New records for the Helius Lepeletier & Serville fauna (Diptera, Limoniidae) of China.

Background: The genus Helius Lepeletier & Serville, 1828 is globally distributed with 232 species and subspecies, of which 25 have been known to occur in China. Amongst the Chinese Helius crane flies, 24 species are distributed in southern China. The species diversity of Helius in other Chinese regions may be severely underestimated due to a lack of investigation. Some investigations on crane flies in Inner Mongolia, China have been initiated by the authors together with other entomologists, with Helius being one of the key targets of attention. New information: Two Helius species, H. (Helius) flavus (Walker, 1856) and H. (H.) gracillimus Alexander, 1938, are added to the Chinese fauna. The two newly-recorded species also represent the first records of the crane fly tribe Elephantomyiini in Inner Mongolia. Re-descriptions and illustrations of the two newly-recorded species are presented.

The genus Platypalpus Macquart (Diptera, Hybotidae, Tachydromiinae) from Inner Mongolia, China, with the description of four new species.

Platypalpus Macquart is reported in Inner Mongolia, China for the first time. Four new species are found: P.flavipilosussp. nov., P.longussp. nov., P.shengisp. nov. and P.shuimogouanussp. nov. This paper provides a description of the four species and a key to the genus in Inner Mongolia.

First Report of Alternaria alternata Causing Leaf Spot on Smooth Bromegrass (Bromus inermis Leyss.) in China.

Smooth bromegrass (Bromus inermis Leyss.) is an important forage crop in northern China. In July 2021, leaf spot symptoms were observed on smooth bromegrass in Ewenki Banner, Hulunbuir, Inner Mongolia. In an area of approximately 0.12 hectares, 95% disease incidence was observed. Ten diseased plants were collected for pathogen isolation. Leaf tissues near the lesions were cut into 5 × 5 mm pieces, surface-disinfested in 75% ethanol for 3 min, and rinsed with sterile distilled water. The pieces were placed on water agar in petri plates and incubated at 25℃ for three days. The resulting colonies were flushed with sterile water and a spore suspension was serially diluted and plated on potato dextrose agar (PDA). A single-spore colony was obtained. Ten isolates were obtained and designated HE1 to HE10. The colony morphology was identical for all isolates, grayish white in color on the upper surface and light black on the underside. The mycelia were light gray and velvety. Conidia were light brown to brown in color and oblate, oblong or oval. The conidial dimensions were typically between 15 to 43 µm by 8 to 9 µm in size. The conidia possessed one to six transverse septa, with slight to distinct constrictions at each division, and zero to two longitudinal septa. These morphological characteristics resembled Alternaria alternata (Fr.) Keissl.. DNA was extracted from three isolates, HE3, HE4 and HE5, using the CTAB method. Polymerase chain reaction (PCR) was performed on the extracted DNA with a set of primers ITS1/ITS4, H31a/H31b, gpd1/gpd2, TEF1-728F/TEF1-986R, and RPB2-5F2/fRPB2-7cR. The amplicon sequences from the three isolates were analyzed using the BLAST in GenBank (https://www.ncbi.nlm.nih.gov/). The results showed a high sequence identity, ranging from 99 to 100%, with the A. alternata strain YTMZ-20-2 across all the genetic markers tested. The strong match reinforced the identification of the strains as A. alternata. The sequences were deposited in GenBank (Table S1). The three fungal isolates were identified as A. alternata based on their morphological and genetic data. To conduct Koch's postulates, the representative isolate HE4 was used. Smooth bromegrass seed was soaked in water for four days and sown in potting soil contained in plastic pots (10 cm diameter × 15 cm height, five seeds/pot) in a greenhouse under a 16-h photoperiod at temperatures between 20 to 25°C and 60% relative humidity. When the plants reached a height of approximately 20 cm, the plants in three pots (replicates) were sprayed with a spore suspension (106 conidial/ml) at 10 ml/pot, and three pots were sprayed with sterile water for control. Five days after inoculation, the plants exhibited leaf spot symptoms similar to those previously described, while the control plants remained unaffected. The causative fungus was successfully re-isolated from the diseased plants and confirmed morphologically and molecularly on its identity as described above. This experiment was independently conducted three times. This is the first report of A. alternata causing leaf spot on smooth bromegrass in China. Since there is risk that the disease could seriously reduce the yield of the forage crop smooth bromegrass, further research is needed.

