Characterization and transcriptomic analysis of a native fungal pathogen against the rice pest Nilaparvata lugens.

The brown planthopper (BPH), Nilaparvata lugens, is one of the most destructive pests of rice. Given the threats posed by insecticide resistance to its control, eco-friendly strategies based on microbial pathogens emerged as a promising biocontrol alternative. In the present study, we isolated a native fungal pathogen against BPH from infected BPH cadavers and preliminarily identified as a strain of Aspergillus fumigatus based on morphological and molecular methods. Laboratory bioassay revealed that this fungal strain was highly virulent to BPH both at nymphal and adult stages, with the median lethal times (LT50) of 7.5 and 5.8 days under high conidial concentration of 1 × 109 conidia mL-1. A genome-wide view of gene expressions in BPH against fungal attack was analyzed by transcriptomic sequencing and consequently a large number of differentially expressed genes that mainly involved in host immune defense and cell detoxification were found. RNAi-mediated knockdown of an upregulated gene encoding a serine protease (NlSPN) could cause a significant decrease in BPH survival. Combination of dsRNA injection and fungal infection showed an additive effect on BPH mortality, which provided clues to develop new pest management strategies against BPH.

Animal DNA Typing and Its Research Progress in Forensic Medicine.

With the advance of molecular biology, DNA analysis technology has been widely applied in forensic science. Non-human DNA analysis can be used in some special cases and has unique forensic value to provide investigation clues and trial basis. Animal DNA typing plays a more prominent role in the detection of all kinds of non-human DNA related cases and is the main content of forensic non-human DNA analysis. This paper reviews the development history, present situation, advantages and disadvantages of animal DNA typing according to its technology, characteristic, challenges facing forensic science application scenarios, and also its future development.

Inter-Alpha-Trypsin Inhibitor Heavy Chain 4 Plays an Important Role in the Development and Reproduction of Nilaparvata lugens.

The brown planthopper, Nilaparvata lugens, is a difficult-to-control insect pest affecting rice yields in Asia. As a structural component of the inter-alpha-trypsin inhibitor (ITI), the inter-alpha-trypsin inhibitor heavy chain (ITIH) has been reported to be involved in various inflammatory or malignant disorders, ovarian development, and ovulation. To reveal the function of ITIH4 in N. lugens, the gene encoding N. lugens ITIH4 (NlITIH4) was cloned and characterized. NlITIH4 contains a signal peptide, a vault protein inter-alpha-trypsin domain, and a von Willebrand factor type A domain. qPCR analysis showed that NlITIH4 was expressed at all developmental stages and in all tissues (fat body, ovary, and gut), with the highest expression in the fat body. Double stranded NlITIH4 (dsNlITIH4) injection clearly led to an RNAi-mediated inhibition of the expression of NlITIH4 and resulted in reduced survival, delayed ovarian development, and reduced egg production and egg hatching. These results indicate that NlITIH4 plays an important role in the development and reproduction of N. lugens.

Whitefly-induced tomato volatiles mediate host habitat location of the parasitic wasp Encarsia formosa, and enhance its efficacy as a bio-control agent.

BACKGROUND: Whitefly Bemisia tabaci is a phloem-feeding insect and causes extensive agricultural damage around the world. Although the parasitic wasp Encarsia formosa is widely used to control B. tabaci on glasshouse tomatoes, low efficiency and discontinuity are frequently recorded. It has been well-documented that herbivore-induced plant volatiles (HIPVs) are important cues in the foraging behavior of the natural enemies of herbivores. However, the volatiles emitted from tomatoes infested by different developmental stages of B. tabaci (nymphs versus adults) have not been compared in terms of their effects on E. formosa attraction. RESULTS: Olfactometer assays with four tomato cultivars revealed that the E. formosa wasps showed a significant attraction to the volatiles from adult-infested plants (except for cv. Castlemart), but not to those from nymph-infested plants. In a close-range habitat, however, the wasps appeared to use visual or tactile cues derived from nymphs for host location. Volatile analyses and behavioral assays showed that wasp attraction was correlated with enhanced ß-myrcene and ß-caryophyllene emissions from adult-infested plants. Furthermore, the use of B. tabaci adult-induced plant cues under glasshouse conditions resulted in a higher parasitism rate by this parasitoid. CONCLUSION: Our findings confirm that E. formosa uses the HIPVs resulting from feeding of B. tabaci adults to locate host habitat. Release of ß-myrcene and ß-caryophyllene from dispensers may enhance the efficacy of E. formosa as a biological control agent against B. tabaci in glasshouse production systems.

