Genome divergence and reproductive incompatibility among populations of Ganaspis near brasiliensis.

During the last decade, the spotted wing drosophila, Drosophila suzukii, has spread from eastern Asia to the Americas, Europe, and Africa. This fly attacks many species of cultivated and wild fruits with soft, thin skins, where its serrated ovipositor allows it to lay eggs in undamaged fruit. Parasitoids from the native range of D. suzukii may provide sustainable management of this polyphagous pest. Among these parasitoids, host-specificity testing has revealed a lineage of Ganaspis near brasiliensis, referred to in this paper as G1, that appears to be a cryptic species more host-specific to D. suzukii than other parasitoids. Differentiation among cryptic species is critical for introduction and subsequent evaluation of their impact on D. suzukii. Here, we present results on divergence in genomic sequences and architecture and reproductive isolation between lineages of Ganaspis near brasiliensis that appear to be cryptic species. We studied five populations, two from China, two from Japan, and one from Canada, identified as the G1 vs G3 lineages based on differences in cytochrome oxidase l sequences. We assembled and annotated the genomes of these populations and analyzed divergences in sequence and genome architecture between them. We also report results from crosses to test reproductive compatibility between the G3 lineage from China and the G1 lineage from Japan. The combined results on sequence divergence, differences in genome architectures, ortholog divergence, reproductive incompatibility, differences in host ranges and microhabitat preferences, and differences in morphology show that these lineages are different species. Thus, the decision to evaluate the lineages separately and only import and introduce the more host-specific lineage to North America and Europe was appropriate.

Modelling potential distribution of the invasive box tree moth across Asia, Europe, and North America.

The box tree moth Cydalima perspectalis (Walker) (Lepidoptera: Crambidae) (BTM) is a native moth throughout eastern Asia, having recently become invasive in Europe (2007) where it feeds on boxwood (= box tree), Buxus spp. The moth rapidly spread across Europe and the Caucasus causing damage to both ornamental and wild Buxus. In 2018, C. perspectalis was found in Toronto, ON, Canada, and has since spread south into the US. To better predict where the moth will establish and have significant impact on ornamental trade in North America, we used most recent scientific literature and distribution points to update the temperature and diapause indices of an existing ecoclimatic CLIMEX model. The model parameters provided a good fit for the potential distribution of BTM compared to its known distribution across eastern Asia and in Europe. Interestingly, our results suggest that the current native distribution in Asia is incomplete and that further expansion is also possible in its introduced range, especially in northern Europe, along the Mediterranean coast of Africa, and eastward to central Russia. In North America, the model predicts that most of North America should be climatically suitable for the moth's establishment, with the exception of Alaska and the northern territories of Canada, as well as higher elevations in the Rocky Mountains and southern hot and dry areas. Our study highlights the importance of the CLIMEX model to assess the risk of BTM spreading in its newly invaded areas, especially North America, and its use to help make decisions in terms of regulatory dispersal restrictions and choice of management options.

Uncovering the Male Presence in Parthenogenetic Marchalina hellenica (Hemiptera: Marchalinidae): Insights into Its mtDNA Divergence and Reproduction Strategy.

Marchalina hellenica (Hemiptera: Marchalinidae), an endemic species in Greece and Turkey, is a major contributor to the annual honey production in its native range. However, in the areas that it invades, lacking natural enemies, it has detrimental effects on pine trees and potentially contributes to tree mortality. Although it was originally reported as thelytokous, males were later reported in Turkey and on several of the islands of Greece. To further disambiguate the exact parthenogenetic reproduction strategy of M. hellenica, we studied the emergence pattern of male individuals in Greece for two consecutive years (2021 and 2022). Furthermore, we examined the genetic variation among 15 geographically distant populations of M. hellenica in Greece using a mitochondrial DNA marker and compared the results with data from Turkey. The findings of this study document the existence of an additional M. hellenica population in its native range that repeatedly produces males, apart from the areas of Greece and Turkey in which they were initially reported, suggesting that males play a major, so far unknown role in the reproduction of this species. The populations in Greece and Turkey exhibited a strong genetic affinity, while human-aided dispersal seems to have obscured the genetic pattern acquired.

Phenology and Potential Fecundity of Neoleucopis kartliana in Greece.

Neoleucopis kartliana Tanasijtshuk (Diptera, Chamaemyiidae) is the most abundant predator of the giant pine scale (GPS), Marchalina hellenica (Hemiptera, Margarodidae) in Greece. GPS is native to Greece and Turkey, where it is not considered a pest of Pinus spp., but a valuable resource for pine honey production. However, its introduction to new areas leads to high population densities of the scale, linked to declines in tree health and insect biodiversity. To assess the potential use of N. kartliana for a classical biological control program in Australia, we studied selected life-history traits of the silver fly, namely its phenology in northern Greece, feeding preferences of adult flies on artificial food sources, and potential fecundity of female flies. The silver fly was present in every site in northern Greece studied and was found to have at least three generations per year in this area. The fly's overall sex ratio was 1:1, and adult females emerged with no or few mature eggs in their ovaries, but egg production was exponential until at least the eighth day after emergence. These findings increase our knowledge about the biology of N. kartliana and aided in the evaluation of the silver fly as a classical biological control agent against invasive GPS in Australia.

