Identification and expression patterns of somatic piRNAs and PIWI genes in Riptortus pedestris (Hemiptera: Alydidae).

RNA interference (RNAi)-based gene silencing is a feasible and sustainable technology for the management of hemipteran pests by double-stranded RNA involvement, including small-interfering RNA, microRNA, and Piwi-interacting RNA (piRNA) pathways, that may help to decrease the usage of chemical insecticides. However, only a few data are available on the somatic piRNAs and their biogenesis genes in Riptortus pedestris, which serves as a significant pest of soybean (Glycine max). In this study, two family members of the PIWI gene were identified and characterized in R. pedestris, containing Argonaute3 (RpAgo3) and Aubergine (RpAub) genes with conserved protein domains, and their clusters were validated by phylogenetic analysis. In addition, they were widely expressed in all developmental stages of the whole body of R. pedestris and had lower expression levels in R. pedestris guts under different rearing conditions based on previous transcriptome sequencing. Furthermore, abundant clean reads were filtered to a total number of 45,998 piRNAs with uridine bias at the first nucleotide (nt) position and 26-32 nt in length by mapping onto the reference genome of R. pedestris according to our previous whole-transcriptome sequencing. Finally, our data revealed that gut bacterial changes were significantly positively or negatively associated with differentially expressed piRNAs among the five comparison groups with Pearson correlation analysis. In conclusion, these findings paved new avenues for the application of RNAi-based biopesticides for broad-spectrum hemipteran pest control.

Shared phylogeographic patterns and environmental responses of co-distributed soybean pests: Insights from comparative phylogeographic studies of Riptortus pedestris and Riptortus linearis in the subtropics of East Asia.

Comparative phylogeographic studies of closely related species sharing co-distribution areas can elucidate the role of shared historical factors and environmental changes in shaping their phylogeographic pattern. The bean bugs, Riptortus pedestris and Riptortus linearis, which both inhabit subtropical regions in East Asia, are recognized as highly destructive soybean pests. Many previous studies have investigated the biological characteristics, pheromones, chemicals and control mechanisms of these two pests, but few studies have explored their phylogeographic patterns and underlying factors. In this study, we generated a double-digest restriction site-associated DNA sequencing (ddRAD-seq) dataset to investigate phylogeographic patterns and construct ecological niche models (ENM) for both Riptortus species. Our findings revealed similar niche occupancies and population genetic structures between the two species, with each comprising two phylogeographic lineages (i.e., the mainland China and the Indochina Peninsula clades) that diverged approximately 0.1 and 0.3 million years ago, respectively. This divergence likely resulted from the combined effects of temperatures variation and geographical barriers in the mountainous regions of Southwest China. Further demographic history and ENM analyses suggested that both pests underwent rapid expansion prior to the Last Glacial Maximum (LGM). Furthermore, ENM predicts a northward shift of both pests into new soybean-producing regions due to global warming. Our study indicated that co-distribution soybean pests with overlapping ecological niches and similar life histories in subtropical regions of East Asia exhibit congruent phylogeographic and demographic patterns in response to shared historical biogeographic drivers.

Identifying a potentially invasive population in the native range of a species: The enlightenment from the phylogeography of the yellow spotted stink bug, Erthesina fullo (Hemiptera: Pentatomidae).

The yellow spotted stink bug (YSSB), Erthesina fullo (Thunberg, 1783) is an important Asian pest that has recently successfully invaded Europe and an excellent material for research on the initial stage of biological invasion. Here, we reported the native evolutionary history, recent invasion history, and potential invasion threats of YSSB for the first time based on population genetic methods [using double digest restriction-site associated DNA (ddRAD) data and mitochondrial COI and CYTB] and ecological niche modelling. The results showed that four lineages (east, west, southwest, and Hainan Island) were established in the native range with a strong east-west differentiation phylogeographical structure, and the violent climate fluctuation might cause population divergence during the Middle and Upper Pleistocene. In addition, land bridges and monsoon promote dispersal and directional genetic exchanging between island populations and neighboring continental populations. The east lineage (EA) was identified as the source of invasion in Albania. EA had the widest geographical distribution among all other lineages, with a star-like haplotype network with the main haplotype as the core. It also had a rapid population expansion history, indicating that the source lineage might have stronger diffusion ability and adaptability. Our findings provided a significant biological basis for fine tracking of invasive source at the lineage or population level and promote early invasion warning of potential invasive species on a much subtler lineage level.

