Natural occurrence of Wolbachia-infected and uninfected Trichogramma species in tomato fields in Portugal

Minute egg parasitoids of the genus Trichogramma (Hymenoptera; Trichogrammatidae) are promising candidates for biological control of lepidopteran pests in tomato in Portugal. This certainly applies to native Trichogramma strains that have thelytokous reproduction, i.e., produce only daughters. In Trichogramma wasps, thelytoky is mostly induced by the intracellular bacterium Wolbachia. In this study, we carried out a field survey of native Trichogramma species in four locations in Ribatejo, the main processing tomato region of Portugal, and determined the prevalence of Wolbachia in those species. Five Trichogramma species were found to emerge from lepidopteran eggs collected in the field, namely Trichogramma bourarache, Trichogramma cordubensis, Trichogramma evanescens, Trichogramma pintoi, and Trichogramma turkestanica. T. evanescens and T. pintoi were by far the dominating species representing, respectively, 64.9 and 26.4% of the trichogrammatids collected. Total natural parasitism rates of the collected lepidopteran eggs by Trichogramma wasps ranged from 28.2 to 64.6%. Three Trichogramma species were found to be infected with Wolbachia, namely T. cordubensis, T. evanescens, and T. turkestanica. All the wasp broods belonging to T. cordubensis were infected, whereas low infection rates were found in T. evanescens (0.9% of the broods) and T. turkestanica (4.5% of the broods). The latter represents the first record of a Wolbachia infection in T. turkestanica. Sequencing of the Wolbachia surface protein, wsp, revealed this Wolbachia infection to be related to other Wolbachia infections in Trichogramma wasps. As Wolbachia-infected thelytokous strains exist for T. evanescens, the most abundant Trichogramma species naturally occurring in the tomato fields of the Ribatejo region, this species offers interesting and powerful options for biological control of lepidopteran pests in processing tomato in this region.     

Trichogramma parasitoids for control of Lepidopteran borers in Taiwan: species,life‐history traits and Wolbachia infections

Trichogramma, polyphagous endoparasitoids of lepidopteran eggs, are used against a variety of crop pests throughout the world including those of sugar cane and corn in south‐eastern Asia. Their ability to be easily and economically reared on factitious hosts and their wide host range have contributed to their widespread use in pest control. The overall aim of this study was to select strains for eventual release in crop areas for control of lepidopteran borer pests of sugar cane and corn. To this end, we identified common Trichogramma species emerging from corn borer egg masses throughout south‐western Taiwan, compared their life‐history characteristics, assessed their thermal limits and identified the Wolbachia infection status of collected Trichogramma parasitoids. Trichogramma ostriniae was the most commonly collected species on corn, with occasional detection of T. chilonis and an unidentified species designated as T. sp. y. Although the sex ratio varied widely between sites, Wolbachia infection was detected only at a single site in one species (T. ostriniae). Wolbachia‐infected T. ostriniae were tolerant to high temperature stress. Trichogramma chilonis had lowest fecundity of the three species tested, and a Wolbachia‐infected T. ostriniae strain had lower fecundity than an uninfected strain. Given the limited availability of distribution and historical data for Trichogramma species in Taiwan, the current study provides a baseline for future work and also highlights the importance of accurately identifying species when establishing colonies of natural enemies for biocontrol.     

Reduced competitive ability due to Wolbachia infection in the parasitoid wasp Trichogramma kaykai

Several hymenopteran parasitoids are infected with parthenogenesis‐inducing (PI) Wolbachia. Infected wasps produce daughters instead of sons from unfertilized eggs. Thus far, little is known about the direct effects of PI Wolbachia on their host's fitness. Here, we report reduced competitive ability due to Wolbachia infection in a minute parasitoid wasp, Trichogramma kaykai Pinto and Stouthamer (Hymenoptera: Trichogrammatidae). Immature survival of infected individuals in a host parasitized by a single infected female, laying a normal clutch of eggs, was lower than those parasitized by a single uninfected individual. When the offspring of infected and uninfected females shared the same host, the infected immatures had significantly lower survival rates than their uninfected counterparts. The survival rate of infected immatures was higher when they competed with other infected immatures from a different infected parent than in competition with uninfected immatures of conspecific wasps. Thus, the host Trichogramma can suffer a substantial reduction in fitness when it is infected with the PI Wolbachia. We discuss why such a reduction is to be expected when populations of infected and uninfected individuals co‐occur, and how the reduced competitive ability of PI Wolbachia influences the spread of the bacteria in the field.     