Evidence for a cryptic parasitoid species reveals its suitability as a biological control agent.

Uncertainty about the taxonomic status and the specificity of a species commonly prevent its consideration as a candidate for biological control of pest organisms. Here we use a combination of molecular analysis and crossing experiments to gather evidence that the parasitoid wasp Ganaspis brasiliensis, a candidate for biological control of the invasive spotted wing drosophila Drosophila suzukii, is a complex of at least two cryptic species. Complementary experiments demonstrate that individuals from one genetic group readily parasitize several drosophila species regardless of their food source while individuals from the other one are almost exclusively specific to larvae feeding in ripening fruits. Because only D. suzukii attacks ripening fruits in its area of invasion, parasitoids from this second group appear to be well suited as a biological control agent. Our study demonstrates the need for a combination of biosystematics with biological and ecological investigations for the development of safe and efficient biological control programs.

Discrimination between Asian populations of the parasitoid wasp Ganaspis cf. brasiliensis using a simple MALDI-TOF MS-based method for use with insects.

The fruit fly Drosophila suzukii has recently become an invasive pest insect of significant economic impact in Europe and the USA. In contrast to other Drosophila species, D. suzukii is able to infest intact fruit by means of a saw-like ovipositor, which allows females to deposit eggs beneath the skin of the fruit. Classical biological control using the parasitoid wasp Ganaspis cf. brasiliensis is currently being researched as an environmentally sustainable option for the control of D. suzukii. In particular, the host specificity of this parasitoid has been assessed for populations from different regions in China and Japan. In order to study the relationship between the differences in specificity and molecular variations, we have adapted a matrix-assisted laser-desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based method, originally developed for use with plant material, to discriminate between example populations of G. cf. brasiliensis. We have employed a combination of principal component analysis and blind-tested comparison between reference sample MALDI-TOF MS spectra and test sample spectra to discriminate, on the basis of the acid-soluble insect protein spectra generated, between four populations of G. cf. brasiliensis (originally collected from Tokyo and Hasuike in Japan and Dali and Ximing in China). MALDI-TOF MS analysis is able to discriminate with 100% accuracy between populations G. cf. brasiliensis. The Chinese populations were observed to be similar, but the Tokyo population is slightly different and the Hasuike population is significantly different from the other populations. The Tokyo population appears more closely related to the Chinese populations than the Hasuike population, even though both originate from Japan.

Impact of rearing temperature on encapsulation and the accumulation of transcripts putatively involved in capsule formation in a parasitized lepidopteran host.

Encapsulation and melanisation are innate immune reactions of insects against foreign intruders such as parasitoids. In an earlier study, we observed that immature life stages of the endoparasitoid Tranosema rostrale (Hymenoptera: Ichneumonidae) parasitizing Choristoneura fumiferana (Lepidoptera: Tortricidae) larvae experienced higher mortality due to encapsulation and melanisation when reared at high (30 °C) than at lower (10 °C, 20 °C) temperatures. Downregulation of T. rostrale polydnavirus genes in parasitized hosts and upregulation of two genes involved in the spruce budworm's melanisation process were identified as likely contributors to parasitoid mortality at high temperature. However, levels of transcripts of genes involved in the spruce budworm's cellular encapsulation process were not measured inasmuch as candidate genes, in the spruce budworm, had not yet been identified. In addition, our assessment of temperature-dependent encapsulation and melanisation of foreign objects in spruce budworm larvae was only partial. To fill these knowledge gaps, we injected Sephadex™ beads into unparasitized spruce budworm larvae and assessed their encapsulation/melanisation after the insects had been held at three different temperatures (10, 20, and 30 °C), and we identified spruce budworm genes putatively involved in the encapsulation process and quantified their transcripts at the same three temperatures, using a qPCR approach. As expected, both encapsulation and melanisation of Sephadex™ beads increased as a function of temperature. At the molecular level, three of the five genes examined (Integrin ß1, Hopscotch, Stat92E) clearly displayed temperature-dependent upregulation. The results of this study further support the hypothesis that a temperature-dependent increase in the encapsulation response of C. fumiferana against T. rostrale is due to the combined effects of reduced expression of polydnavirus genes and enhanced expression of host immune genes.

High temperature induces downregulation of polydnavirus gene transcription in lepidopteran host and enhances accumulation of host immunity gene transcripts.