Mitogenomes Provide Insights into the Species Boundaries and Phylogenetic Relationships among Three Dolycoris Sloe Bugs (Hemiptera: Pentatomidae) from China.

(1) Background: The three sloe bugs, Dolycoris baccarum, Dolycoris indicus, and Dolycoris penicillatus, are found in the Chinese mainland and are morphologically similar. The species boundaries and phylogenetic relationships of the three species remain uncertain; (2) Methods: In this study, we generated multiple mitochondrial genomes (mitogenomes) for each of the three species and conducted comparative mitogenomic analysis, species delimitation, and phylogenetic analysis based on these data; (3) Results: Mitogenomes of the three Dolycoris species are conserved in nucleotide composition, gene arrangement, and codon usage. All protein-coding genes (PCGs) were found to be under purifying selection, and the ND4 evolved at the fastest rate. Most species delimitation analyses based on the COI gene and the concatenated 13 PCGs retrieved three operational taxonomic units (OTUs), which corresponded well with the three Dolycoris species identified based on morphological characters. A clear-cut barcode gap was discovered between the interspecific and intraspecific genetic distances of the three Dolycoris species. Phylogenetic analyses strongly supported the monophyly of Dolycoris, with interspecific relationship inferred as (D. indicus + (D. baccarum + D. penicillatus)); (4) Conclusions: Our study provides the first insight into the species boundaries and phylogenetic relationships of the three Dolycoris species distributed across the Chinese mainland.

Limits of mitochondrial genes in delimiting species within a Carbula species complex (Hemiptera: Pentatomidae).

Molecular data has become a powerful tool for species delimitation, particularly among those that present limited morphological differences; while the mitochondrial genome, with its moderate length, low cost of sequencing and fast lineage sorting, has emerged as a practical data set. Due to the limited morphological differences among the closely related species of Carbula Stål 1865, the species boundaries between Carbula abbreviata (Motschulsky, 1866), Carbula humerigera (Uhler, 1860), and Carbula putoni (Jakovlev, 1876) have remained particularly unclear. In this study, we applied two phylogenetic reconstruction methods to two data sets (mitogenome and COI) to assess the phylogeny of Carbula distributed in Asia, and five species delimitation methods to determine the boundaries between East Asian Carbula species. Our phylogenetic analyses showed Carbula to be paraphyletic; the seven known species distributed within East Asia to form a single monophyletic group, and within this, C. abbreviata, C. humerigera, C. putoni and middle-type to comprise a C. humerigera species complex. Our results show that mitogenome data alone, while effective in the differentiation of more distantly related Carbula species, is not sufficient to accurately delimit the species within this newly described complex.

Integrated analysis of miRNA profiles and gut bacterial changes in Altica viridicyanea following antibiotic treatment.