Effects of host-egg ages on host selection and suitability of four Chinese Trichogramma species,egg parasitoids of the rice striped stem borer,Chilo suppressalis

The striped stem borer, Chilo suppressalis (Walker), is one of the most economically important rice pests worldwide. However, biological control of this pest using natural-enemy insects has been rarely documented to date. With the objective of screening suitable candidate species for controlling the striped stem borer, we investigated the effect of the age of host eggs on the host selection and suitability by four indigenous Trichogramma species on their native host, C. suppressalis. The results indicated that the differently aged eggs of C. suppressalis were all accepted by T. japonicum, T. dendrolimi and T. chilonis, and there was a clear tendency to parasitize older eggs less under no-choice and choice conditions. The number of parasitized host eggs by T. ostriniae also decreased with the increasing host age in the no-choice test, but more 2-day-old host eggs were parasitized in the choice test. When 0-, 2-day-old eggs were offered, T. dendrolimi, T. japonicum and T. chilonis exhibited similar parasitism ability, whereas T. ostriniae appeared to have a stronger ability to attack the older host eggs (4-day-old). Trichogramma japonicum developed and emerged on parasitized C. suppressalis eggs of all ages tested, while showing a better adaptation to younger host eggs with significantly faster developmental time, higher survival and more female progeny on 0-day-old eggs. No adults for each of the other three Trichogramma species emerged from parasitized 4-day-old host eggs, and they had similar developmental time, survival and female progeny on parasitized 0-, 2-day-old host eggs with an exception of female progeny for T. chilonis. On 0-day-old host eggs, T. japonicum developed faster and T. ostriniae had lower progeny survival than the other three Trichogramma species evaluated, respectively. The current study provides useful information to select suitable Trichogramma species for controlling the striped stem borer, C. suppressalis.     

Molecular characterization of Iranian Trichogrammatids (Hymenoptera: Trichogrammatidae) and their Wolbachia endosymbiont

During 2009–2010, a field survey of native Trichogramma species was carried out in six provinces of Iran, including Khorasan Razavi, Tehran, Mazandaran, Guilan, Golestan, and Qom. In this study, a molecular method for identifying Trichogramma and for determining the prevalence of Wolbachia in those species was used. Based on ITS2 (internal transcribed spacer 2) sequence, 14 populations were identified as the species T. embryophagum, T. evanescens, or T. brassicae. Wolbachia infection in these Trichogrammatids was detected using wsp gene sequencing. The highest infection rates in Trichogramma were found in Mazandaran and Golestan provinces. There was no evidence of infection in Trichogramma species in Guilan and Qom provinces. Of the three infected populations, two populations of T. evanescens were infected with only one Wolbachia strain from sib subgroup and one population was superinfected. Here, we report the first data on molecular characterization of Iranian Trichogrammatids and their Wolbachia-endosymbionts.     

Successful horizontal transfer of Wolbachia symbionts between Trichogramma wasps

Rickettsial symbionts of the genus Wolbachia, harboured by many arthropod species, are implicated in feminization, cytoplasmic incompatibility and parthenogenesis phenomena. These symbionts induce thelytokous parthenogenesis in some egg parasitoids of the Trichogramma genus. In our study of these minute wasps, purified Wolbachia from an infected species, T. pretiosum, were transferred by microinjection into in vitro developed pupae of an uninfected species, T. dendrolimi. We believe this to be the first successful transfer of Wolbachia in parasitoids. The presence or absence of Wolbachia was determined using DAPI staining, PCR and ftsZ gene sequencing. An ftsZ gene fragment from microinjected T. dendrolimi was shown to be identical to that of T. pretiosum, confirming that transfer was successful. Wolbachia were still present in the recipient species 26 generations after the transfer, although only partial induction of thelytoky was observed. Therefore, in Trichogramma, density of symbionts or symbiont–host interactions may be involved in the expression of parthenogenesis. The successful transfer of the symbiont within the Trichogrammatidae, a group of insects used in biological control strategies, could increase their agronomic importance by manipulating their reproductive system.     