Brandt's vole hole detection and counting method based on deep learning and unmanned aircraft system.

Rodents are essential to the balance of the grassland ecosystem, but their population outbreak can cause major economic and ecological damage. Rodent monitoring is crucial for its scientific management, but traditional methods heavily depend on manual labor and are difficult to be carried out on a large scale. In this study, we used UAS to collect high-resolution RGB images of steppes in Inner Mongolia, China in the spring, and used various object detection algorithms to identify the holes of Brandt's vole (Lasiopodomys brandtii). Optimizing the model by adjusting evaluation metrics, specifically, replacing classification strategy metrics such as precision, recall, and F1 score with regression strategy-related metrics FPPI, MR, and MAPE to determine the optimal threshold parameters for IOU and confidence. Then, we mapped the distribution of vole holes in the study area using position data derived from the optimized model. Results showed that the best resolution of UAS acquisition was 0.4 cm pixel-1, and the improved labeling method improved the detection accuracy of the model. The FCOS model had the highest comprehensive evaluation, and an R2 of 0.9106, RMSE of 5.5909, and MAPE of 8.27%. The final accuracy of vole hole counting in the stitched orthophoto was 90.20%. Our work has demonstrated that UAS was able to accurately estimate the population of grassland rodents at an appropriate resolution. Given that the population distribution we focus on is important for a wide variety of species, our work illustrates a general remote sensing approach for mapping and monitoring rodent damage across broad landscapes for studies of grassland ecological balance, vegetation conservation, and land management.

Identification of Crucial Genes and Regulatory Pathways in Alfalfa against Fusarium Root Rot.

Fusarium root rot, caused by Fusarium spp. in alfalfa (Medicago sativa L.), adversely impacts alfalfa by diminishing plant quality and yield, resulting in substantial losses within the industry. The most effective strategy for controlling alfalfa Fusarium root rot is planting disease-resistant varieties. Therefore, gaining a comprehensive understanding of the mechanisms underlying alfalfa's resistance to Fusarium root rot is imperative. In this study, we observed the infection process on alfalfa seedling roots infected by Fusarium acuminatum strain HM29-05, which is labeled with green fluorescent protein (GFP). Two alfalfa varieties, namely, the resistant 'Kangsai' and the susceptible 'Zhongmu No. 1', were examined to assess various physiological and biochemical activities at 0, 2, and 3 days post inoculation (dpi). Transcriptome sequencing of the inoculated resistant and susceptible alfalfa varieties were conducted, and the potential functions and signaling pathways of differentially expressed genes (DEGs) were analyzed through gene ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Meanwhile, a DEG co-expression network was constructed though the weighted gene correlation network analysis (WGCNA) algorithm. Our results revealed significant alterations in soluble sugar, soluble protein, and malondialdehyde (MDA) contents in both the 'Kangsai' and 'Zhongmu No. 1' varieties following the inoculation of F. acuminatum. WGCNA analysis showed the involvement of various enzyme and transcription factor families related to plant growth and disease resistance, including cytochrome P450, MYB, ERF, NAC, and bZIP. These findings not only provided valuable data for further verification of gene functions but also served as a reference for the deeper explorations between plants and pathogens.

Functional Characterization of Pheromone Receptors in the Beet Webworm, Loxostege sticticalis (Lepidoptera: Pyralidae).

Lepidopteran insects mainly rely on sex pheromones to complete sexual communications. Pheromone receptors (PRs) are expressed on the olfactory receptor neurons (ORNs) of the sensilla trichodea and play an essential role in sexual communication. Despite extensive investigations into the mechanisms of peripheral recognition of sex pheromones in Lepidoptera, knowledge about these mechanisms in L. sticticalis remains limited. In this study, five candidate LstiPRs were analyzed in a phylogenetic tree with those of other Lepidopteran insects. Electroantennography (EAG) assays showed that the major sex pheromone component E11-14:OAc elicited a stronger antennal response than other compounds in male moths. Moreover, two types of neurons in sensilla trichodea were classified by single sensillum recordings, of which the "a" neuron specifically responded to E11-14:OAc. Five candidate PRs were functionally assayed by the heterologous expression system of Xenopus oocytes, and LstiPR2 responded to the major sex pheromone E11-14:OAc. Our findings suggest that LstiPR2 is a PR sensitive to L. sticticalis's major sex pheromone compound, E11-14:OAc. Furthermore, this study offers valuable insights into the sexual communication behavior of L. sticticalis, forming a foundation for further analysis of the species' central nervous system.