The gut bacteria involves in insect homeostasis by playing essential roles in host physiology, metabolism, innate immunity, and so forth. microRNAs (miRNAs) are endogenous small noncoding RNAs that posttranscriptionally regulate gene expression to affect immune or metabolic processes in insects. For several non-model insects, the available knowledge on the relationship between changes in the gut bacteria and miRNA profiles is limited. In this study, we investigated the gut bacterial diversity, composition, and function from Altica viridicyanea feeding on normal- and antibiotic-treated host plants using 16S rRNA amplicon sequencing; antibiotics have been shown to affect the body weight and development time in A. viridicyanea, suggesting that the gut bacteria of the normal sample were more diverse and abundant than those of the antibiotic-fed group, and most of them were involved in various physical functions by enrichment analysis. Furthermore, we executed small RNA transcriptome sequencing using the two experimental groups to obtain numerous sRNAs, such as piRNAs, siRNAs, and known and novel miRNAs, by data mapping and quality control, and furthermore, a total of 224 miRNAs were identified as significantly differentially expressed miRNAs, of which some DEMs and their target genes participated in immune- and metabolism-related pathways based on GO and KEGG annotation. Besides, regarding the regulatory roles of miRNA and target genes, a interaction network of DEM-target gene pairs from eight immune- or metabolism-related signaling pathways were constructed. Finally, we discovered that DEMs from above pathways were significantly positively or negatively correlated with gut bacterial alterations following antibiotic treatment. Collectively, the observations of this study expand our understanding of how the disturbance of gut bacteria affects miRNA profiles in A. viridicyanea and provide new valuable resources from extreme ranges for future studies on the adaptive evolution in insects.

The lncRNA-mediated ceRNA network of Altica viridicyanea is involved in the regulation of the Toll/Imd signaling pathway under antibiotic treatment.

Long noncoding RNAs (lncRNAs) play significant roles in the regulation of mRNA expression or in shaping the competing endogenous RNA (ceRNA) network by targeting miRNA. The insect gut is one of the most important tissues due to direct contact with external pathogens and functions in the immune defense against pathogen infection through the innate immune system and symbionts, but there are limited observations on the role of the lncRNA-involved ceRNA network of the Toll/Imd pathway and correlation analysis between this network and bacterial microbiota in the Altica viridicyanea gut. In this research, we constructed and sequenced six RNA sequencing libraries using normal and antibiotic-reared samples, generating a total of 17,193 lncRNAs and 26,361 mRNAs from massive clean data by quality control and bioinformatic analysis. Furthermore, a set of 8,539 differentially expressed lncRNAs (DELs) and 13,263 differentially expressed mRNAs (DEMs), of which related to various immune signaling pathways, such as the Toll/Imd, JAK/STAT, NF-κB, and PI3K-Akt signaling pathways, were obtained between the two experimental groups in A. viridicyanea. In addition, numerous GO and KEGG enrichment analyses were used to annotate the DELs and their target genes. Moreover, six Toll family members and nineteen signal genes from the Toll/Imd signaling pathway were identified and characterized using online tools, and phylogenetic analyses of the above genes proved their classification. Next, a lncRNA-miRNA-mRNA network of the Toll/Imd pathway was built, and it contained different numbers of DEMs in this pathway and related DELs based on prediction and annotation. In addition, qRT-PCR validation and sequencing data were conducted to show the expression patterns of the above DELs and DEMs related to the Toll/Imd signaling pathway. Finally, the correlated investigations between DELs or DEMs of the Toll/Imd signaling pathway and most changes in the gut bacterial microbiota revealed significantly positive or negative relationships between them. The present findings provide essential evidence for innate immune ceRNAs in the beetle gut and uncover new potential relationships between innate immune pathways and the gut bacterial microbiota in insects.

New data on mitogenomes of Thienemanniella Kieffer, 1911 (Diptera: Chironomidae, Orthocladiinae).

The mitochondrial genome (mitogenome) has been widely used as a powerful marker in phylogenetic and evolutionary studies of various Dipteran groups. However, only a few mitogenomes from the Thienemanniella genus have been reported till now. Furthermore, there is still indeterminacy in the phylogenetic relationships of the genus Thienemanniella. In this study, mitogenomes of five Thienemanniella species were sequenced and analyzed newly. Combined with the published mitogenome of Thienemanniella nipponica, the obtained results showed that mitogenomes of Thienemanniella were conserved in structure, and all genes were observed to be arranged in the same gene order as the ancestral mitogenome. Nucleotide composition varied significantly among different genes, and the control region displayed the highest A + T content. All protein coding genes are subjected to purification selection, and the fastest evolving gene is ATP8. Maximum likelihood and Bayesian inference analyses showed the phylogeny of Thienemanniella which was supported in five topologies. Our present study provides valuable insight into the phylogenetic relationships of Thienemanniella species.