The genetics and evolution of obligate reproductive parasitism in Trichogramma pretiosum infected with parthenogenesis-inducing Wolbachia

Parthenogenesis-inducing (PI) Wolbachia belong to a class of intracellular symbionts that distort the offspring sex ratio of their hosts toward a female bias. In many PI Wolbachia-infected species sex ratio distortion has reached its ultimate expression-fixation of infection and all-female populations. This is only possible with thelytokous PI symbionts as they provide an alternative form of reproduction and remove the requirement for males and sexual reproduction. Many populations fixed for PI Wolbachia infection have lost the ability to reproduce sexually, even when cured of the infection. We examine one such population in the species Trichogramma pretiosum. Through a series of backcrossing experiments with an uninfected Trichogramma pretiosum population we were able to show that the genetic basis for the loss of female sexual function could be explained by a dominant nuclear effect. Male sexual function had not been completely lost, though some deterioration of male sexual function was also evident when males from the infected population (created through antibiotic curing of infected females) were mated to uninfected females. We discuss the dynamics of sex ratio selection in PI Wolbachia-infected populations and the evolution of non-fertilizing mutations.     

Iranian <Emphasis Type="Italic">Trichogramma</Emphasis>: ITS2 DNA characterization and natural <Emphasis Type="Italic">Wolbachia</Emphasis> infection

Egg parasitoids of the genus Trichogramma (Hymenoptera: Trichogrammatidae) are important natural enemies of many lepidopterous pests in agricultural and forest areas. Because the use of indigenous Trichogramma populations/species can significantly increase the biological control efficiency, the characterization of endemic populations and precise species identification is important. In this study, Trichogramma wasps were collected from parasitized eggs of several lepidopteran pests in Northern and Central Iran. Seven Trichogramma species were identified based on the morphology and the nucleotide sequence, size and restriction profile (PCR-RFLP) of the internal transcribed spacer 2 (ITS2) region of the rDNA of T. brassicae Bezdenko, T. cacoeciae Marchal, T. embryophagum Hartig, T. evanescens Westwood, T. euproctidis Girault, T. pintoi Voegelé and T. tshumakovae Sorokina. This is the first report of T. euproctidis in Iran. Additionally, natural populations were surveyed for the prevalence of Wolbachia. Two populations of T. brassicae were found to be infected by a single Wolbachia strain which was identified by using a multi-locus sequencing approach.     

Male rescue maintains low frequency parthenogenesis-inducing <Emphasis Type="Italic">Wolbachia</Emphasis> infection in <Emphasis Type="Italic">Trichogramma</Emphasis> populations

Parthenogenesis-inducing (PI) Wolbachia bacteria are reproductive parasites that cause infected (W ⁺) female haplodiploid parasitoids to produce daughters without fertilization by males. Theoretically, PI Wolbachia infection should spread to fixation within Trichogramma populations as males are no longer required to produce female offspring. Infections in some naturally occurring Trichogramma populations are, however, maintained at frequencies ranging from 4 to 26%. Here we describe discrete equation models to examine if the PI Wolbachia infection in Trichogramma populations can be maintained at relatively low frequencies by mating regularity. Model outcomes suggest the probability of W ⁺ females mating could stabilize Wolbachia infection frequency at low levels in Trichogramma populations. The primary mechanism maintaining low-level PI Wolbachia infection in Trichogramma populations is reducing the survivorship from egg to adult in infected relative to uninfected females. The model successfully demonstrates that the relatively low PI Wolbachia infection frequency in host populations can be maintained by fertilization, or male rescue, of infected eggs, which avoids potentially hazardous gamete duplication that occurs during Wolbachia-induced parthenogenesis.     