First Report of Leaf Spot Disease Caused by Epicoccum nigrum on Smooth Bromegrass (Bromus inermis) in China.

Smooth bromegrass (Bromus inermis Leyss.) is an excellent forage species widely distributed in Gansu, Qinghai, Inner Mongolia, and other provinces of China (Gong et al. 2019). In July 2021, typical leaf spot symptoms were observed on the leaves of smooth bromegrass plants in Ewenki Banner of Hulun Buir, China (49°5'8″N, 119°44'28″E, alt. 622.5 m). Approximately 90% of plants were affected, with symptoms apparent throughout the plant but mainly concentrated on the lower middle leaves. We collected 11 plants to identify the causal pathogen of leaf spot on smooth bromegrass. Samples (5×5 mm) of symptomatic leaves were excised and surface-sanitized with 75% ethanol for 3 min, rinsed three times with sterile distilled water, and incubated on water agar (WA) at 25℃ for three days. The lumps were cut along the edges and transplanted to potato dextrose agar (PDA) for subculture. After two purification cultures, ten strains, termed HE2 to HE11, were collected. The front side of the colony morphology was cottony or woolly, the center was greyish-green, circled with greyish-white color, with reddish pigmentation on the reverse. The conidia were globose or subglobose, yellow-brown or dark brown, with surface verrucae, and 23.89±3.76×20.28±3.23 µm (n = 50) in size. The morphological characteristics of the mycelia and conidia of the strains mtched those of Epicoccum nigrum (El-Sayed et al. 2020). The primers ITS1/ITS4 (White et al. 1991), LROR/LR7 (Rehner and Samuels 1994), 5F2/7cR (Sung et al. 2007), and TUB2Fd/TUB4Rd (Woudenberg et al. 2009) were used to amplify and sequence four phylogenic loci (ITS, LSU, RPB2 and ß-tubulin), respectively. The sequences of ten strains have been deposited in GenBank, and the detailed accession numbers were shown in Table S1. BLAST analysis of these sequences showed 99-100%, 96-98%, 97-99% and 99-100% homology with the E. nigrum strain in the ITS, LSU, RPB2 and TUB sequenced regions, respectively. The sequences of ten test strains and other Epicoccum spp. strains obtained from GenBank were aligned by ClustalW by MEGA (version 11.0) software. After a series of alignment, cutting and splicing, the phylogenetic tree was constructed by the neighbor-joining method with 1000 bootstrap replicates based on the ITS, LSU, RPB2, and TUB sequences. The test strains were clustered together with E. nigrum, with branch support rate of 100%. Combined with morphological and molecular biological characteristics, ten strains were identified as E. nigrum. For the pathogenicity test, the seeds of smooth bromegrass were soaked for four days and then sown into six pots (10 cm diameter × 15 cm height) and kept in a greenhouse under a 16-h photoperiod with temperatures of 20-25°C and 60% relative humidity. Microconidia of the strain produced on wheat bran medium after 10 days were washed with sterile deionized water, filtered through three layers of sterile cheese cloth, quantified, and the concentration adjusted to 1 × 106 microconidia/ml with a hemocytometer. When the plants had grown to a height of about 20 cm, the leaves of plants in three pots were sprayed with the spore suspension, 10 mL per pot, while the remaining three pots were inoculated with sterile water and served as controls (LeBoldus and Jared 2010). The inoculated plants were cultured in an artificial climate box under a 16-h photoperiod with temperatures of 24°C and 60% relative humidity. Brown spots were apparent on the leaves of the treated plants after five days, whereas the leaves of the controls remained healthy. The same E. nigum strain were re-isolated from the inoculated plants and identified by the morphological and molecular techniques described above. To our knowledge, this is the first report of leaf spot disease caused by E. nigrum on smooth bromegrass in China, as well as in the world. Infection with this pathogen could reduce the yield and quality of smooth bromegrass production. For this reason, strategies for the management and control of this disease should be developed and implemented.