Changes in the gut bacterial community affect miRNA profiles in Riptortus pedestris under different rearing conditions.

Insects possess complex and dynamic gut microbial system, which contributes to host nutrient absorption, reproduction, energy metabolism, and protection against stress. However, there are limited data on interactions of host-gut bacterial microbiota through miRNA (microRNA) regulation in a significant pest, Riptortus pedestris. Here, we performed the 16S rRNA amplicon sequencing and small RNA sequencing from the R. pedestris gut under three environmental conditions and antibiotic treatment, suggesting that we obtained a large amount of reads by assembly, filtration and quality control. The 16S rRNA amplicon sequencing results showed that the abundance and diversity of gut bacterial microbiota were significantly changed between antibiotic treatment and other groups, and they are involved in metabolism and biosynthesis-related function based on functional prediction. Furthermore, we identified different numbers of differentially expressed unigenes (DEGs) and differentially expressed miRNAs (DEMs) based on high-quality mappable reads, which were enriched in various immune-related pathways, including Toll-like receptor, RIG-I-like receptor, NOD-like receptor, JAK/STAT, PI3K/Akt, NF-κB, MAPK signaling pathways, and so forth, using GO and KEGG enrichment analysis. Later on, the identified miRNAs and their target genes in the R. pedestris gut were predicted and randomly selected to construct an interaction network. Finally, our study indicated that alterations in the gut bacterial microbiota are significantly positively or negatively associated with DEMs of the Toll/Imd signaling pathway with Pearson correlation analysis. Taken together, the results of our study lay the foundation for further deeply understanding the interactions between the gut microbiota and immune responses in R. pedestris through miRNA regulation, and provide the new basis for pest management in hemipteran pests.

Mechanical characteristics analysis and control algorithm for floating raft system with mass variation.

The ship floating raft system adopts the integrated design of large liquid tanks and rafts, which can optimize the arrangement in the cabin and increase the intermediate mass of the system to achieve efficient vibration isolation of equipment. One of the major challenges is that the change of liquid mass in the tank will cause displacement of the raft, which will change the modal characteristics of the system and affect the stability of the vibration isolation system performance. This paper establishes a mechanical analysis model of a floating raft system under time-varying liquid mass conditions. Taking a ship variable mass floating raft system as the research object, the effect of mass change on the characteristics of raft displacement, isolator load distribution, and modal frequency of the vibration isolation system is analyzed. The analysis shows that when the liquid tank goes from full load to no-load state, its mass change accounts for 40% of the total mass of the raft, which will cause a large displacement of the raft and change the low order modal frequency of the system, bringing the risk of equipment safety and vibration isolation performance degradation. Therefore, an adaptive variable load control method is proposed to realize the raft attitude balance and load equalization optimization under the variable mass condition of the floating raft air spring system. The test results show that the proposed control method can automatically adapt to the large mass gradual change from full load to no load of the liquid tank on the raft, and control the displacement of the raft structure from about 10 mm to 1.5 mm, which effectively ensures the stability of the air spring system performance.

Mitochondrial introgression and mito-nuclear discordance obscured the closely related species boundaries in Cletus Stål from China (Heteroptera: Coreidae).