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.     

Biodiversity of Wolbachia and of their effects in Trichogramma (Hymenoptera: Trichogrammatidae)

Hymenopteran egg parasitoids belonging to the Trichogramma genus are infected by endosymbionts of the Wolbachia genus distributed into 2 supergroups and 4 groups according to the sequence of the wsp gene. The symbionts are variously distributed according to host populations and species and induce, in most cases, thelytokous parthenogenetic reproduction and, sometimes strongly, increase fecundity. Nevertheless, after an interspecific artificial transfer, no effect on reproduction has so far been recorded. Wolbachia often have a negative effect, or no effect at all, on host fitness. Positive effects have, however, been recorded especially in completely infected species. Symbionts show a variable efficiency of vertical transmission and a variable sensitivity to high temperature in relation to host species.     

Experimental comparison of laboratory reared vs. wild-typeTrichogramma confusum [Hym.: Trichogrammatidae] I. Fertility,fecundity and longevity

A comparative study was made of the fertility, fecundity and longevity ofTrichogramma confusum Vigg. reared in the laboratory on eggs ofCorcyra cephalonica St., vs. a wild type population of the same species. Laboratory reared females showed a significantly higher degree of sterility than wild-type females while sterility amongst males did not differ significantly. Under laboratory conditions, laboratory reared females lived longer than wild-type females but produced fewer progeny as indicated by life-table data suggesting that their “effective” life is shorter. The high precentage of sterility amongst laboratory reared females should be taken into account when making inundative field releases. As size of the host may influence the fecundity and behaviour of laboratory rearedTrichogramma, the use of a suitable host which produces larger eggs would have to be considered in any mass-breeding programme.     

Effects of cold shock on host egg parasitization by females of Trichogramma buesi Voegele (Hymenoptera, Trichogrammatidae)

The effects of cold shock (4°C for 18–24 h) on survival of Trichogramma buesi females, their fecundity, and the inclination to parasitize grain moth eggs were studied under laboratory conditions. Cold shock did not result in any significant change in the survival rate, whereas the fraction of females that parasitized grain moth eggs and the fecundity of these females slightly decreased. However, females which had already started oviposition before cold shock infested grain moth eggs much more frequently. In most insect species studied, cold shock results in a sharp decrease or even disruption of the effect of experience on the subsequent behavior. Thus, the results of this study suggest that “the effect of acquired experience” (a tendency to continue infestation of the particular host species) in Trichogramma females is based not on learning or not only on learning but on some other, possibly hormonal mechanisms.     

Decrease of memory retention in a parasitic wasp: an effect of host manipulation by Wolbachia?

Several factors, such as cold exposure, aging, the number of experiences and viral infection, have been shown to affect learning ability in different organisms. Wolbachia has been found worldwide as an arthropod parasite/mutualist symbiont in a wide range of species, including insects. Differing effects have been identified on physiology and behavior by Wolbachia. However, the effect of Wolbachia infection on the learning ability of their host had never previously been studied. The current study carried out to compare learning ability and memory duration in 2 strains of the parasitoid Trichogramma brassicae: 1 uninfected and 1 infected by Wolbachia. Both strains were able to associate the novel odors with the reward of an oviposition into a host egg. However, the percentage of females that responded to the experimental design and displayed an ability to learn in these conditions was higher in the uninfected strain. Memory duration was longer in uninfected wasps (23.8 and 21.4 h after conditioning with peppermint and lemon, respectively) than in infected wasps (18.9 and 16.2 h after conditioning with peppermint and lemon, respectively). Memory retention increased in response to the number of conditioning sessions in both strains, but memory retention was always shorter in the infected wasps than in the uninfected ones. Wolbachia infection may select for reduced memory retention because shorter memory induces infected wasps to disperse in new environments and avoid competition with uninfected wasps by forgetting cues related to previously visited environments, thus increasing transmission of Wolbachia in new environments.     