Non-genetic factors affecting the meat quality and flavor of Inner Mongolian lambs: A review.

The Inner Mongolia Autonomous Region ranks first among the five major pastoral areas in terms of lamb breeding of China. The Inner Mongolia Autonomous Region has a vast territory, with many famous grasslands and thousands of forage plants and multiple local high-quality lamb breeds. After hundreds of years of artificial breeding and improvement, Mongolian sheep have developed many varieties. Different diets, feeding and treatment methods have effects on the production performance, lipid deposition and flavor composition of mutton sheep. Therefore, understanding the relationship among Inner Mongolian lamb, meat quality, and flavor will improve the production of high-quality mutton. The regulation of meat quality and flavor will have a profound impact on the deep processing and income-generating capabilities of mutton. Non-genetic factors affect the quality and flavor of mutton, which are more intuitive than genetic factors. In this review, we cover the contributions made by scientists to explore and improve the quality and flavor of Inner Mongolia lambs through non-genetic means, compare the differences between grazing and drylot-feeding in detail, and summarize some feed additives. We hope that based on our review, we can provide some inspiration to improve the meat quality of Mongolian sheep.

Resistance to Beta-cypermethrin, Azadirachtin, and Matrine, and Biochemical Characterization of Field Populations of Oedaleus asiaticus (Bey-Bienko) in Inner Mongolia, Northern China.

Oedaleus asiaticus (Bey-Bienko) is an economically devastating locust species found in grassland and pastoral areas of the Inner Mongolia region of northern China. In this study, resistance to three frequently used insecticides (beta-cypermethrin, matrine, and azadirachtin) was investigated in six field populations of O. asiaticus using the leaf-dip bioassay method. The inhibitory effects of synergists and the activities of detoxification enzyme activities in the different populations were determined to explore potential biochemical resistance mechanisms. The results showed that the field populations SB (resistance ratio [RR] = 7.85), ZB (RR = 5.64), and DB (RR = 6.75) had developed low levels of resistance to beta-cypermethrin compared with a susceptible control strain. Both the SB (RR = 5.92) and XC (RR = 6.38) populations had also developed low levels of resistance against matrine, with the other populations remaining susceptible to both beta-cypermethrin and matrine. All field populations were susceptible to azadirachtin. Synergism analysis showed that triphenyl phosphate (TPP) and diethyl-maleate (DEM) increased the toxicity of beta-cypermethrin significantly in the SB population, while the synergistic effects of TPP, piperonyl butoxide (PBO), and DEM on the toxicity of matrine were higher in SB (SR 3.86, 4.18, and 3.07, respectively) than in SS (SR 2.24, 2.86, and 2.29, respectively), but no synergistic effects of TPP, PBO, and DEM on azadirachtin were found. Biochemical assays showed that the activities of carboxylesterases (CarEs) and glutathione-S-transferases (GSTs) were significantly raised in all field populations of O. asiaticus, with a significant positive correlation observed between beta-cypermethrin resistance and CarE activity. The activities of cytochrome P450 monooxygenases (P450) and multi-function oxidases (MFO) were elevated in all six field populations, and P450 activity displayed strong positive correlations with the three insecticides. Our findings suggest that resistance to beta-cypermethrin in O. asiaticus may be mainly attributed to elevated CarE and GST activities, while P450 plays an important role in metabolizing matrine and azadirachtin. Our study provides insights that will help improve insecticide resistance management strategies.

Transcriptome Analysis of Beet Webworm Shows That Histone Deacetylase May Affect Diapause by Regulating Juvenile Hormone.

The beet webworm (Loxostege sticticalis L.) is an important agricultural pest and can tolerate harsh environmental conditions by entering diapause. The diapause mechanism of beet webworm is unknown. Therefore, we conducted a transcriptomic study of the process from diapause induction to diapause release in beet webworms. The results revealed 393 gene modules closely related to the diapause of beet webworm. The hub gene of the red module was the HDACI gene, which acts through histone deacetylase (HDAC) enzymes. HDAC enzyme activity was regulated by the light duration and influenced the JH content under induced beet webworm diapause conditions (12 h light:12 h dark). In addition, transcriptomic data suggested that circadian genes may not be the key genes responsible for beet webworm diapause. However, we showed that the photoperiod affects HDAC enzyme activity, and HDAC can regulate the involvement of JH in beet webworm diapause. This study provided a new module for studying insect diapause and links histone acetylation and diapause at the transcriptome level.

