Effect of fluorescent brighteners on the insecticidal activity of Bacillus thuringiensis var. kurstaki and LdMNPV on Lymantria dispar asiatica in Korea.

Stilbene-based fluorescent brighteners (FBs) have been demonstrated to improve the insecticidal activities of entomopathogenic viruses; however, there is limited information regarding their effect on entomopathogenic bacteria. We conducted this study to investigate the effect of two FBs (FB 28 and FB 71) on the insecticidal activities of Bacillus thuringiensis var. kurstaki (Btk) and Lymantria dispar multiple nuclear polyhedrosis virus (LdMNPV) on Lymantria dispar asiatica. FB 28 and Btk combination at low concentration (1.6 × 102 IU/mL) increased the mortality, whereas FB 71 and Btk combination at intermediate and high concentrations (1.6 × 103 and 1.6 × 104 IU/mL) slightly reduced the mortality compared with that with Btk alone. The lethal time was also shorter with combinations of Btk and FB 28 than with FB 71. Both FB 28 and FB 71 increased the mortality in combination with LdMNPV at all concentrations (3 × 102 , 3 × 104 , and 3 × 106 polyhedral occlusion bodies/mL compared with that with LdMNPV alone. Our results suggest that FBs improve the insecticidal activities of Btk and LdMNPV, and their activities depend on their interactions with the midgut structures of the host insect species.

Population dynamics and biorational management of sucking insect vectors on chili (Capsicum annuum L.) in Bangladesh.

To evaluate population fluctuations in relation to weather parameters and biorational management of sucking insect vectors in chili (Capsicum annuum L.), we conducted a study at the experimental field of Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, in 2020 and 2021. It has been shown in this study that sucking insects (aphids, jassids, whiteflies, and thrips) were active throughout the study period. The highest count of sucking insect vectors (24.67 aphids, 13.72 whitefly, and 56.56 thrips) in March and (14.83 jassid) in April was recorded at average temperatures of 34-36°C and 31°C, respectively. There was a positive correlation between pest abundance and temperature, relative humidity, and rainfall for all insects, with the exception of a negative correlation between whiteflies and temperature and rainfall. The results of linear regression models showed that abiotic factors contribute to pest abundance levels, with 100R2 values of 14.9 (thrips), 46.3 (jassids), 7.1 (whiteflies), and 0.67 (aphids); the results were statistically significant for all models in the case of thrips, jassids, and whiteflies, but not significant in the case of aphids. The most effective treatment was spinosad 45SC, a bacterium-derived pesticide recommended for the control of sucking insect vector complexes in chili. The results from the spinosad-treated plot, in terms of insect counts and corresponding mortality rates, were as follows: aphids (3.68), 68.89%; jassids (3.52), 72.01%; whiteflies (3.00), 66.69%; and thrips (3.40), 69.20%. The results of this study will aid in developing predictive models of different control agents against sucking insect vectors in vegetable crops.

Control efficacy of azadirachtin on the fall armyworm, Spodoptera frugiperda (J. E. Smith) by soil drenching.

The fall armyworm (FAW), Spodoptera frugiperda, is an important agricultural pest species native to the Western Hemisphere and has recently invaded to Africa and Asia. Owing to the development of pesticide resistance and environmental contamination, ecofriendly pesticides are desirable for FAW control. Azadirachtin is a plant-derived natural pesticide with low toxicity to humans and the natural environment. Azadirachtin is primarily applied by foliar spraying; however, this approach lowers the efficacy of controlling target insects owing to photodegradation and might give a harmful effect on nontarget beneficial insects. Thus, we investigated whether applying azadirachtin to soil improves FAW control and its toxicity to corn plants. Soil drainage of azadirachtin exhibited no phytotoxic effects on corn plants but significantly reduced the larval body weight and delayed the developmental period of each larval instar of FAW. Applying 10, 15, and 20 ppm azadirachtin to soil inhibited larval growth by 68%, 76%, and 91%, respectively. Furthermore, the survival rate of FAW gradually decreased when larvae were fed azadirachtin-treated corn leaves. Collectively, this is the first study suggesting the systemic efficacy of azadirachtin by soil drenching against FAW.

Biology, distribution, and management of invasive South American tomato leafminer, Tuta absoluta (Meyrick) (Lepidoptera; Gelechiidae), in Asia.