Accurate taxonomy and delimitation are of great importance for pest control strategies and management programs. Here, we focus on Cletus (Insecta: Hemiptera: Coreidae), which includes many crop pests. The species boundaries still conflict and only cytochrome c oxidase subunit I (COI) barcoding has been previously used for molecular studies. We generated new mitochondrial genome and nuclear genome-wide SNPs to explore the species boundaries of 46 Cletus samples from China using multiple species delimitation approaches. All results recovered a monophyly with high support, except for two closely related species in clade I - C. punctiger and C. graminis. Mitochondrial data demonstrated admixture in clade I, while genome-wide SNPs unambiguously identified two separate species, which were confirmed by morphological classification. Inconsistent nuclear and mitochondrial data indicated mito-nuclear discordance. Mitochondrial introgression is the most likely explanation, and more extensive sampling and more comprehensive data are needed to ascertain a pattern. Accurate species delimitation will shed light on species status; thus, an accurate taxonomy is of particular concern, as there is a pressing need to implement precise control of agricultural pests and to perform further research on diversification.

Out of East Asia: Early Warning of the Possible Invasion of the Important Bean Pest Stalk-Eyed Seed Bug Chauliops fallax (Heteroptera: Malcidae: Chauliopinae).

The short stay at the beginning of the invasion process is a critical time for invasive species identification and preventing invasive species from developing a wider distribution and significant economic impact. The stalk-eyed seed bug Chauliops fallax is an important agricultural pest of soybean and was first reported to occur outside East Asia. Here, we reported the native evolutionary history, recent invasion history, and potential invasion threats of C. fallax for the first time based on population genetic methods and ecological niche modelling. The results showed that four native East Asian genetic groups (EA, WE, TL, and XZ) were well supported, showing an east-west differentiation pattern consistent with the geographical characteristics of three-step landforms in China. Two main haplotypes existed: Hap1 might have experienced a rapid northwards expansion process after the LGM period, and Hap5 reflected local adaptation to the environment in southeastern China. The Kashmir sample was found to come from the recent invasion of populations in the coastal areas of southern China. Ecological niche modelling results suggested that North America has a high risk of invasion, which might pose a serious threat to local soybean production. In addition, with future global warming, the suitable habitat in Asia will move towards the higher latitude region and gradually deviate from the soybean planting area, which indicates the threat of C. fallax to soybean production in Asia will decrease in the future. The results could provide new insights into the monitoring and management of this agricultural pest in the early invasion stage.

Taxonomic review of Manocoreini with description of a new species from China (Hemiptera, Heteroptera, Coreidae).

In the present paper, all seven species of Manocoreini are reviewed, and a new species Manocoreushsiaoisp. nov. is described from Guangxi, China. Photographs of habitus of all species, and detailed structures of the new species and type species of Manocoreus Hsiao, 1964 are provided. All species of Manocoreini of the world are keyed. A distribution map of all species is also provided.

Climate change impacts on the potential worldwide distribution of the soybean pest, Piezodorus guildinii (Hemiptera: Pentatomidae).

The redbanded stink bug, Piezodorus guildinii (Westwood, 1837), is a highly destructive soybean pest native to the Neotropical Region. In the past 60 yr, P. guildinii has been observed to expand its distribution in North and South America, causing significant soybean yield losses. In order to predict the future distribution direction of P. guildinii and create an effective pest control strategy, we projected the potential global distribution of P. guildinii using 2 different emission scenarios, Shared Socioeconomic Pathways 126 and 585, and 3 Earth system models, with the maximum entropy niche model (MaxEnt). Then, the predicted distribution areas of P. guildinii were jointly analyzed with the main soybean-producing areas to assess the impact for different soybean region. Our results showed that temperature is the main environmental factor limiting the distribution of P. guildinii. Under present climate conditions, all continents except Antarctica have suitable habitat for P. guildinii. These suitable habitats overlap with approximately 45.11% of the total global cultivated soybean areas. Moreover, P. guildinii was predicted to expand its range in the future, particularly into higher latitudes in the Northern hemisphere. Countries, in particular the United States, where soybean is widely available, would face a management challenge under global warming. In addition, China and India are also high-risk countries that may be invaded and should take strict quarantine measures. The maps of projected distribution produced in this study may prove useful in the future management of P. guildinii and the containment of its disruptive effects.