Development of a new attract‐and‐kill technology for Oriental fruit moth control using insecticide‐impregnated fabric

Various physiological effects of Wolbachia infection have been reported in invertebrates, but the impact of this infection on behavior and the consequences of these behavioral modifications on fitness have rarely been studied. Here, we investigate the effect of Wolbachia infection on the estimation of host nutritive resource quality in a parasitoid wasp. We compare decision‐making in uninfected and Wolbachia‐infected strains of Trichogramma brassicae Bezdenko (Hymenoptera: Trichogrammatidae) on patches containing either fresh or old host eggs. For both strains, fresh eggs were better hosts than older eggs, but the difference was smaller for the infected strain than for the uninfected strain. Oviposition behavior of uninfected wasps followed the predictions of optimal foraging theory. They behaved differently toward high‐ vs. low‐quality hosts, with more hosts visited and more ovipositions, fewer high‐quality hosts used for feeding or superparasitism, and a sex ratio that was more biased toward females in patches containing high‐quality hosts than in patches containing low‐quality ones. Uninfected wasps also displayed shorter acceptance and rejection times in high‐quality hosts than in hosts of lower quality. In contrast, infected wasps were less efficient in evaluating the nutritive quality of the host (fresh vs. old eggs) and had a reduced ability to discriminate between unparasitized and parasitized hosts. Furthermore, they needed more energy and therefore engaged in host feeding more often. This study highlights possible decision‐making manipulation by Wolbachia, and we discuss its consequences for Wolbachia fitness.     

Suitability assessment of three Trichogramma species in the control of Mythimna separata (Lepidoptera: Noctuidae)

Mythimna separata (Walker) is a serious cosmopolitan pest. Trichogramma species have been used worldwide as biological control agents of lepidopteran pests. However, no data on the control efficiency of Trichogramma species on this pest have been reported. We evaluated the suitability of M. separata eggs for three Trichogramma species, Trichogramma dendrolimi Matsumura, T. ostriniae Pang et Chen and T. japonicum Ashmead, and compared the suitability with that of Corcyra cephalonica (Stainton) eggs. The parasitism rate, development time, emergence rate, female proportion, body size and egg load of female offspring indicated that the M. separata egg is a suitable host for the three Trichogramma species. On the other hand, the non‐emergence rate and the relationship between egg load and body size reflected that M. separata eggs are less suitable for the three Trichogramma species than C. cephalonica eggs. Egg resorption occurred in T. japonicum 4 days after emergence.     

Assessing European Egg Parasitoids as a Mean of Controlling the Invasive South American Tomato Pinworm Tuta absoluta

The South American tomato pinworm (Tuta absoluta) has recently invaded Europe and is rapidly spreading in the Afro-Eurasian continent where it is becoming a major pest on tomato crops. Laboratory tests were undertaken to evaluate the potential of 29 European strains of Trichogramma parasitoids to control T. absoluta. In addition to the host itself, the host plant (tomato) was used during the laboratory tests in order to increase the chance of selecting the best parasitoid strains. Trichogramma females were placed with T. absoluta eggs on a tomato leaflet in tubes. We compared the parasitism of T. absoluta by the various Trichogramma species tested to the Trichogramma species currently commercially available for the pest control in Europe, i.e. Trichogramma achaeae. Thereafter, the more promising strains were tested on a larger scale, in mesocosm (i.e. cages in greenhouses) and in greenhouse compartments to evaluate efficiency of laboratory selected strains under cropping conditions. The most efficient strain from the laboratory screening trials did not perform as efficiently under the greenhouse conditions. We discuss differences in parasitism levels among species and strains and among the different scales tested in the experiments, as well as implications of these results for further screening for biocontrol agents.     

