Ostrinia Scapulalis Research Articles

Reaction of Imagos of the Adzuki Bean Borer Ostrinia Scapulalis to Light Stimuli in a Wind Tunnel

Reaction of Imagos of the Adzuki Bean Borer Ostrinia Scapulalis to Light Stimuli in a Wind Tunnel

Effect of quorum sensing inducers and inhibitors on male-killing Wolbachia, the endosymbiont of the adzuki bean borer, Ostrinia scapulalis (Lepidoptera: Crambidae)

Effect of quorum sensing inducers and inhibitors on male-killing Wolbachia, the endosymbiont of the adzuki bean borer, Ostrinia scapulalis (Lepidoptera: Crambidae)

The response of adult adzuki been borer <i>Ostrinia scapulalis</i> to light stimuli in a wind tunnel

The attraction of nocturnal insects to light is a known phenomenon with unknown physiological mechanisms. Moths of the pyralid family are characterized by the most pronounced response to light, but the spectral preferences of these insects have not been studied. The adzuki been borers Ostrinia scapulalis, which live on dicotyledonous plants, are the ancestral form for the Asian and European corn borers, widespread pests of corn. Using a wind tunnel modified to deliver light stimuli, we tested the responses of males and females to light stimuli with wavelengths of 532, 440 and 365 nm at 2 lux, capable of stimulating photoreceptors of compound eyes (with maximum sensitivity of 352, 413, 480 and 530 nm) as well as simple ocelli, whose sensitivity had the main peak in ultraviolet and additional one in the green spectrum (360 and 520 nm). It turned out that ultraviolet was the most attractive stimulus. A small percentage of insects responded to green light, but showed a freezing reaction near the light source. The least attractive was blue light, which also caused freezes. Thus, the flight of O. scapulalis adults is induced by short-wavelength light, the response is more consistent with the sensitivity of the ocelli than the compound eyes; blue and green light leads to a masking effect.

Cell-based analysis reveals that sex-determining gene signals in Ostrinia are pivotally changed by male-killing Wolbachia.

Wolbachia, a maternally transmitted bacterium, shows male-killing, an adaptive phenotype for cytoplasmic elements, in various arthropod species during the early developmental stages. In lepidopteran insects, lethality of males is accounted for by improper dosage compensation in sex-linked genes owing to Wolbachia-induced feminization. Herein, we established Ostrinia scapulalis cell lines that retained sex specificity per the splicing pattern of the sex-determining gene doublesex (Osdsx). We found that Wolbachia transinfection in male cell lines enhanced the female-specific splice variant of Osdsx (OsdsxF ) while suppressing the male-specific variant (OsdsxM ), indicating that Wolbachia affects sex-determining gene signals even in vitro. Comparative transcriptome analysis isolated only two genes that behave differently upon Wolbachia infection. The two genes were respectively homologous to Masculinizer (BmMasc) and zinc finger-2 (Bmznf-2), male-specifically expressed sex-determining genes of the silkworm Bombyx mori that encode CCCH-type zinc finger motif proteins. By using cultured cells and organismal samples, OsMasc and Osznf-2 were found to be sex-determining genes of O. scapulalis that are subjected to sex-specific alternative splicing depending upon the chromosomal sex, developmental stage, and infection status. Overall, our findings expound the cellular autonomy in insect sex determination and the mechanism through which sex is manipulated by intracellular selfish microbes.

Evolution of the Sex Pheromone Communication System in Ostrinia Moths.

Simple SummaryMoths typically rely on sex pheromone communication to find a mate. This involves the production of species-specific sex pheromones by females (the signaller) and the corresponding selective detection by conspecific males (the receiver). A key question in the evolution of the pheromone communication system is how the female signals can diversify and still be tracked by the receivers over the process of speciation. The genus Ostrinia, which comprises 20 species worldwide including several well-recognised agricultural pests, is an attractive model in the study of the evolution of sex pheromone communication, as the closely related species and strains provide an ideal example of ongoing speciation. This review presents a comprehensive overview of the research on pheromone communication in different Ostrinia species over the past four decades, from the identity and biosynthesis of pheromones in the females to the molecular and neuronal basis of the pheromone perception in males. The evolutionary insights from these studies are discussed and the directions for future research are outlined.It remains a conundrum in the evolution of sexual communication how the signals and responses can co-ordinate the changes during speciation. The genus Ostrinia contains several closely related species as well as distinctive strains with pheromone polymorphism and represents an example of ongoing speciation. Extensive studies in the genus, especially in the species the European corn borer O. nubilalis (ECB), the Asian corn borer O. furnacalis (ACB) and the adzuki bean borer O. scapulalis (ABB), have provided valuable insights into the evolution of sex pheromone communication. This review presents a comprehensive overview of the research on pheromone communication in different Ostrinia species over the past four decades, including pheromone identification and biosynthesis, the ligand profiles of pheromone receptor (PR) genes, the physiology of peripheral olfactory sensory neurons (OSNs) and the projection pattern to the antennal lobe. By integrating and comparing the closely related Ostrinia species and strains, it provides an evolutionary perspective on the sex pheromone communication in moths in general and also outlines the outstanding questions that await to be elucidated by future studies.