Chromosome-level genome of the bean bug Megacopta cribraria in native range, provides insights into adaptation and pest management.

Megacopta cribraria, a bean pest causing tremendous economic losses in Asia, was discovered in North America in 2009. Although M. cribraria has become the focus of research on biological invasion and pest management, the lack of genomic resources limits in-depth studies. Here, we report the first chromosome-level genome of M. cribraria using Illumina, PacBio, and Hi-C data. The assembled genome size was 699.65 Mb, with a contig N50 of 1.43 Mb and a scaffold N50 of 109.27 Mb. >97.51 % of bases were successfully anchored to six chromosomes. Through genome annotation, a total of 13,308 coding genes were predicted, 96.3 % of which were successfully accessed function. Expanded gene families were involved in proteolysis, protein metabolism and nitrogen metabolism reflected the underlying genome basis for host adaptation during evolution. Transcriptome analysis revealed different gene expression patterns in antenna, mouthpart, head, leg, wing, and carcass body of the adult M. cribraria, respectively. Moreover, the expression profiles of the odorant receptor genes indicated the potential target genes for pest control. The high-quality chromosome-level genome will benefit further research on the adaptation, evolution, and population genetics of the M. cribraria that will assist in the pest management and tracking the biological invasion routes.

The first discovery of Tc1 transposons in yeast.

Background: Identification of transposons without close homologs is still a difficult task. IS630/Tc1/mariner transposons, classified into a superfamily, are probably the most widespread DNA transposons in nature. Tc1/mariner transposons have been discovered in animals, plants, and filamentous fungi, however, not in yeast. Results: In the present study, we report the discovery of two intact Tc1 transposons in yeast and filamentous fungi, respectively. The first one, named Tc1-OP1 (DD40E), represents Tc1 transposons in Ogataea parapolymorpha. The second one, named Tc1-MP1 (DD34E), represents Tc1 transposons in the Rhizopodaceae and Mucoraceae families. As a homolog of Tc1-OP1 and Tc1-MP1, IS630-AB1 (DD34E) was discovered as an IS630 transposon in Acinetobacter spp. Conclusion: Tc1-OP1 is not only the first reported Tc1 transposon in yeast, but also the first reported nonclassical Tc1 transposon. Tc1-OP1 is the largest of IS630/Tc1/mariner transposons reported to date and significantly different from others. Notably, Tc1-OP1 encodes a serine-rich domain and a transposase, extending the current knowledge of Tc1 transposons. The phylogenetic relationships of Tc1-OP1, Tc1-MP1 and IS630-AB1 indicated that these transposons had evolved from a common ancestor. Tc1-OP1, Tc1-MP1 and IS630-AB1 can be used as reference sequences to facilitate the identification of IS630/Tc1/mariner transposons. More Tc1/mariner transposons will be identified in yeast, following our discovery.

The anthropogenic effect of land use on population genetics of Malcus inconspicuus.

Since the beginning of the Holocene era, human activities have seriously impacted animal habitats and vegetative environments. Species that are dependent on natural habitats or with narrow niches might be more severely affected by habitat changes. Malcus inconspicuus is distributed in subtropical China and highly dependent on the mountain environment. Our study investigated the role of the mountainous landscape in the historical evolution of M. inconspicuus and the impact of Holocene human activities on it. A phylogeographical approach was implemented with integrative datasets including double-digest restriction site-associated DNA (ddRAD), mitochondrial data, and distribution data. Three obvious clades and an east-west phylogeographical pattern were found in subtropical China. Mountainous landscape has "multifaceted" effects on the evolutionary history of M. inconspicuus, it has contributed to population differentiation, provided glacial refuges, and provided population expansion corridors during the postglacial period. The effective population size (Ne) of M. inconspicuus showed a sharp decline during the Holocene era, which revealed a significantly negative correlation with the development of cropland in a hilly area at the same time and space. It supported that the species which are highly dependent on natural habitats might undergo greater impact when the habitat was damaged by agricultural activities and we should pay more attention to them, especially in the land development of their distribution areas.