Introgressive hybridization between two non-native apple snails in China: widespread hybridization and homogenization in egg morphology.

BACKGROUND: Apple snails from the genus Pomacea have spread widely in paddy fields and other wetlands of southern China since their introduction in the 1980s. Pomacea spp. are commonly identified using mitochondrial COI sequences. However, sequencing the nuclear elongation factor 1-alpha (EF1α) gene revealed genetic introgression between field populations of P. canaliculata and P. maculata, which produce surviving hybrids in laboratory crossbreeding experiments. RESULTS: In this study, we sequenced 1054 EF1α clones to design specific primers and established a fast and accurate multiplex polymerase chain reaction (PCR) method for genotyping EF1α. Combined with genotyping P. canaliculata and P. maculata based on mitochondrial COI and nuclear EF1α, we revealed the genetic introgression patterns of 30 apple snail populations in China. Purebred and hybrid individuals of P. canaliculata were widely distributed, while pure maculata-EF1α type was detected only in a few individuals identified as P. canaliculata based on COI sequences. Each egg clutch had one to three genetic patterns, indicating multiple paternity or segregation in the progeny of hybrids. The higher percentages of hybrids in both wild populations and progeny than the homozygotes indicated a potential heterosis in the apple snail populations. Additionally, egg size and clutch size of the apple snails became homogeneous among the non-native populations exhibiting introgression hybridization. CONCLUSION: Our findings emphasize the value of apple snails as a model to study the mechanisms and impacts of introgressive hybridization on fitness traits. © 2020 Society of Chemical Industry.

Comparative mitogenome analysis on species of four apple snails (Ampullariidae: Pomacea).

The genus Pomacea contains most of the economically important Ampullariid apple snails. Five Pomacea species were reported introduced out of their native ranges, including three highly invasive species, i.e. P. maculata, P. canaliculata and an unidentified species, Pomacea sp. In this study, the mitogenome of P. maculata was determined, which carried typical gene set of metazoan mitogenomes and shared the same gene rearrangement of Ampullariidae mitogenomes. The control region of the P. maculata has a 13-bp inverted repeat unit. We compared the mitogenome of P. maculata with P. canaliculata, Pomacea sp. and the aquatic pet species, P. diffusa. The three highly invasive species showed high sequence similarity of PCGs and RNAs. atp8 and nad2 showed the lowest similarity and the highest Ka/Ks ratios, indicating that both genes have potential for studying species identification and populations genetics in apple snails. All PCGs have the Ka/Ks ratios <1, indicating the existence of purifying selection in Pomacea species. We reconstructed phylogenetic trees using 14 Caenogastropda species based on sequences of PCGs and rRNAs using Bayesian inference. Pomacea maculata grouped with other Ampullariids and was most closely related to Pomacea sp. The mitogenome of P. maculata provides useful genetic resource for exploring the genetics and evolution of P. maculata and other apple snails.

[Relationship between changes of herbaceous plants and Oncomelania hupensis snail distribution under walnut forest of inhibition of snails in mountainous regions of Yunnan Province].

OBJECTIVE: To explore the relationship between the changes of herbaceous plants and Oncomelania hupensis snail distribution under the walnut forest of inhibition of snails in mountainous regions of Yunnan Province. METHODS: The experimental field was established at Sanying Village of Eryuan County, Yunnan Province, where the "Flourishing Forest and Controlling Snails Project" was implemented. The different stand ages (2, 4, 6, 8, 10 years)of walnut forest in experimental groups were selected based on the method of space replacing time, and the non-stocked land was served as a control group. The growth of forest, change of snails, number, biomass, overcast, height of the herbaceous plant and the soil moisture were investigated. RESULTS: The crown closure of 6-year-old walnut forest of inhibition of snails was 0.65. There were 11 species of herbaceous plant belonging to 11 genera, 6 families in 10-year-old forest and its crown closure was 0.77. Compared with the control group, the numbers of families, genera, and species of the 10-year-old forest were decreased by 64.71%, 69.44%, and 77.08%, and the biomass, overcast, and height of it decreased by 12.63%, 19%, and 22.18%, respectively. The soil moisture content (0-20 cm) monthly changes were increased obviously with the increase of stand age. There were no snails besides the control group and 2-year-old walnut forest. Compare with the control group, the occurrence rate of frames with living snails in the 2-year-old walnut forest was decreased by 50%, which was 1.25%. The density of living snails was decreased by 60.16%. CONCLUSIONS: The construction of walnut forest of inhibition of snails in mountainous regions of Yunnan Province are suitable for controlling the growth of herbaceous plants and altering the environment of snails. If the coalescence intercropped with crops is carried out, it is not only beneficial to the construction of good ecological environment, but also improves the utilization efficiencies of land, light, and thermal resource, and the income of peasants.