Complete Mitogenomic Structure and Phylogenetic Implications of the Genus Ostrinia (Lepidoptera: Crambidae).

To understand mitogenome characteristics and reveal phylogenetic relationships of the genus Ostrinia, including several notorious pests of great importance for crops, we sequenced the complete mitogenomes of four species: Ostrinia furnacalis (Guenée, 1854), Ostrinia nubilalis (Hübner, 1796), Ostrinia scapulalis (Walker, 1859) and Ostrinia zealis (Guenée, 1854). Results indicate that the four mitogenomes—O. furnacalis, O. nubilalis, O. scapulalis, and O. zealis—are 15,245, 15,248, 15,311, and 15,208 bp in size, respectively. All four mitogenomes are comprised of 37 encoded genes and a control region. All 13 protein-coding genes (PCGs) initiate with ATN and terminate with TAN, with the exception of cox1 that starts with CGA, and cox1, cox2, and nad5 that terminate with an incomplete codon T. All transfer RNA genes (tRNAs) present the typical clover-leaf secondary structure except for the trnS1 (AGN) gene. There are some conserved structural elements in the control region. Our analyses indicate that nad6 and atp6 exhibit higher evolution rates compared to other PCGs. Phylogenetic analyses based on mitogenomes using both maximum likelihood (ML) and Bayesian inference (BI) methods revealed the relationship (O. palustralis + (O. penitalis + (O. zealis + (O. furnacalis + (O. nubilalis + O. scapulalis))))) within Ostrinia.

Susceptibility of three species of the genus Ostrinia (Lepidoptera: Crambidae) to Nosema pyrausta (Microsporidia: Nosematida)

Microsporidia are obligate intracellular parasites that affect the population density of many insect pests. In particular, infection with Nosema pyrausta is one of the major mortality factors for the European corn borer Ostrinia nubilalis, the Asian corn borer Ostrinia furnacalis and the adzuki bean borer Ostrinia scapulalis. The purpose of the work is to compare the susceptibility to N. pyrausta and pathogenesis of three species of moths of the genus Ostrinia. Studies conducted over 2 years have shown that in all three species of host insects under laboratory conditions, both during oral infection and transovarian transmission of infection (in the daughter generations of experimentally infected insects), only diplokaryotic spores formed corresponding to the main morphotype of the genus Nosema. Mean lethal time increased with instar of larvae used for infection but didn’t differ between the three species. The rates of transovarial transmission of N. pyrausta were also similar. Thus, all the insect species examined may equally participate in the parasite persistence in nature and serve as model laboratory hosts for parasitological research and mass propagation of the microsporidium.

Transcriptomic response of female adult moths to host and non-host plants in two closely related species