South American tomato leafminer, Tuta absoluta (Meyrick, 1917) (Lepidoptera: Gelechiidae), is native to South America, but is a major invasive and quarantine pest species in Europe, Africa, and Asia. It causes extensive damage of up to 100% yield loss in tomatoes (Solanum lycopersicum) in open and greenhouse conditions. Since its first invasion in Spain in 2006, it has spread rapidly into many countries in the Mediterranean and Western Europe and further invaded Africa and Asia. In Asia, it was first recorded in August 2009 in Turkey and spread to most South and East Asian countries. In this study, we reviewed existing work on the biology and distribution of T. absoluta in Asia, as well as the damage it causes. This review will help to develop efficient management tactics as well as establish quarantine and phytosanitary precautions in uninvaded countries.

Identification of begomoviruses associated with the insect vector Bemisia tabaci and various host plants on Java Island, Indonesia.

Begomoviruses are economically important plant viruses and are transmitted by Bemisia tabaci which is a complex of various cryptic species. However, it is uncertain whether most begomoviruses that infect host plants are transmitted by B. tabaci at a similar rate. We compared the begomovirus profiles that were detected in a total of 37 whitefly populations and 52 host plants on Java Island, Indonesia. Seven begomovirus species were detected in B. tabaci at different rates: pepper yellow leaf curl Indonesia virus (PepYLCIV, 56.8%), tomato yellow leaf curl Kanchanaburi virus (TYLCKaV, 46.0%), tomato leaf curl New Delhi virus (ToLCNDV, 21.6%), squash leaf curl China virus (SLCCNV, 21.6%), ageratum yellow vein China virus (AYVCNV, 2.7%), mungbean yellow mosaic India virus (MYMIV, 2.7%), and okra enation leaf curl virus (OELCuV, 2.7%). The begomoviruses were detected at different rates in three cryptic species of B. tabaci. In addition, six begomovirus species were detected in the various host plants at different rates: PepYLCIV (67.3%), TYLCKaV (53.9%), ToLCNDV (13.5%), MYMIV (11.5%), AYVCNV (3.9%), and Tomato yellow leaf curl Thailand virus (TYLCTHV) (1.9%). By comparing the virus presence between whiteflies and plants, five begomoviruses (AYVCNV, MYMIV, PepYLCIV, ToLCNDV, and TYLCKaV) were detected in both samples, but their sequence similarity was highly variable depending on the begomovirus themselves; TYLCKaV was highest (99.4%-100%) than any other viruses. Our study suggests B. tabaci acquire begomoviruses at different rates from plants. This study provides important information on the potential variation in the begomovirus transmission mechanism.

Effects of Lymantria dispar multiple nucleopolyhedrovirus and Bacillus thuringiensis var. kurstaki on different larval instars of Lymantria dispar asiatica.

Outbreaks of Lymantria dispar asiatica (the Asian spongy moth; Lepidoptera: Erebidae) occur sporadically, causing widespread damage to forest and fruit trees. Owing to the development of pesticide resistance and environmental contamination, biopesticides, including L. dispar multiple nucleopolyhedrovirus (LdMNPV) and Bacillus thuringiensis var. kurstaki (Btk), can significantly contribute to controlling overall larval stage of this species. Although both pathogens are highly effective at the larval stage, their effects on different instar stages have not been investigated. In this study, we analyzed the mortality and lethality in different L. dispar asiatica instars exposed to single or combined pathogen treatments. Treatments with low or medium LdMNPV concentrations induced lower mortality and had higher LT50 values at the 4th and 5th instars compared with other instars, whereas high LdMNPV treatments induced high mortality in all instars, with higher LT50 values at later instars. Treatment with Btk induced a rapid 100% mortality in all instars, with higher LT50 values for the later instars. The combination of LdMNPV and Btk delayed the killing time compared with the effects of single treatments, with the effect being more pronounced in the 1st and 5th instar stage than at other stages at low Btk concentrations. Our findings indicate that the pathogenic effects of LdMNPV and Btk on L. dispar asiatica differ according to larval stage, thereby providing novel insights into enhancing the biological control efficacy of these agents against L. dispar asiatica in the field.

Genetic diversity of cryptic species of Bemisia tabaci in Asia.