Obtaining cellulose binding and hydrolyzing activity of a family 11 hybrid xylanase by fusion with xylan binding domain.

The xylan binding domain (XBD) and linker sequences (LS) from thermostable and thermophilic Thermomonospora fusca xylanase A (TfxA) was fused to the carboxyl-terminus of a family 11 hybrid xylanase ATx. The constructed chimera (ATxX) was successfully expressed in Pichia pastoris, partially purified to homogeneity, and then characterized in detail. After 96-h 0.25% methanol induction, the xylanase and cellulose activity of ATxX from pPATxX1 transformant culture medium supernatant were 452.1 U/mg and 19.3 U/mg, respectively. SDS-PAGE analysis revealed that the molecular mass of ATxX was about 33.01 kDa. 3.7% ATxX was bound after incubation with 1% microcrystal cellulose at 25 °C for 3 h, while the ATx did not show cellulose binding-hydrolyzing ability. These results suggested that ATx obtained cellulose binding and hydrolyzing ability by fusing with XBD and LS. Enzymatic studies showed that the temperature and pH optimum of the ATxX xylanase activity were 60 °C and pH 5.0, respectively, which were the same as that of ATx. The temperature and pH optimum of the ATxX cellulase activity were 60 °C and pH 6.0, respectively. The major hydrolytic products released by ATxX from birchwood xylan were xylotriose and xylohexaose. Xylooligosaccharides from xylobiose to xylohexaose could be hydrolyzed by ATxX. Mode of action analysis showed that the chimeric ATxX was an endo-acting enzyme. The XBD and LS plays an important role in the binding and hydrolyzing of xylanase to insoluble substrates.

[Snail control effect and eco-economical benefit of forest for snail control and schistosomiasis prevention in mountainous regions].

OBJECTIVE: To evaluate the snail control effect and eco-economical benefit of the forest for snail control and schistosomiasis prevention in mountainous regions. METHODS: From 2006 to 2010, the retrospective survey, field inspection and family interview were adopted to collect the data of snail status, and the economic income and ecological environment of local region in rice paddy, seedling bed, irrigation channel and kaleyard channel in Sanying Village, Eryuan County, Yunnan Province, where the Flourishing Forest and Controlling Snails Project was implemented. RESULTS: After the implementation of the project, compared to 2006, the snail areas in rice paddy, seedling bed, irrigation channel and kaleyard channel in 2010 reduced by 70.5%, 91.0%, 49.7%, and 58.8%, respectively; the occurrence rates of frames with living snails dropped by 81.6%, 97.2%, 84.4% and 87.9%, respectively; and the average densities of living snails dropped by 79.0%, 97.8%, 86.0% and 93.6%, respectively. The economic income of farmers and ecological environment improved obviously. CONCLUSIONS: The forest for snail control and schistosomiasis prevention in mountainous areas not only has an obvious effect on snail control, but also can yield significant economic and ecological benefits.

Expression of recombinant Bacillus licheniformis xylanase A in Pichia pastoris and xylooligosaccharides released from xylans by it.

The mature peptide of Bacillus licheniformis xylanase A (BlxA) was successfully expressed in Pichia pastoris under the control of AOX1 promoter. After 96-h 0.25% methanol induction, the activity of recombinant B. licheniformis xylanase A (reBlxA) in culture supernatant was 122.9 U/mg. Enzymatic properties assays showed that the optimum temperature and pH for reBlxA were 60 degrees C and pH 6.0, respectively. When treated at 70 degrees C, pH 6.0 for 2 min, the residual activities of the reBlxA were 76%. Over 80% of reBlxA activity was retained after treatment of the enzyme by preincubation over a pH range of 5.0-9.0 for 1h at 25 degrees C. High performance liquid chromatography (HPLC) analysis revealed that xylotriose (X3) was the main hydrolysis product released from birchwood xylan and wheat bran insoluble xylan by reBlxA. The mode of action studies showed that reBlxA was an endo-acting xylanase and xylobiose (X2), xylotriose, xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) could be hydrolyzed by it. This is the first report on the expression of reBlxA in yeast and on determining and quantifying the hydrolysis products released from xylans by reBlxA.