Identification and Functional Characterization of Sex Pheromone Receptors in the Oriental Fruit Moth, Grapholita molesta (Lepidoptera: Tortricidae).

The oriental fruit moth, Grapholita molesta, is a worldwide pest that damages Rosaceae fruit trees. Sex pheromones play an important role in controlling this pest; however, the corresponding chemosensation mechanism is currently unknown. In this study, 60 candidate odorant receptors, including eight pheromone receptors (PRs), were identified by antennal transcriptome analysis. Expression profiles indicated that most PRs were highly expressed in the males, except GmolOR21 and GmolOR22, which were specifically expressed in the females. Among them, GmolOR2 was identified in response to the main sex pheromone Z8-12:OAc and E8-12:OAc, and its in vivo function was confirmed by RNA interference analysis. Electrophysiological analysis showed that the males had a significantly reduced sensitivity to the main pheromones after the knockdown of GmolOR2. Our research makes a better understanding of pheromone chemoreception and provides a theoretical basis to developing novel, efficient, and environmentally friendly insect attractants.

Fusarium incarnatum-equiseti Species Complex Causing Root Rot Disease on Leymus chinensis in China.

Leymus chinensis (Trin.) Tzvel. is a rhizomatous grass widely grown in the grasslands of Eurasia. With strong fertility and stress resistance, L. chinensis makes an excellent pasture and mowing grass, contributing to animal husbandry and thus playing an important role in the local economy of the northern grassland area in China (Baoyin et al. 2014). During August to September 2019, diseased roots of L. chinensis were collected from an artificially planted grassland (40°47'44" N, 111°43'58″ E, alt. 1049 m) in Shaerqin County, Hohhot, China. Infected plants were scattered across the field with disease incidence up to 2%. Symptoms observed were wilted plants and rotten roots. In order to identify the causal pathogen of root rot on L. chinensis, symptomatic pieces (5 × 5 mm) of grass roots were excised and surface sterilized with 75% ethanol for 3-5 s followed by 1% NaClO for 2-3 min, rinsed three times with sterile distilled water, and placed on water agar and incubated at 25°C for 3 days. The mycelia were cut and transferred onto potato dextrose agar (PDA) for subculture. A fungus was consistently isolated, and a strain, named LCH054, was obtained by hyphal tip culture. Culture developed as white and fluffy aerial mycelia, with diffused pink pigment on the reverse side of PDA after culturing at 25℃ for 7 days. A culture of LCH054 was transferred to carnation leaf agar (CLA) (Li et al. 2014) and incubated at 25°C for 10 days. Microconidia were absent but macroconidia were produced. Macroconidia were hyaline, sickle-shaped, and had 4 to 7 septa, 19.8 to 63.6 (mean 43.8) × 1.8 to 5.7 (mean 3.2) µm (n = 100). Chlamydospores were ellipsoidal or subglobose, with thick walls in clumps or chains. All morphological characteristics of LCH054 resembled Fusarium equiseti (Leslie and Summerell 2006). The primers of the internal transcribed spacer (ITS) region (White et al. 1990) and translation elongation factor 1α gene (TEF-1α) (O'Donnell et al. 1998) were used to amplify the isolate, and the fragments were sequenced. BLASTn search in the NCBI database using the ITS and TEF-1α sequences revealed 99 to 100% similarities with F. equiseti. BLAST analysis of the ITS and TEF-1α sequencies in the FUSARIUM-ID database showed them to have 99.21% (500 bp out of 504 bp) and 99.52% (622 bp out of 625 bp) similarities with the Fusarium incarnatum-equiseti species complex (FIESC) (strain NRRL 45997) (O'Donnell et al. 2009), respectively. The ITS and TEF1-α sequences were deposited in GenBank as accession numbers MT937067 and MT947530, respectively. The strain LCH054 was identified as a member of the FIESC based on morphological and molecular characteristics. For the pathogenicity test, one hundred of L. chinensis seeds were planted into five pots (12 cm [diameter]) × 15 cm [high]) and kept in a greenhouse under a 16-h photoperiod with temperatures of 20-25°C and 40% relative humidity. The conidial suspension of LCH054 was prepared by washing 7-day old fungal culture grown on CLA medium using sterile deionized water. Conidia were filtered through three layers of sterile cheese cloth, counted, and adjusted to 1 × 105 conidia/ml with a hemocytometer. Forty 1-month-old healthy plants (four pots) were inoculated with 400 ml of conidia suspension using the root drenching method, whereas the inoculum was replaced with 100 ml sterile water on control plants (one pot). Fourteen days after inoculation, all inoculated plants showed the typical symptoms of root rot identical to those observed in the field, whereas the control plants remained healthy. LCH054 was re-isolated from the inoculated plants and identified by the morphological and molecular approaches as described above. To the best of our knowledge, this is the first report of root rot caused by F. incarnatum-equiseti on L. chinensis in China as well as worldwide. The presence of the pathogen could cause significant economic losses in L. chinensis production. For this reason, strategies for the management and control of this disease should be developed and implemented.