Nocturnal parasitism of moth eggs by Trichogramma wasps

Parasitoid wasps of the genus Trichogramma are used worldwide as biological control agents against lepidopteran pests. Trichogramma wasps develop inside eggs of a wide range of host species, most of them moths. They are generally considered as diurnal insects. Here, we investigated whether Trichogramma wasps can also successfully parasitise host eggs at night under controlled laboratory conditions. Eggs of the moth Ephestia kuehniella were offered under dark conditions (scotophase) to females of Trichogramma brassicae and Trichogramma evanescens either from 9:00 PM to 9:00 AM or from 11:00 AM to 5:00 PM at four different temperatures (5°C, 10°C, 15°C and 20°C). Both species are known to parasitise E. kuehniella eggs in the photophase during daytime. The results show that T. brassicae did not parasitise eggs in the scotophase at night and only very few in the artificially induced scotophase during daytime from 10°C to 20°C. In contrast, T. evanescens parasitised more eggs in the dark both at night and artificially induced scotophase during daytime. Parasitism in the scotophase already started at 5°C, with more eggs being parasitised and more offspring being produced at higher temperatures. T. evanescens displayed higher parasitism activity in the induced scotophase during daytime than in the scotophase at night. The present study suggests that Trichogramma are capable of successfully parasitising host eggs at night, even at low temperatures, but that nocturnal activity with respect to parasitism varies between wasp species.     

Temperature-Dependent Parasitism,Survival, and Longevity of Five Species of Trichogramma Westwood (Hymenoptera: Trichogrammatidae) Associated with Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae)

Egg parasitoids of the genus Trichogramma Westwood play an important role in the control of the velvetbean caterpillar Anticarisa gemmatalis Hübner in soybean crops in Southern Brazil. The effectiveness of Trichogramma species as biocontrol agents is dependent of several factors, but their adaptation to the climatic conditions of the region where they will be released is one of the most important. In this context, this study evaluated the effects of five constant temperatures ranging from 14 to 30°C on parasitism, age-specific survival, progeny production and longevity of Trichogramma pretiosum Riley, Trichogramma atopovirilia Oatman & Platner, Trichogramma acacioi Brun, Moraes & Soares, Trichogramma lasallei Pinto, and Trichogramma rojasi Nagaraja & Nagarkatti. We demonstrate that temperature differently affected the species of Trichogramma investigated when using eggs of the velvetbean caterpillar. We also demonstrate that T. pretiosum and T. atopovirilia are better adapted to a wide range of temperatures than the other species evaluated, and are therefore better suited as biocontrol agents for applied biological control programs of A. gemmatalis.     

Effect of fertilized,unfertilized, and UV‐irradiated hosts on parasitism and suitability for Trichogramma parasitoids

Trichogramma spp. (Hymenoptera: Trichogrammatidae) parasitoids have been commonly used as biological control agents in insect pest management. Host quality is believed to influence parasitism, host preference, and suitability for parasitoids. To date, limited studies have compared the parasitism of Trichogramma parasitoids on fertilized, unfertilized, and sterilized host eggs. Hence, we studied the performance of three Trichogramma egg parasitoids, Trichogramma japonicum Ashmead, Trichogramma chilonis Ishii, and Trichogramma leucaniae Pang & Chen, on fertilized, unfertilized, and ultraviolet (UV)‐irradiated fertilized (UVF) eggs of rice moth, Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae). In a no‐choice test, T. japonicum and T. leucaniae parasitized significantly more fertilized or UVF than unfertilized hosts, and T. chilonis parasitized significantly more UVF than either fertilized or unfertilized hosts. In a choice test, all three Trichogramma parasitoids parasitized UVF hosts the most, and unfertilized hosts the least. There were similar percentages of adult emergence and female progeny among fertilized, unfertilized, and UVF hosts for all three Trichogramma parasitoids, except that T. japonicum had significantly lower adult emergence on fertilized hosts. We also found that all three Trichogramma parasitoids developed slower on unfertilized hosts. Regardless of host treatments, T. leucaniae had the longest developmental time and T. chilonis had the shortest. We conclude that Trichogramma parasitoids prefer parasitizing UVF eggs of C. cephalonica without negative effects on their emergence and sex allocation.