopods, and symbionts of two thelytokous Trichogramma wasp species, belong to a monophyletic group of related symbionts, some of which have previously been detected in several insects exhibiting cytoplasmic incompatibility. Three molecular lineages can be identified on the basis of 16S as well as 23S sequences. Although they are only known as endocellular symbionts, Wolbachia spread by horizontal transfer across host lineages as evidenced by their diversification which occurred long after that of their hosts, and by the non-congruence of the phylogenetic relationships of symbionts and their hosts. Indeed, symbionts of two different lineages have been found in the same host species, whereas closely related endosymbionts are found in distinct insect orders. Isopod endosymbionts form a separate lineage, and they can determine feminization as well as cytoplasmic incompatibility. The ability to determine cytoplasmic incompatibility, found in all lineages, is probably ancestral to this group.

Temperature-dependent differences in biological traits between two strains of Trichogramma cacoeciae (Hymenoptera: Trichogrammatidae)

Parasitoids’ efficiency in controlling pests depends not only on their ability to parasitize their hosts but also on how much they are adapted to climatic conditions (notably temperature) of the area where they are planned to be released. In addition, the optimal conditions for production of parasitoids used for inundative releases like Trichogramma spp. may also vary largely as a function of strains. Using the parasitoid Trichogramma cacoeciae Marchal as biological model, we studied how temperature affects important parasitoid efficiency-related biological traits under laboratory conditions. Emergence, mortality rates and fecundity of two strains of T. cacoeciae, one originating from France (Alsace) and the other one from Tunisia (Degache), were compared at constant temperatures of 15, 20, 25 and 30°C. The parasitoids of the French strain showed highest fecundity at 25°C with wasps that had been reared at 20 or 25°C. The Tunisian strain showed the highest fecundity at 25°C, but only when wasps were previously reared at 25 or 30°C. For both strains, the highest mortality occurred among wasps that had laid eggs at 30°C. Emergence rates were relatively high at all temperatures, although the French strain did better at 15–25°C and the Tunisian one at 20–30°C. Because of the differences in biological traits of these two T.cacoeciae strains in relation to the temperature, a judicious choice must be made among the various strains when using this species in biological control programs.

Effects of repeated thermic and antibiotic treatments on a Trichogramma (Hym., Trichogrammatidae) symbiont

Each individual of all populations of the thelytokous species Trichogramma cordubensis is infected by Wolbachia. Populations are composed of females, a few rare males and some intersexual or gynandromorphic individuals often capable of normal reproduction as females. Antibiotic (minocycline) and high temperature (30°C) treatments inactivate Wolbachia and induce male production. After these treatments, the sex ratio depending on the remaining frequency of thelytoky shows a genetic variability. Effect of cycles with and without treatments (minocycline or 30°C) on the thelytokous reproduction of ‘individuals’ (one ‘individual’ being one Trichogramma+n Wolbachia) was studied. The resistance of thelytokous reproduction to 30°C but not to minocycline, probably due to Wolbachia genes, increased. It is concluded that potentialities of adaptation to high temperature exist in Wolbachia and can explain a part of the spreading of these symbionts among host populations.

A molecular key to the common species of Trichogramma of the Mediterranean region.

A molecular key for the identification of common Trichogramma (Hymenoptera: Trichogrammatidae) species found in agricultural settings around the Mediterranean is developed based on the sequence of the internal transcribed spacer 2 of the ribosomal cistron. Using the size of the ITS2 PCR product and restriction fragment length polymorphisms of the amplicon, ten Trichogramma species (T. bourarachae Pintureau and Babault, T. brassicae Bezdenko, T. cacoeciae Marchal/T. embryophagum Hartig, T. cordubensis Vargas and Cabello, T. dendrolimi Matsumura, T. euproctidis Girault, T. evanescens Westwood, T. nerudai Pintureau and Gerding, T. oleae Voegelé and Pointel, and T. pintoi Voegelé) can be distinguished.

Use of tetracycline in larval diet to study the effect of Wolbachia on host fecundity and clarify taxonomic status of Trichogramma species in cured bisexual lines.