Functional characterization of pheromone receptor candidates in codling moth Cydia pomonella (Lepidoptera: Tortricidae).

Sex pheromones serve a critical role in Lepidopterans finding mates. Male moths perceive and react to sex pheromones emitted by conspecific females through a delicate pheromone communication system. Pheromone receptors (PRs) are the key sensory elements at the beginning of that process. The codling moth (Cydia pomnonella) is an important pome fruit pest globally and a serious invasive species in China. Pheromone-based techniques have been used successfully in monitoring and controlling this species. We conducted ribonucleic acid sequencing analysis of the codling moth antennal transcriptome and identified 66 odorant receptors (ORs) in a population from Xinjiang province, China, of which 14 were PRs, including two novel PRs (CpomOR2e and CpomOR73). Four PRs that contain full-length open reading frames (CpomOR1, OR2a, OR5, OR7) and four PRs with ligands that have not been reported previously (CpomOR1, OR2a, OR5, OR7) were selected to deorphanize in the heterologous Xenopus oocyte expression system. Specifically, we found that CpomOR2a and CpomOR5 responded to (E,E)-8, 10-dodecadien-1-yl acetate (codlemone acetate). Furthermore, CpomOR5 (EC50 = 1.379 × 10-8 mol/L) was much more sensitive to codlemone acetate than CpomOR2a (EC50 = 1.663 × 10-6 mol/L). Since codlemone acetate is an important component of C. pomonella sex pheromone, our results improve the current understanding of pheromone communication in codling moths and will be helpful for the development of pest management strategies.

A chromosome-level genome assembly of Cydia pomonella provides insights into chemical ecology and insecticide resistance.

The codling moth Cydia pomonella, a major invasive pest of pome fruit, has spread around the globe in the last half century. We generated a chromosome-level scaffold assembly including the Z chromosome and a portion of the W chromosome. This assembly reveals the duplication of an olfactory receptor gene (OR3), which we demonstrate enhances the ability of C. pomonella to exploit kairomones and pheromones in locating both host plants and mates. Genome-wide association studies contrasting insecticide-resistant and susceptible strains identify hundreds of single nucleotide polymorphisms (SNPs) potentially associated with insecticide resistance, including three SNPs found in the promoter of CYP6B2. RNAi knockdown of CYP6B2 increases C. pomonella sensitivity to two insecticides, deltamethrin and azinphos methyl. The high-quality genome assembly of C. pomonella informs the genetic basis of its invasiveness, suggesting the codling moth has distinctive capabilities and adaptive potential that may explain its worldwide expansion.

The vitellogenin receptor has an essential role in vertical transmission of rice stripe virus during oogenesis in the small brown plant hopper.