BackgroundDivergent selection has been shown to promote speciation in many taxa and especially in phytophagous insects. In the Ostrinia species complex, the European corn borer (ECB) and adzuki bean borer (ABB) are two sibling species specialized to different host plants. The first is a well-known maize pest, whereas the second is a polyphagous species associated with various dicotyledons. Their specialization to host plants is driven by morphological, behavioral and physiological adaptations. In particular, previous studies have shown that ECB and ABB display marked behavior with regard to plant choice during oviposition, involving specific preference and avoidance mechanisms. In this study, our goal was to identify the mechanisms underlying this host-plant specialization in adult females through an analysis of their gene expression. We assembled and annotated a de novo reference transcriptome and measured differences in gene expression between ECB and ABB females, and between environments. We related differentially expressed genes to host preference behavior, and highlighted the functional categories involved. We also conducted a specific analysis of chemosensory genes, which are considered to be good candidates for host recognition before oviposition.ResultsWe recorded more differentially expressed genes in ECB than in ABB samples, and noticed that the majority of genes potentially involved in the host preference were different between the two species. At the functional level, the response to plant environment in adult females involved many processes, including the metabolism of carbohydrates, lipids, proteins, and amino acids; detoxification mechanisms and immunity; and the chemosensory repertoire (as expected). Until now, most of the olfactory receptors described in Ostrinia spp. had been tested for their putative role in pheromone recognition by males. Here we observed that one specific olfactory receptor was clearly associated with ECB’s discrimination between maize and mugwort conditions, highlighting a potential new candidate involved in plant odor discrimination in adult females.ConclusionsOur results are a first step toward the identification of candidate genes and functions involved in chemosensory processes, carbohydrate metabolism, and virus and retrovirus dynamics. These candidates provide new avenues for research into understanding the role of divergent selection between different environments in species diversification.

Prevalence Rates of the Endosymbiotic Bacterium of the Wolbachia Genus in Natural Populations of Ostrinia nubilalis and Ostrinia scapulalis (Lepidoptera: Pyraloidea: Crambidae) in Southwestern Russia

Analysis of the prevalence rates of the endosymbiotic bacterium of the Wolbachia genus in the populations of corn borers of the Ostrinia genus was carried out. Samples of genomic DNA were extracted from field-collected larvae. The PCR analysis for the presence of Wolbachia infection was performed independently using three diagnostic loci: 16SrRNA, gatB, and fbpA. A positive signal for Wolbachia was obtained for 13.5% of the Ostrinia nubilalis larvae (N = 141) and 31.9% of the Ostrinia scapulalis larvae (N = 138). The infection rate ranged from 2.9% (N = 34) to 65.8% (N = 38) in different geographical populations. At the same time, a significantly higher infection rate of O. scapulalis than O. nubilalis was observed in three out of four habitats. It was also found that the Wolbachia infection was more common for O. scapulalis individuals feeding on hemp than for those collected from mugwort.

Larval transcriptomic response to host plants in two related phytophagous lepidopteran species: implications for host specialization and species divergence

BackgroundMost phytophagous insects have morphological, behavioral and physiological adaptations allowing them to specialize on one or a few plant species. Identifying the mechanisms involved in host plant specialization is crucial to understand the role of divergent selection between different environments in species diversification, and to identify sustainable targets for the management of insect pest species. In the present study, we measured larval phenotypic and transcriptomic responses to host plants in two related phytophagous lepidopteran species: the European corn borer (ECB), a worldwide pest of maize, and the adzuki bean borer (ABB), which feeds of various dicotyledons. Our aim was to identify the genes and functions underlying host specialization and/or divergence between ECB and ABB.ResultsAt the phenotypic level, we observed contrasted patterns of survival, weight gain and developmental time between ECB and ABB, and within ECB and ABB reared on two different host plants. At the transcriptomic level, around 8% of the genes were differentially expressed (DE) between species and/or host plant. 70% of these DE genes displayed a divergent pattern of expression between ECB and ABB, regardless of the host, while the remaining 30% were involved in the plastic response between hosts. We further categorized plastic DE genes according to their parallel or opposite pattern between ECB and ABB to specifically identify candidate genes involved in the species divergence by host specialization. These candidates highlighted a comprehensive response, involving functions related to plant recognition, digestion, detoxification, immunity and development. Last, we detected viral, bacterial, and yeast genes whose incidence contrasted ECB and ABB samples, and maize and mugwort conditions. We suggest that these microorganism communities might influence the survival, metabolism and defense patterns observed in ECB and ABB larvae.ConclusionsThe comprehensive approach developed in the present study allowed to identify phenotypic specialization patterns and underlying candidate molecular mechanisms, and highlighted the putative role of microorganisms in the insect-host plant interaction. These findings offer the opportunity to pinpoint specific and sustainable molecular or physiological targets for the regulation of ECB pest populations.

Parallel evolution of behaviour during independent host-shifts following maize introduction into Asia and Europe.