Bemisia tabaci is a species complex consisting of various genetically different cryptic species worldwide. To understand the genetic characteristics and geographic distribution of cryptic species of B. tabaci in Asia, we conducted an extensive collection of B. tabaci samples in ten Asian countries (Bangladesh, Indonesia, Japan, Korea, Myanmar, Nepal, Philippines, Singapore, Taiwan, and Vietnam) from 2013 to 2020 and determined 56 different partial sequences of mitochondrial cytochrome oxidase subunit I (COI) DNA. In addition, information on 129 COI sequences of B. tabaci identified from 16 Asian countries was downloaded from the GenBank database. Among the total 185 COI sequences of B. tabaci, the sequence variation reached to 19.68%. In addition, there were 31 cryptic species updated from 16 countries in Asia, that is, Asia I, Asia I India, Asia II (1-13), Asia III, Asia IV, Asia V, China 1-6, MEAM (1, 2, K), MED, Australia/Indonesia, Japan (1 and 2). Further, MED cryptic species consisted of 2 clades, Q1 and Q2. This study provides updated information to understand the genetic variation and geographic diversity of B. tabaci in Asia.

Developing extreme heat acclimation in Bemisia tabaci Mediterranean (Hemiptera: Aleyrodidae).

Bemisia tabaci Mediterranean (MED) is a highly invasive cryptic species complex found in the world's tropical, subtropical, and temperate regions. It is a severe pest of various crops and a vector of plant pathogenic viruses, particularly geminiviruses. Thermal acclimation of insects is a critical for the survival in unfavorable temperature condition. We observed that great survival rate of B. tabaci MED at the uncontrolled greenhouse which had fluctuating temperature condition (FTC) from 10°C to 60°C in spring and summer season. Our study showed that while B. tabaci MED reared under FTC for 10 weeks from April to June, its survival rate was gradually increased when heat shock was treated 50°C for 0.5 h. In contrast, the same heat shock treatment was lethal in the colony reared under constant temperature condition (CTC) at the controlled insectary. After being acclimated, the lethal temperatures LT50 , LT95 , and LT100 under CTC were 47.7°C, 50.1°C, and 50.3°C, whereas those under FTC were 59.8°C, 62.7°C, and 63.0°C, respectively. In addition, we observed that the transcript levels of three investigated heat shock protein (HSP) genes (hsp20, hsp70, and hsp90) were lower under FTC than under CTC. This study suggests that B. tabaci MED retains high heat acclimation ability, making it tolerant of extreme thermal conditions.

Identification of entomopathogenic fungus Metarhizium rileyi infested in fall armyworm in the cornfield of Korea, and evaluation of its virulence.

The fall armyworm (FAW) Spodoptera frugiperda is an important invasive pest in Africa and Asia. It is a polyphagous pest with at least 353 recorded host plant species, including corn. Chemical control of this pest is unsuccessful because of a developed resistance and harmful effects on the environment. Entomopathogenic fungi are potential biological control agents for FAW. In this study, the native strain of Metarhizium rileyi (KNU-Ye-1), collected from a cornfield at Yeongcheon, Korea, was identified by morphological and molecular characterization. The susceptibility of the fourth-instar larvae of FAW to the native strain M. rileyi was examined in the laboratory. The results showed that the Korean strain of M. rileyi (KNU-Ye-1) was highly virulent to FAW larvae, causing 89% mortality 7 days posttreatment. Therefore, M. rileyi (KNU-Ye-1) identified in this study is highly valuable for the biological control of FAW in the field.

Identification of begomoviruses from different cryptic species of Bemisia tabaci in Bangladesh.

Bemisia tabaci is a global species complex consisting of at least 40 cryptic species. It is also a vector for at least 100 species of begomovirus, many of which cause severe crop damage. The relationship between begomoviruses and cryptic species of the B. tabaci species complex, however, remains unclear. Our previous study [13] was identified four cryptic species (Asia I, Asia II 1, Asia II 5, and Asia II 10) of B. tabaci from Bangladesh. Using those 110 whitefly samples, vector-based PCR analysis identified 8 different begomovirus species: BYVMV, BGYVV, OELCV, SLCCV, SLCV, TbCSV, ToLCBV, and ToLCNDV. The overall rate of virus infection was 26.4%, and BYVMV and ToLCNDV were the most frequently detected in the B. tabaci vector. Virus infection rates for Asia I, Asia II 1, Asia II 5, and Asia II 10 were 22.4% (15/67), 35% (7/20), 27.3% (6/22), and 100% (1/1), respectively. Each cryptic species infected multiple virus species, but SLCCV, TbCSV, and BGYVV were each only detected in, Asia I, Asia II 1, and Asia II 5, respectively. This study demonstrates the geographic distribution of various begomoviruses in Bangladesh and their relationships with cryptic species of B. tabaci.