Phylogeny and historical biogeography of the water boatmen (Insecta: Hemiptera: Heteroptera: Nepomorpha: Corixoidea).

The water boatmen of Corixoidea, a group of aquatic bugs with more than 600 extant species, is one of the largest superfamilies of Nepomorpha. Contrary to the other nepomorphan lineages, the Corixoidea are most diverse in the Laurasian remnant Holarctic region. To explicitly test whether the present-day Holarctic distribution of diverse corixids is associated with the arising of the Laurasian landmass that was separated from Gondwana, we investigated the phylogeny, divergence times and historical biogeography of Corixoidea based on morphological and molecular characters sampled from 122 taxa representing all families, subfamilies, tribes and approximately 54 % of the genera. Our results were largely congruent with the phylogenetic relationships within the established nepomorphan phylogenetic context. The fossil calibrated chronogram, diversification analysis and ancestral ranges reconstruction indicated that Corixoidea began to diversify in Gondwana in the late Triassic approximately at 224 Ma and the arising of the most diverse subfamily Corixinae in Corixidae in the Holarctic region was largely congruent with the time of separation of Laurasia from Gondwana. The large-scale expansion of the temperate and cold zones on the northward-moving Laurasian landmass after the breakup of the Pangea provided new aquatic niches and ecological opportunities for promoting rapid diversification for the Holarctic corixid lineage.

Heissophila macrotheleae Schuh, 2006, the first record of the subfamily Heissophilinae from China (Hemiptera: Heteroptera: Plokiophilidae).

Heissophila Schuh, 2006, a monotypic genus of Heissophilinae (Hemiptera: Plokiophilidae), is newly recorded from Oriental China based on Heissophila macrotheleae Schuh, 2006 collected from Yunnan Province, China. The diagnosis of the species is expanded based on the newly collected materials. In addition, a DNA barcode of H. macrotheleae and host spider are provided. Photographs of the co-existing web-loving bugs and the natural habitat are also present.

Species boundary and phylogeographical pattern provide new insights into the management efforts of Megacopta cribraria (Hemiptera: Plataspidae), a bean bug invading North America.

BACKGROUND: Correct identification together with information on distribution range, geographical origin and evolutionary history are the necessary basis for the management and control of invasive species. The bean bug Megacopta cribraria is a crucial agricultural pest of soybean. Recently, M. cribraria has invaded the United States and spread rapidly, causing severe reductions in soybean yields. However, the species boundary and phylogeographical pattern of this invasive bean bug are still unclear. RESULTS: The results of different species delimitation methods (Automatic Barcode Gap Discovery, Assemble Species by Automatic Partitioning, Bayesian Poisson Tree Processes and Bayesian Phylogenetics and Phylogeography) strongly demonstrated that M. cribraria and Megacopta punctatissima represent the same species. M. punctatissima should not be considered a distinct species but rather a variety of M. cribraria. Phylogenetic analyses revealed three well-supported clades (Southeast Asia [SEA], East Asia continent [EAC] and Japan [JA]) with distinct geographical structures in the M. cribraria-M. punctatissima complex. The SEA clade was at the base of the phylogenetic tree, and the sister relationship between the EAC clade and JA clade was strongly supported. The split between the EAC clade and JA clade occurred at approximately 0.71 Ma, corresponding to the submergence period of the East China Sea land bridge. CONCLUSION: This study clarified the species boundary between M. cribraria and its closely related species and revealed the phylogeographical pattern and evolutionary history of M. cribraria. The species delimitation and phylogeography results achieved in this study could provide new insights into the monitoring and management of this agricultural pest. © 2022 Society of Chemical Industry.