Maize was introduced into opposite sides of Eurasia 500 years ago, in Western Europe and in Asia. This caused two host‐shifts in the phytophagous genus Ostrinia; O. nubilalis (the European corn borer; ECB) and O. furnacalis (the Asian corn borer; ACB) are now major pests of maize worldwide. They originated independently from Dicot‐feeding ancestors, similar to O. scapulalis (the Adzuki bean borer; ABB). Unlike other host‐plants, maize is yearly harvested, and harvesting practices impose severe mortality on larvae found above the cut‐off line. Positive geotaxis in the ECB has been proposed as a behavioural adaptation to harvesting practices, allowing larvae to move below the cut‐off line and thus escape harvest mortality. Here, we test whether the same behavioural adaptation evolved independently in Europe and in Asia. We sampled eight genetically differentiated ECB, ACB and ABB populations in France and China and monitored geotaxis through the entire larval development in artificial stacks mimicking maize stems. We find that all ECB and ACB populations show a similar tendency to move down during the latest larval stages, a behaviour not observed in any European or Asian ABB population. The behaviour is robustly expressed regardless of larval density, development mode or environmental conditions. Our results indicate that maize introduction triggered parallel behavioural adaptations in Europe and Asia, harvest selection presumably being the main driver.

Prevalence rates of Wolbachia endosymbiotic bacterium in natural populations of Ostrinia Nubilalis and Ostrinia Scapulalis (Lepidoptera: Pyraloidea: Crambidae) in South-Western Russia

Background. Endosymbiotic bacteria of the genus Wolbachia are widespread in arthropods and often cause reproductive abnormalities in lepidopteran insects, including corn borers of the genus Ostrinia. Wolbachia-Ostrinia is a promising model for studies of parasite-host interactions yet parasite prevalence in natural insect host populations remains unknown.
 Materials and Methods. Molecular genetic screening and statistical analysis is applied to evaluate prevalence rates of Wolbachia in sympatric populations of two corn borer species. Individual genomic DNA samples were extracted from last instar larvae collected in nature from different forage plants. For each sample of DNA showing positive signal with insect-specific primers the detection is performed using three diagnostic loci of Wolbachia: 16SrRNA, gatB and fbpA.
 Results. Wolbachia-positive signal is obtained for 13.5% larvae of Ostrinia nubilalis (N = 141) and 31.9% larvae of Ostrinia scapulalis (N = 138). In different localities the Wolbachia prevalence ranged from 2.9% (N = 34) to 65.8% (N = 38). Significantly higher rates of Wolbachia prevalence in insects from mugwort and hemp (O. scapulalis) as compared to those from corn (O. nubilalis) are revealed in three out of four localities.
 Conclusions. Endosymbiotic bacteria of the genus Wolbachia are revealed in natural populations of corn borers for the first time for Eastern Europe. The prevalence rates can be high and this should be taken into consideration when reproductive isolation is examined in population of these hosts as well as establishment of laboratory cultures is performed.

High regional genetic diversity and lack of host-specificity in Ostrinia nubilalis (Lepidoptera: Crambidae) as revealed by mtDNA variation.

The European corn borer (Ostrinia nubilalis) infests a wide array of host plants and is considered one of the most serious pests of maize in Europe. Recent studies suggest that individuals feeding on maize in Europe should be referred to O. nubilalis (sensu nov.), while those infesting dicots as Ostrinia scapulalis (sensu nov.). We test if the clear genetic distinctiveness among individuals of O. nubilalis living on maize vs. dicots is tracked by mitochondrial DNA (mtDNA). We used fragments of COI and COII genes of 32 individuals traditionally recognized as O. nubilalis collected on three host plants, maize, mugwort and hop, growing in different parts of Poland. In addition, we reconstructed the mtDNA phylogeny of Ostrinia species based on our data and sequences retrieved from GenBank to assess host and/or biogeographic patterns. We also compared haplotype variation found in Poland (east-central Europe) with other regions (Anatolia, Eastern Europe, Balkans, Far East, North America). Our study showed high mtDNA diversity of O. nubilalis in Poland in comparison with other regions and revealed rare haplotypes likely of Asian origin. We did not find distinct mtDNA haplotypes in larvae feeding on maize vs. dicotyledonous plants. Phylogenetic analyses showed an apparent lack of mtDNA divergence among putatively distinct lineages belonging to the O. nubilalis group as identical haplotypes are shared by Asian and European individuals. We argue that human-mediated dispersal, hybridization and sporadic host jumps are likely responsible for the lack of a geographic pattern in mtDNA variation.