Differential responses between a vector species Bemisia tabaci and a nonvector species Trialeurodes vaporariorum following ingestion of tomato yellow leaf curl virus.

In transmitting plant viruses, insect vectors undergo physiological and behavioral alterations. The whitefly Bemisia tabaci is a vector of tomato yellow leaf curl virus (TYLCV), causing severe damages to various horticultural crop plants. To determine whether whitefly alteration is specific to vector species, the responses to TYLCV ingestion were compared between B. tabaci and Trialeurodes vaporariorum, a nonvector for TYLCV. The two species were reared on TYLCV-infected and noninfected tomato, a host of TYLCV, and their longevity and fecundity were determined while rearing in either tomato or eggplant, a nonhost of TYLCV. TYLCV-ingested B. tabaci increased their developmental rates but reduced fecundity when they were reared in either tomato or eggplant compared with those of TYLCV-free ones. In contrast, TYLCV-ingested T. vaporariorum did not show any of the aforementioned changes when reared on both plant species. In addition, TYLCV-ingested B. tabaci increased their levels of three heat shock protein genes ( hsp20, hsp70, and hsp90) against thermal stress, whereas TYLCV-ingested T. vaporariorum did not. The presence of TYLCV virions was identified in two colonies of both species via polymerase chain reaction analysis. TYLCV was detected in the whole body, saliva, and eggs of B. tabaci, while TYLCV was detected only in the whole body but not in the saliva and eggs of T. vaporariorum. The present results strongly indicated that TYLCV specifically manipulate physiological processes of the vector species, B. tabaci.

Insecticidal efficacy of three benzoate derivatives against Aphis gossypii and its predator Chrysoperla carnea.

Plant-derived benzoates are known to have insecticidal and repellent activities, however, these effects have been evaluated thoroughly in only a few groups of insects. In this study, the insecticidal activities of three commercially available benzoates, methyl benzoate (MB), ethyl benzoate (EB), and vinyl benzoate (VB), were assessed against the cotton aphid, Aphis gossypii Glover, and its lacewing predator, Chrysoperla carnea Stephens. MB showed the highest contact toxicity against nymphs and adults of A. gossypii, as compared to those of EB and VB. Treatments with 1% MB, EB, and VB were 100.0%, 93.0% and 60.0% effective, respectively, against third-instar nymphs and 100.0%, 69.0%, and 39.0% effective, respectively, against adults of A. gossypii, as evaluated 24 h after application. A mixture of MB + EB showed higher efficacy than other benzoate combinations against A. gossypii. The efficacy of MB, EB, and VB against A. gossypii on cucumber plants under greenhouse conditions was 93.7%, 68.5%, and 56.6%, respectively. In addition, treatments with 1% MB, EB, and VB were 20.0%, 24.0%, and 12.0% effective, respectively, against first-instar larvae and 6.7%, 13.3%, and 6.7% effective, respectively, against adults of C. carnea at 24 h after treatment. Our results showed that the tested benzoates were less toxic to the predator C. carnea than to the pest A. gossypii. In addition, the aphid population was significantly lower in a treatment combining predation by C. carnea followed by use of 1% MB, compared with treatments of either 1% MB or predators alone. This study suggests that plant-derived benzoates are potential biopesticides for aphid control and are compatible with natural enemies in integrated pest management.

Induction of stress- and immune-associated genes in the Indian meal moth Plodia interpunctella against envenomation by the ectoparasitoid Bracon hebetor.