Endoparasitoids face the challenge of overcoming the immune reaction of their hosts, which typically consists of encapsulation and melanisation of parasitoid eggs or larvae. Some endoparasitic wasps such as the solitary Tranosema rostrale (Hymenoptera: Ichneumonidae) that lay their eggs in larvae of the spruce budworm, Choristoneura fumiferana (Lepidoptera: Tortricidae), have evolved a symbiotic relationship with a polydnavirus (PDV), which in turn helps them suppress the host's immune response. We observed an increase in mortality of immature T. rostrale with increasing temperature, and we tested two hypotheses about the mechanisms involved: high temperatures (1) hamper the expression of T. rostrale PDV genes and (2) enhance the expression of spruce budworm immunity-related genes. Dissections of parasitized spruce budworm larvae reared at 30°C revealed that most parasitoid eggs or larvae had died as a result of encapsulation and melanisation by the host. A qPCR analysis of T. rostrale PDV (TrIV) gene expression showed that the transcription of several TrIV genes in host larvae was downregulated at high temperature. On the other hand, encapsulation, but not melanisation, of foreign bodies in spruce budworm larvae was enhanced at high temperatures, as shown by the injection of Sephadex™ beads into larvae. However, at the molecular level, the transcription of genes related to spruce budworm's melanisation process (prophenoloxidase 1 and 2) was upregulated. Our results support the hypothesis that a temperature-dependent increase of encapsulation response is due to the combined effects of reduced expression of TrIV genes and enhanced expression of host immune genes.

Developmental and reproductive responses of the spruce budworm (Lepidoptera: Tortricidae) parasitoid Tranosema rostrale (Hymenoptera: Ichneumonidae) to temperature.

The temperature-dependent development and survival of immatures, as well as adult longevity and potential fecundity of the endoparasitoid Tranosema rostrale (Hymenoptera: Ichneumonidae) parasitizing spruce budworm Choristoneura fumiferana (Lepidoptera: Tortricidae) larvae was investigated under laboratory conditions at several constant temperatures ranging from 5 to 30°C. Maximum likelihood modeling approaches were used to estimate thermal responses in development, survival, and longevity. A model describing the effect of temperature on potential fecundity of the parasitoid was also developed taking oogenesis and oosorption into account. In-host and pupal development rates of the parasitoid increased with temperature up to 25°C, and decreased thereafter. Immature survival was highest below 20°C, and rapidly decreased at higher temperatures. Adult longevity decreased exponentially with increasing temperature for both males and females. Highest potential fecundity was reached at 10°C. Considering survival and potential fecundity, the parasitoid seems best adapted to cool temperatures below 20°C. Simulations of the life-history traits under variable temperature regimes indicate that temperature fluctuations decrease survival and increase realised fecundity compared to constant temperatures. The temperature-dependent fecundity model developed can be applied to other non-host-feeding synovigenic parasitoids. The equations and parameter estimates provided in this paper can be used to build comprehensive models predicting the seasonal phenology of this parasitoid and spruce budworm parasitism under changing climatic conditions.

Seasonal Parasitism and Host Instar Preference by the Spruce Budworm (Lepidoptera: Tortricidae) Larval Parasitoid Tranosema rostrale (Hymenoptera: Ichneumonidae).

The seasonal pattern of parasitism by a parasitoid can be influenced by many factors, such as interspecific competition and host instar preference. We conducted field and laboratory experiments to describe the seasonal pattern of parasitism of spruce budworm Choristoneura fumiferana (Clemens) larvae by Tranosema rostrale (Brischke), and to investigate whether this pattern can be explained by interaction with other parasitoid species, or by host instar preference. Larval survival, developmental time, sex ratio, and adult size of T. rostrale developing in different host instars were also measured to further assess the potential importance of host instar on parasitoid life history. Parasitism by T. rostrale increased over the season, reaching the highest rate during the fourth-instar larva, and then decreased again until the sixth-instar. At the same time, parasitism by another parasitoid, Elachertus cacoeciae (Howard), increased over the season, and multiparasitism with T. rostrale suggests potential competition between these two parasitoids. Tranosema rostrale showed no host instar preference when third- to sixth-instar larvae were exposed simultaneously in a manipulative field experiment. The proportion of females emerging from spruce budworm larvae increased over the season; however, no difference in sex ratio was observed in the manipulative field experiment. Only male pupal development time and adult size were marginally increased in fifth-instar spruce budworm larvae. We conclude that T. rostrale's seasonal phenology or competition with E. cacoeciae, but not host instar preference, were possibly responsible for the observed seasonal pattern of parasitism.

Influence of partial cutting on parasitism of endemic spruce budworm (Lepidoptera: Tortricidae) populations.

Silvicultural treatments such as thinning have been suggested as management tools against the spruce budworm, Choristoneura fumiferana (Clemens) (Lepidoptera: Tortricidae). Among other things, parasitoids are also proposed to be influenced by silvicultural procedures, but the effect of thinning on spruce budworm's natural enemies has not been tested yet. In this study, the influence of partial cutting on parasitism of endemic spruce budworm populations has been investigated in mature balsam fir-white birch forests. Two intensities of partial cutting (25 and 40% stand basal area reduced) were conducted in 2009 and parasitism of introduced spruce budworm larvae and pupae was determined during the 3 yr after these treatments. Pupal parasitism was too low for comparison between treatments. However, 2 yr after treatments, parasitism of the fourth- and fifth-instar larvae was significantly reduced in plots with both intensities of partial cutting, which was attributed to the parasitoid Tranosema rostrale (Brischke). Three years after treatments, no significant influence of partial cutting on parasitism of spruce budworm larvae was found. This study suggests that the influence of partial cutting on parasitism of endemic spruce budworm populations is not consistent, but that under certain circumstances parasitism is reduced by partial cutting.