Comparison of the ability to catabolize DIMBOA, a maize antibiotic, between Ostrinia furnacalis and Ostrinia scapulalis (Lepidoptera: Crambidae), with reference to their hybrids

Maize contains 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), which functions as a feeding deterrent, growth inhibitor, and toxin against many herbivorous insects. The Asian corn borer Ostrinia furnacalis (Guenee) (Lepidoptera: Crambidae) feeds on maize, whereas the sympatric congener O. scapulalis (Walker) does not. In laboratory assays, the addition of 0.3 mg/g of DIMBOA to an artificial diet markedly affected the survival of O. scapulalis larvae but not that of O. furnacalis larvae. Hybrids of O. furnacalis and O. scapulalis, crossed in both directions, tolerated DIMBOA to the same extent as O. furnacalis, indicating that this tolerance was conferred by a single or a few autosomal genes that are dominant to those of O. scapulalis. In vitro, DIMBOA was rapidly catabolized when incubated with the homogenate of the digestive tract of O. furnacalis in the presence of uridine diphosphate (UDP)-glucose. The UDP-glucose-dependent DIMBOA-catabolizing activities of the homogenate of the digestive tracts of O. scapulalis and hybrids correlated with their tolerance; low in O. scapulalis and high in the hybrids. These results reconfirmed that UDP-glucosyltransferase (UGT) or other UDP-dependent enzymes are involved in the catabolism of DIMBOA in O. furnacalis; however, consistent with our previous findings, DIMBOA-glucoside, the expected product of UGT, was not detected in the products of in vitro assays.

Effects of diapause on post‐diapause reproductive investment in the moth Ostrinia scapulalis

AbstractDiapause is a strategy used by many insect species to survive adverse environmental conditions. However, diapause incurs costs that may have adverse effects on post‐diapause development and reproduction. We herein investigated the effects of diapause on the post‐diapause reproductive investment of males and females in a multivoltine moth, the adzuki bean borer, Ostrinia scapulalis (Walker) (Lepidoptera: Crambidae). We found that (1) post‐diapause males and females were smaller and had lower mating success than non‐diapause individuals, (2) post‐diapause females had lower fecundity and shorter longevity than non‐diapause females, (3) post‐diapause males transferred similar numbers of eupyrene and apyrene sperm as non‐diapause males, (4) the fecundity and longevity of non‐diapause females mated with post‐diapause males and those mated with non‐diapause males were not significantly different, and (5) no significant relationship was found between diapause duration (short and long) and post‐diapause reproductive investments in both males and females. These results suggest that post‐diapause males did not reduce reproductive investment in spite of the cost of diapause, and the significant decline in reproductive output in post‐diapause females was due to their reduced body weight and longevity, which appeared to be direct consequences of the cost of diapause.

Host specialization involving attraction, avoidance and performance, in two phytophagous moth species.

Host specialization plays a key role in the extreme diversification of phytophagous insects. Whereas proximate mechanisms of specialization have been studied extensively, their consequences for species divergence remain unclear. Preference for, and performance on hosts are thought to be a major source of divergence in phytophagous insects. We assessed these major components of specialization in two moth species, the European corn borer (ECB) and the Adzuki bean borer (ABB), by testing their oviposition behaviour in different conditions (choice or no-choice set-ups) and their performances, by reciprocal transplant at the larval stage on the usual host and an alternative host plant. We demonstrated that both ABB and ECB have a strong preference for their host plants for oviposition, but that relative larval performances on the usual host and an alternative host differed according to the experiment and the trait considered (weight or survival). Finally, we show for the first time that the preference for maize in ECB conceals a strong avoidance of mugwort. The differences in performance, attraction and avoidance between ECB and ABB are discussed in the light of the underlying mechanisms and divergence process.