Envenomation is an important process in parasitism by parasitic wasps; it suppresses the immune and development of host insects. However, the molecular mechanisms of host responses to envenomation are not yet clear. This study aimed to determine the transcription-level responses of the Indian meal moth Plodia interpunctella against envenomation of the ectoparasitoid Bracon hebetor. Quantitative real-time reverse-transcription PCR was used to determine the transcriptional changes of 13 selected genes, which are associated with development, metabolism, stress, or immunity, in the feeding and wandering fifth instar larvae over a 4-day period after envenomation. The effects of envenomation on the feeding-stage larvae were compared with those of starvation in the transcriptional levels of the 13 genes. Most selected genes were altered in their expression by either envenomation or starvation. In particular, a heat shock protein, hsp70, was highly upregulated in envenomated larvae in both the feeding and wandering stages as well as in starved larvae. Further, some genes were upregulated by envenomation in a stage-specific manner. For example, hsp25 was upregulated after envenomation in the feeding larvae, but hsp90 and an immune-associated gene, hemolin, were upregulated in the wandering larvae. However, both envenomation and starvation resulted in the downregulation of genes associated with development and metabolism. Taken together, P. interpunctella upregulated stress- and immune-responsive genes, but downregulated genes associated with development and metabolism after envenomation. This study provides important information for understanding the molecular mechanisms of host responses to parasitism.

Anti-Inflammatory Activity of Crude Venom Isolated from Parasitoid Wasp, Bracon hebetor Say.

Pest control in the agricultural fields, a major concern globally, is currently achieved through chemical or biological methods. Chemical methods, which leave toxic residue in the produce, are less preferred than biological methods. Venoms injected by stings of various wasps that kill the pest is considered as the examples of the biological method. Although several studies have investigated the biological control of pests through these venoms, very few studies have reported the effects of these venoms on mammalian cells. Bracon hebetor, an ectoparasitoid of the order Hymenoptera, is having a paramount importance in parasitizing various lepidopterous larvae including Plodia interpunctella also called as Indianmeal moth (IMM). Since it is biologically controlled by B. hebetor venom, therefore in our study, herein for the first time, we report the anti-inflammatory activities of the venom from B. hebetor (BHV). We developed a septic shock mice model for in vivo anti-inflammatory studies and RAW 264.7 cells for in vitro studies. Our results clearly demonstrate that BHV can dose dependently abrogate the nitric oxide (NO) production and suppress the levels of proinflammatory mediators and cytokines without posing any cytotoxicity via the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways.

Tomato yellow leaf curl virus Can Overwinter in Stellaria aquatica, a Winter-Hardy TYLCV-Reservoir Weed.

Tomato yellow leaf curl virus (TYLCV), one of the most serious plant viruses in tropical and subtropical regions, is transmitted to host plants by the vector insect Bemisia tabaci. In order to control TYLCV, it is important to identify weed hosts for overwintering TYLCV. Stellaria aquatica, a winter-hardy weed, was found growing with TYLCV-infected tomato plants in greenhouse production. TYLCV was detected in S. aquatica plants by polymerase chain reaction and Southern blot hybridization analysis. The intergenic region nucleotide sequences amplified from TYLCV-infected tomato plants, TYLCV-viruliferous whiteflies, and S. aquatica were identical. During winter (December to February), TYLCV-viruliferous whiteflies and TYLCV-infected tomato plants were removed or absent from greenhouses. However, S. aquatica plants were observed over a period of 10 months from August to May in such greenhouses, and TYLCV was consistently detected in some of these plants. To investigate the transmission of TYLCV from TYLCV-infected S. aquatica plants to healthy tomato plants by whiteflies, TYLCV-infected S. aquatica plants were transplanted to pots in cages with nonviruliferous whiteflies and healthy tomato plants. After 4 weeks, tomato plants developed typical TYLCV disease symptoms, and TYLCV was detected in both whiteflies and tomato plants. These results show that S. aquatica can act as a winter-hardy reservoir for TYLCV, and suggest that this weed could play an important role in overwintering of TYLCV in tomato greenhouses.

Lamium amplexicaule (Lamiaceae): a weed reservoir for tomato yellow leaf curl virus (TYLCV) in Korea.

After the first identification of tomato yellow leaf curl virus (TYLCV) in the southern part of Korea in 2008, TYLCV has rapidly spread to tomato farms in most regions of Korea. From 2008 to 2010, a survey of natural weed hosts that could be reservoirs of TYLCV was performed in major tomato production areas of Korea. About 530 samples were collected and identified as belonging to 25 species from 11 families. PCR and Southern hybridization were used to detect TYLCV in samples, and replicating forms of TYLCV DNA were detected in three species (Achyranthes bidentata, Lamium amplexicaule, and Veronica persica) by Southern hybridization. TYLCV transmission mediated by Bemisia tabaci from TYLCV-infected tomato plants to L. amplexicaule was confirmed, and TYLCV-infected L. amplexicaule showed symptoms such as yellowing, stunting, and leaf curling. TYLCV from infected L. amplexicaule was also transmitted to healthy tomato and L. amplexicaule plants by B. tabaci. The rate of infection of L. amplexicaule by TYLCV was similar to that of tomato. This report is the first to show that L. amplexicaule is a reservoir weed host for TYLCV.

