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Coordinated surveillance, vaccination and public information efforts have brought the Chinese rabies epizootic under control, but significant numbers of fatalities are still reported annually with some cases occurring in previously rabies free regions. Tibet has remained virtually rabies free for 16 years, but since 2015 one human rabies case has been reported each year. To better understand the origins of these cases, we sequenced three human samples and an additional sample isolated from a dog in 2012. Three genomes were sequenced from brain samples: human case 1 (reported in 2015), human case 3 (2017), and the 2012 dog case. For human case 2 (2016), the rabies N gene was sequenced from a limited saliva sample. Phylogenetic analysis shows that Case 1 (CXZ1501H) and the dog case (CXZ1201D) belong to China IV lineage (equivalent to Arctic-like-2 in global rabies), suggesting an association with a wildlife spillover event. However, Case 2 (CXZ1601H) is placed within the dominant lineage China I, and was most similar with recent strains from neighboring Yunnan province, indicating the current epizootic has finally reached Tibet. Most surprisingly however, was the finding that Case 3 (CXZ1704H) is distinct from other Chinese isolates. This isolate is placed in the Indian Subcontinent clade, similar to recent Nepal strains, indicating that cross-border transmission is a new source for rabies infections. Thus, the complex mixture of the rabies epizootic in Tibet represents a major new challenge for Tibet and national rabies control.

BACKGROUND Uridine diphosphate‐N‐acetylglucosamine (UDP‐GlcNAc) diphosphorylase (UAP) catalyzes the formation of UDP‐GlcNAc, the precursor for the production of chitin in ectodermally derived epidermal cells and midgut, for GlcNAcylation of proteins and for generation of glycosyl‐phosphatidyl‐inositol anchors in all tissues in Drosophila melanogaster. RESULTS: Here, we identified a putative HvUAP gene in Henosepilachna vigintioctopunctata. Knockdown of HvUAP at the second‐, third‐ and fourth‐instar stages impaired larval development. Most resultant HvUAP hypomorphs showed arrested development at the third‐, fourth‐instar larval or prepupal stages, and became paralyzed, depending on the age when treated. Some HvUAP‐silenced larvae had weak and soft scoli. A portion of HvUAP‐depleted beetles formed misshapen pupae. No HvUAP RNA interference pupae successfully emerged as adults. Dissection and microscopic observation revealed that knockdown of HvUAP affected gut growth and food ingestion, reduced cuticle thickness, and negatively affected the formation of newly generated cuticle layers during ecdysis. Furthermore, HvUAP deficiency inhibited development of the tracheal respiratory system and thinned tracheal taenidia. CONCLUSION: The phenotypical defects in HvUAP hypomorphs suggest that HvUAP is involved in the production of chitin. Moreover, our findings will enable the development of a double‐stranded RNA‐based pesticide to control H. vigintioctopunctata. © 2021 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Henosepilachna vigintioctopunctata is one of the most serious insect pests to a large number of nightshades and cucurbits. RNA interference (RNAi) triggered by double-stranded RNA (dsRNA) offers a reduced risk approach to control the beetle. Identification of amenable target genes and determination of appropriate life stage for dsRNA treatment are two critical steps in order to improve RNAi efficiency. In the present paper, we identified three vATPase genes, namely HvvATPaseC, HvvATPaseE and HvvATPaseH. We found that the three transcripts were widely expressed in the eggs, first- to fourth-instar larvae, prepupae, pupae and adults. They were abundantly transcribed in the hindgut and Malpighian tubules, in contrast to the epidermis and fat body. Three days' ingestion of dsvATPaseC, dsvATPaseE and dsvATPaseH by the fourth-instar larvae significantly decreased corresponding transcript level by 90.1, 88.9 and 97.2%, greatly reduced larval fresh weight by 28.0, 29.9 and 28.0%, and caused 66.7, 100 and 78.7% larval lethality respectively. Comparably, 3 days' exposure of the third-instar larvae to dsvATPaseC significantly reduced HvvATPaseC mRNA level by 89.5%, decreased approximately 80% of the larval fresh weight, and killed 100% of the treated larvae. Therefore, the three vATPase genes, especially HvvATPaseE, are potential amenable target genes and young larvae are more susceptible to dsRNA. Our findings will enable the development of the dsRNA-based pesticide to control H. vigintioctopunctata. [ABSTRACT FROM AUTHOR]

Henosepilachna vigintioctopunctata is a serious insect pest which attacks a large number of nightshades and cucurbits in Asian countries, Brazil and Australia. Prolonged application of traditional pesticides has caused environmental pollution and exerted deleterious effects on human health. Finding new approaches with high target specificity and low environmental contamination has become an urgent task. RNA interference (RNAi) induced by double‐stranded RNA (dsRNA) is expected to be applicable to managing this pest. Here we evaluated the effects of Escherichia coli‐expressed dsRNAs targeting ecdysone receptor (EcR) gene via dietary delivery in laboratory and foliar spraying in a greenhouse. The target transcript was successfully knocked down when the 4th‐instar larvae had fed on potato foliage dipped with dsEcR in a laboratory bioassay. Around 85% of the HvEcR RNAi larvae remained as prepupae or became abnormal pupae, and failed to emerge into adults. Ingestion of dsEcR‐immersed foliage by the 3rd‐instar larvae effectuated a comparable RNAi response and brought about more severe defects: all the resultant larvae arrested development, remained as prepupae and finally died. For assay in the greenhouse, a dsEcR‐contained E. coli suspension was directly sprayed to the foliage of greenhouse‐growing potato plants and the 3rd‐ and 4th‐instar larvae were transferred to the leaves. High RNAi efficacy was obtained and identical RNAi phenotypes were observed in treated larvae. In addition, spraying dsEcR reduced leaf damage. Our results indicate a possibility of practical application of dsEcR as an environmentally friendly RNA pesticide to control H. vigintioctopunctata larvae. [ABSTRACT FROM AUTHOR]

Post-embryonic development of insects is highly dependent on ecdysteroid hormone 20-hydroxyecdysone. Halloween gene spookier ( spok, cyp307a2) has been documented to be involved in ecdysteroidogenesis in Drosophila melanogaster and Bombyx mori. We describe here the cloning and characterization of Halloween gene spookier ( Lsspok, Lscyp307a2) in the small brown planthopper Laodelphax striatellus, a hemipteran insect species. LsSPOK has three insect-conserved P450 motifs, that is, Helix-K, PERF motif and heme-binding domain. Temporal and spatial expression patterns of Lsspok were evaluated by quantitative polymerase chain reaction. Through the fouth-instar and the early fifth-instar stages, Lsspok showed two expression peaks in the second- and fifth-day fourth-instar nymphs, and two troughs in the first-day fourth and fifth instars. On day 5 of the fourth-instar nymphs, Lsspok clearly had a high transcript level in the thorax where prothoracic glands were located. Dietary introduction of double-stranded RNA of Lsspok in the nymph stage successfully knocked down the target gene, decreased expression level of ecdysone receptor ( LsEcR) gene, caused nymphal lethality and delayed development. Ingestion of 20-hydroxyecdysone in Lsspok-dsRNA-exposed nymphs did not increase Lsspok expression level, but almost completely rescued the LsEcR mRNA level and relieved the negative effects on survival and development. Thus, our data suggest that the ecdysteroidogenic pathway is conserved in insects and LsSPOK is responsible for specific steps in ecdysteroidogenesis in L. striatellus. [ABSTRACT FROM AUTHOR]