Endosymbionts of the genus Wolbachia were efficiently cured from Trichogramma species by incorporating 0.02% tetracycline into the artificial diet used to rear larvae. Use of this technique yielded stable cured lines (bisexual and arrhenotokous lines) in which no Wolbachia organisms were detected by PCR for up to 14 generations after curing. Four cured strains of Trichogramma pretiosum showed a significantly lower total fecundity compared to their Wolbachia-infected counterpart. However, the fecundity of a single cured strain of Trichogramma evanescens was similar to its Wolbachia-infected counterpart. These differences in the effect on fecundity may be due to differences between the Wolbachia strains infecting T. pretiosum or T. evanescens, providing additional evidence for the hypothesis that a specific interaction exists between some Trichogramma species and their Wolbachia symbionts. Tetracycline in larval diet was also used to generate bisexual strains of Trichogramma oleae and Trichogramma cordubensis so that these species could be crossed with the closely related species, respectively, T. pretiosum and T. evanescens, to test their compatibility. These crosses showed a lack of compatibility, validating maintenance of these as distinct species.

Addition of wsp sequences to the Wolbachia phylogenetic tree and stability of the classification.

Abstract.Wolbachia are symbiotic bacteria altering reproductive characters of numerous arthropods. Their most recent phylogeny and classification are based on sequences of the wsp gene. We sequenced wsp gene from six Wolbachia strains infecting six Trichogramma species that live as egg parasitoids on many insects. This allows us to test the effect of the addition of sequences on the Wolbachia phylogeny and to check the classification of Wolbachia infecting Trichogramma. The six Wolbachia studied are classified in the B supergroup. They confirm the monophyletic structure of the B Wolbachia in Trichogramma but introduce small differences in the Wolbachia classification. Modifications include the definition of a new group, Sem, for Wolbachia of T. semblidis and the merging of the two closely related groups, Sib and Kay. Specific primers were determined and tested for the Sem group.

Effects of the Host Species and the Number of Parasitoids per Host on the Size of Some Trichogramma Species (Hymenoptera: Trichogrammatidae)

The size of some Trichogramma spp. adults and especially the ovipositor length depends on the species, but is also related to the host species and to the number of parasitoids per host. The length is greater in T. evanescens than in T. pretiosum itself greater than in T. exiguum, but the width is similar in the three species. For T. evanescens, the size obtained in Mamestra brassicae host when three or four insects emerged is similar to that obtained in Ephestia kuehniella host when singly parasitized. The size of the ovipositor is important because it may influence the possibility of in vitro egg laying in artificial host eggs. A shorter or a narrower ovipositor could cause difficulties in egg-laying into artificial host eggs composed of a membrane of unsuitable thickness.

Effect of photoperiod experienced by parents on diapause induction in Trichogramma cacoeciae

In the genus Trichogramma, the prepupal stage can survive the cold season in diapause. However, optimal conditions for the induction of this cessation of development during the process of mass production of the parasitoid in a biological control program depend on the species. In Trichogramma cacoeciae Marchal (Hymenoptera: Trichogrammatidae), diapause is induced more easily if the parents are reared under short‐day conditions (L10:D14), and if the temperature is 10 °C rather than 13 °C. However, the effect of parental photoperiod on diapause induction is weaker at lower temperatures (10 °C). Following diapause induction, individuals can be stored at 3 °C for several months, up to 1 year. Non‐optimal conditions led to the establishment of a quiescent state in some or all individuals. In such cases, it was necessary to reduce the storage period to 1 or 2 months only, to prevent high mortality rates and low fecundity.

Enzyme polymorphism in some African, American and Asiatic Trichogramma and Trichogrammatoidea species (Hymenoptera: Trichogrammatidae)

Electrophoretic analysis of three enzyme systems (esterases, MDH and SOD) was performed on 28 strains of Trichogramma (16 species) and Trichogrammatoidea (two species) from Africa, America and Asia. The band patterns were described, compared and interpreted from a genetic point of view. These biochemical characteristics, together with morphological ones, were used to group the species. The revision of all the groups and the construction of a phylogenetic tree were subsequently undertaken.

Effects of endosymbiotic Wolbachia on the diapause in Trichogramma hosts and effects of the diapause on Wolbachia

Abstract Used as an agent in biological control, Trichogramma which has been infected by endosymbiotic bacteria belonging to the genus Wolbachia and inducing the thelytokous mode of reproduction, increases rearing productivity in commercial insectaries. These insectaries can now make use of Trichogramma diapause to spread out and optimise their production. We therefore studied the effect of Wolbachia on the diapause in Trichogramma evanescens. The symbionts show no beneficial or harmful effect on the proportion of emergence in diapausing individuals or on their fecundity. Therefore, infected strains can easily be used in crop protection. On the other hand, low temperatures exert a slightly negative effect on Wolbachia, as detected from the proportion of females in the offspring of symbiotic diapausing females.