Sweet pepper confirmed as a reservoir host for tomato yellow leaf curl virus by both agro-inoculation and whitefly-mediated inoculation.

Tomato yellow leaf curl virus (TYLCV), a member of the genus Begomovirus, has a single-stranded DNA genome. TYLCV can induce severe disease symptoms on tomato plants, but other hosts plants such as cucurbits and peppers are asymptomatic. A full-length DNA clone of a Korean TYLCV isolate was constructed by rolling-circle amplification from TYLCV-infected tomatoes in Korea. To assess relative susceptibility of sweet pepper varieties to TYLCV, 19 cultivars were inoculated with cloned TYLCV by agro-inoculation. All TYLCV-infected sweet peppers were asymptomatic, even though Southern hybridization and polymerase chain reaction analysis showed TYLCV genomic DNA accumulation in roots, stems, and newly produced shoots. Southern hybridization indicated that TYLCV replicated and moved systemically from agro-inoculated apical shoot tips to roots or newly produced shoots of sweet peppers. Whitefly-mediated inoculation experiments showed that TYLCV can be transmitted to tomatoes from TYLCV-infected sweet peppers. Taken together, these results indicate that sweet pepper can be a reservoir for TYLCV in nature.

Genetic Diversity and DNA Barcoding of Thrips in Bangladesh.

Thrips are economically important pests, and some species transmit plant viruses that are widely distributed and can damage vegetables and cash crops. Although few studies on thrips species have been conducted in Bangladesh, the variation and genetic diversity of thrips species remain unknown. In this study, we collected thrips samples from 16 geographical locations throughout the country and determined the nucleotide sequences of the mitochondrial cytochrome c oxidase subunit 1 (mtCOI) gene in 207 thrips individuals. Phylogenetic analysis revealed ten genera (Thrips, Haplothrips, Megalothrips, Scirtothrips, Frankliniella, Dendrothripoides, Astrothrips, Microcephalothrips, Ayyaria, and Bathrips) and 19 species of thrips to inhabit Bangladesh. Among these, ten species had not been previously reported in Bangladesh. Intraspecific genetic variation was diverse for each species. Notably, Thrips palmi was the most genetically diverse species, containing 14 haplotypes. The Mantel test revealed no correlation between genetic and geographical distances. This study revealed that thrips species are expanding their host ranges and geographical distributions, which provides valuable insights into monitoring the diversity of and control strategies for these pests.

A comprehensive framework for the delimitation of species within the Bemisia tabaci cryptic complex, a global pest-species group.

Identifying cryptic species poses a substantial challenge to both biologists and naturalists due to morphological similarities. Bemisia tabaci is a cryptic species complex containing more than 44 putative species; several of which are currently among the world's most destructive crop pests. Interpreting and delimiting the evolution of this species complex has proved problematic. To develop a comprehensive framework for species delimitation and identification, we evaluated the performance of distinct data sources both individually and in combination among numerous samples of the B. tabaci species complex acquired worldwide. Distinct datasets include full mitogenomes, single-copy nuclear genes, restriction site-associated DNA sequencing, geographic range, host speciation, and reproductive compatibility datasets. Phylogenetically, our well-supported topologies generated from three dense molecular markers highlighted the evolutionary divergence of species of the B. tabaci complex and suggested that the nuclear markers serve as a more accurate representation of B. tabaci species diversity. Reproductive compatibility datasets facilitated the identification of at least 17 different cryptic species within our samples. Native geographic range information provides a complementary assessment of species recognition, while the host range datasets provide low rate of delimiting resolution. We further summarized different data performances in species classification when compared with reproductive compatibility, indicating that combination of mtCOI divergence, nuclear markers, geographic range provide a complementary assessment of species recognition. Finally, we represent a model for understanding and untangling the cryptic species complexes based on the evidence from this study and previously published articles.