BACKGROUND: The small brown plant hopper (SBPH), Laodelphax striatellus Fallén, is one of the most destructive pests on rice. This pest transmits rice stripe virus (RSV) both horizontally and vertically, leading to major yield and economic losses in rice production. However, the way that RSV particles enter oocytes of SBPH remains largely unknown. Thus, identification of key factors involved in the interaction between SBPH and RSV in the ovary is crucial. RESULTS: Transcriptome of non-viruliferous (NV) or high viruliferous (HV) SBPH ovaries at 24 and 48 h of emergence was sequenced. Differentially expressed genes analysis showed that vitellogenin receptor was significantly highly expressed in the ovary of the HV SBPH strains compared to NV strains. Quantitative real-time polymer chain reaction showed that the vitellogenin receptor in L. striatellus (LsVgR) was highly expressed in the ovaries of female adults and maintained a high level of expression at the early stage of ovary development. By using RNA interference, the expression of LsVgR in the ovaries of the HV strain was significantly decreased by 98.1%. RSV titer was reduced by 60.9% as quantified by viral RNA3 intergenic region and the transcripts of nucleocapsid protein gene (CP) reduced by 46.3%. The numbers of offspring hatched were significantly reduced in dsRNA-treated groups. The transcripts of CP were not affected by silencing LsVgR, whereas the abundance of RNA-dependent RNA polymerase increased by 15-fold in the member of surviving progenies. CONCLUSION: Our results suggest that vitellogenin receptor participates in regulating RSV replication during oogenesis. © 2018 Society of Chemical Industry.

Cry64Ba and Cry64Ca, Two ETX/MTX2-Type Bacillus thuringiensis Insecticidal Proteins Active against Hemipteran Pests.

Genetically modified crops that express insecticidal Bacillus thuringiensis (Bt) proteins have become a primary approach for control of lepidopteran (moth) and coleopteran (beetle) pests that feed by chewing the plants. However, the sap-sucking insects (Hemiptera) are not particularly susceptible to Bt toxins. In this study, we describe two Cry toxins (Cry64Ba and Cry64Ca) from Bt strain 1012 that showed toxicity against two important hemipteran rice pests, Laodelphax striatellus and Sogatella furcifera Both of these proteins contain an ETX/MTX2 domain and share common sequence features with the ß-pore-forming toxins. Coexpression of cry64Ba and cry64Ca genes in the acrystalliferous Bt strain HD73- resulted in high insecticidal activity against both hemipteran pests. No toxicity was observed on other pests such as Ostrinia furnacalis, Plutella xylostella, or Colaphellus bowringi Also, no hemolytic activity or toxicity against cancer cells was detected. Binding assays showed specific binding of the Cry64Ba/Cry64Ca toxin complex to brush border membrane vesicles isolated from L. striatellus Cry64Ba and Cry64Ca are Bt Cry toxins highly effective against hemipteran pests and could provide a novel strategy for the environmentally friendly biological control of rice planthoppers in transgenic plants.IMPORTANCE In Asia, rice is an important staple food, whose production is threatened by rice planthoppers. To date, no effective Bacillus thuringiensis (Bt) protein has been shown to have activity against rice planthoppers. We cloned two Bt toxin genes from Bt strain 1012 that showed toxicity against small brown planthoppers (Laodelphax striatellus) and white-backed planthoppers (Sogatella furcifera). To our knowledge, the proteins encoded by the cry64Ba and cry64Ca genes are the most efficient insecticidal Bt Cry proteins with activity against hemipteran insects reported so far. Cry64Ba and Cry64Ca showed no toxicity against some lepidopteran or coleopteran pests. These two proteins should be able to be used for integrated hemipteran pest management.

Genome Sizes of Nine Insect Species Determined by Flow Cytometry and k-mer Analysis.

The flow cytometry method was used to estimate the genome sizes of nine agriculturally important insects, including two coleopterans, five Hemipterans, and two hymenopterans. Among which, the coleopteran Lissorhoptrus oryzophilus (Kuschel) had the largest genome of 981 Mb. The average genome size was 504 Mb, suggesting that insects have a moderate-size genome. Compared with the insects in other orders, hymenopterans had small genomes, which were averagely about ~200 Mb. We found that the genome sizes of four insect species were different between male and female, showing the organismal complexity of insects. The largest difference occurred in the coconut leaf beetle Brontispa longissima (Gestro). The male coconut leaf beetle had a 111 Mb larger genome than females, which might be due to the chromosome number difference between the sexes. The results indicated that insect invasiveness was not related to genome size. We also determined the genome sizes of the small brown planthopper Laodelphax striatellus (Fallén) and the parasitic wasp Macrocentrus cingulum (Brischke) using k-mer analysis with Illunima Solexa sequencing data. There were slight differences in the results from the two methods. k-mer analysis indicated that the genome size of L. striatellus was 500-700 Mb and that of M. cingulum was ~150 Mb. In all, the genome sizes information presented here should be helpful for designing the genome sequencing strategy when necessary.