Misdirection of dosage compensation underlies bidirectional sex-specific death in Wolbachia-infected Ostrinia scapulalis

Endosymbiotic bacteria of the genus Wolbachia often manipulate the reproductive system of their hosts to propagate themselves in host populations. Ostrinia scapulalis moths infected with Wolbachia (wSca) produce female-only progeny (sex chromosomes: ZW), whereas females cured of the infection by antibiotic treatment produce male-only progeny (ZZ). The occurrence of female- and male-only progeny has been attributed to the specific death of the opposite sex during embryonic and larval development. In this bidirectional sex-specific lethality, embryos destined to die express a phenotypic sex opposite to their genotypic sex. On the basis of these findings, we suggested that wSca carries a genetic factor that feminizes the male host, the W chromosome of the host has lost its feminizing function, and discordance between the genotypic and phenotypic sexes underlies this sex-specific death. In the present study, we examined whether the failure of dosage compensation was responsible for this sex-specific mortality. Quantitative PCRs showed that Z-linked gene expression levels in embryos destined to die were not properly dosage compensated; they were approximately two-fold higher in the male progeny of wSca-infected females and approximately two-fold lower in the female progeny of infected-and-cured females. These results support our hypothesis that misdirection of dosage compensation underlies the sex-specific death.

A short, high-temperature treatment of host larvae to analyze Wolbachia–host interactions in the moth Ostrinia scapulalis

Maternally inherited endosymbiotic bacteria of the genus Wolbachia cause various reproductive alterations in their hosts. Wolbachia induces male-specific death during embryonic and larval stages in the moth Ostrinia scapulalis. To investigate how the density of Wolbachia affects their performance in the host, we attempted to reduce its density using a short, high-temperature treatment of the host at the larval stage. Individuals cured of infection as well as sexual mosaics, which harbor Wolbachia, were obtained by this method in the next generation. The sex of uninfected offspring was exclusively male, similar to that of the offspring of larvae treated with antibiotics. A strong correlation was found between Wolbachia density in female moths and the sex ratio of their progeny. These results suggest that a short, high-temperature treatment at the larval stage reduced the density of Wolbachia in the adult stage, and, hence, inhibited interference with the host’s development in the next generation. Since the direct effects of the heat treatment on Wolbachia were transient, this method may be useful for specifying the critical time for interference by Wolbachia in host development.

Female Condition-dependent Allocation of Nuptial Gifts by Males in the Moth Ostrinia scapulalis (Lepidoptera: Crambidae)

ABSTRACT The males of many insect species transfer a spermatophore, i.e., a proteinaceous capsule containing sperm, to females during copulation, and this may also function as a nuptial gift. If production of the spermatophore is costly and variations in the quality of females are large, males may strategically allocate their investment based on the quality of the mate to maximize their own reproductive success. We examined the size and protein content of spermatophores transferred to females of different ages and body sizes, and also to water-deprived and water-replete females in the moth Ostrinia scapulalis (Walker). Males transferred a spermatophore of a smaller size or with less protein to older females, smaller females, and water-deprived females. These results indicated that O. scapulalis males manipulated their reproductive investment based on the conditions of the mate. We also demonstrated that older males varied their resource allocation to a greater extent in response to female conditions than ...

‘Becoming a species by becoming a pest’ or how two maize pests of the genus Ostrinia possibly evolved through parallel ecological speciation events

New agricultural pest species attacking introduced crops may evolve from pre-existing local herbivores by ecological speciation, thereby becoming a species by becoming a pest. We compare the evolutionary pathways by which two maize pests (the Asian and the European corn borers, ACB and ECB) in the genus Ostrinia (Lepidoptera, Crambidae) probably diverged from an ancestral species close to the current Adzuki bean borer (ABB). We typed larval Ostrinia populations collected on maize and dicotyledons across China and eastern Siberia, at microsatellite and mitochondrial loci. We found only two clusters: one on maize (as expected) and a single one on dicotyledons despite differences in male mid-tibia morphology, suggesting that all individuals from dicotyledons belonged to the ABB. We found evidence for migrants and hybrids on both host plant types. Hybrids suggest that field reproductive isolation is incomplete between ACB and ABB. Interestingly, a few individuals with an 'ABB-like' microsatellite profile collected on dicotyledons had 'ACB' mtDNA rather than 'ABB-like' mtDNA, whereas the reverse was never found on maize. This suggests asymmetrical gene flow directed from the ACB towards the ABB. Hybrids and backcrosses in all directions were obtained in no-choice tests. In laboratory conditions, they survived as well as parental strain individuals. In Xinjiang, we found ACB and ECB in sympatry, but no hybrids. Altogether, our results suggest that reproductive isolation between ACB and ABB is incomplete and mostly prezygotic. This points to ecological speciation as a possible evolutionary scenario, as previously found for ECB and ABB in Europe.