Aspergillus leaf spot of field bindweed (Convolvulus arvensis L.) caused by Aspergillus niger in China.

Leaf spot was found on field bindweed (Convolvulus arvensis L.) in Shihezi City, Xinjiang Province, China, during the summer of 2015. Pathogens were isolated from the infected leaves of field bindweed and identified as Aspergillus niger based on morphological and molecular analyses (internal transcribed spacer rDNA and ß-tubulin gene). A pathogenicity test confirmed that Aspergillus niger caused the healthy leaves of field bindweed to become diseased. To our knowledge, this is the first report of field bindweed infected naturally by A. niger.

Diapause induction and termination in the small brown planthopper, Laodelphax striatellus (Hemiptera: Delphacidae).

The small brown planthopper, Laodelphax striatellus (Fallén) enters the photoperiodic induction of diapause as 3rd or 4th instar nymphs. The photoperiodic response curves in this planthopper showed a typical long-day response type with a critical daylength of approximately 11 h at 25 °C, 12 h at 22 and 20 °C and 12.5 h at 18 °C, and diapause induction was almost abrogated at 28 °C. The third stage was the most sensitive stage to photoperiod. The photoperiodic response curve at 20 °C showed a gradual decline in diapause incidence in ultra-long nights, and continuous darkness resulted in 100% development. The required number of days for a 50% response was distinctly different between the short- and long-night cycles, showing that the effect of one short night was equivalent to the effect of three long nights at 18 °C. The rearing day length of 12 h evoked a weaker intensity of diapause than did 10 and 11 h. The duration of diapause was significantly longer under the short daylength of 11 h than it was under the long daylength of 15 h. The optimal temperature for diapause termination was 26 and 28 °C. Chilling at 5 °C for different times did not shorten the duration of diapause but significantly lengthened it when chilling period was included. In autumn, 50% of the nymphs that hatched from late September to mid-October entered diapause in response to temperatures below 20 °C. The critical daylength in the field was between 12 h 10 min and 12 h 32 min (including twilight), which was nearly identical to the critical daylength of 12.5 h at 18 °C. In spring, overwintering nymphs began to emerge in early March-late March when the mean daily temperature rose to 10 °C or higher.

Use of a dietary exposure system for screening of insecticidal compounds for their toxicity to the planthopper Laodelphax striatellus.

We developed a dietary exposure assay for screening insecticidal compounds for their toxicity and for assessing the side effects of insecticidal proteins produced by genetically engineered (GE) plants on the planthopper Laodelphax striatellus Fallén. The fitness bioassay confirmed that the diet fulfills the requirements to be used in the dietary exposure system. To validate the efficacy of the dietary exposure system, nymphs of L. striatellus were fed diets treated with different concentrations of an inorganic stomach poison, potassium arsenate (PA), or a cysteine protease inhibitor, E-64. The results showed that with increasing concentrations of E-64, the larval development time was prolonged, the adult weight was reduced and the survival rate of L. striatellus was decreased. Similarly the survival rates of L. striatellus consistently decreased with increasing PA content in the diet. The data indicate that the dietary exposure assay is able to detect the effects of insecticidal compounds on L. striatellus. Subsequently, this assay was successfully used for assessing the potential toxicity of Cry2Aa. The results showed that L. striatellus larvae were not negatively affected when fed the artificial diet containing purified Cry2Aa at 300 µg/g diet. In the assay, the stability and bioactivity of crystal (Cry) proteins in the food sources were confirmed by enzyme-linked immunosorbent assay and sensitive-insect bioassays. These results show that L. striatellus is not sensitive to Cry2Aa. We conclude that the dietary exposure system is valid and useful for assessing the toxicity of insecticidal compounds produced by GE plants on planthoppers.