Key role of the ryanodine receptor I4790K mutation in mediating diamide resistance in Plutella xylostella.

The diamondback moth Plutella xylostella, a global insect pest of cruciferous vegetables, has evolved resistance to many classes of insecticides including diamides. Three point mutations (I4790M, I4790K, and G4946E) in the ryanodine receptor of P. xylostella (PxRyR) have been identified to associate with varying levels of resistance. In this study, we generated a knockin strain (I4790K-KI) of P. xylostella, using CRISPR/Cas9 to introduce the I4790K mutation into PxRyR of the susceptible IPP-S strain. Compared to IPP-S, the edited I4790K-KI strain exhibited high levels of resistance to both anthranilic diamides (chlorantraniliprole 1857-fold, cyantraniliprole 1433-fold) and the phthalic acid diamide flubendiamide (>2272-fold). Resistance to chlorantraniliprole in the I4790K-KI strain was inherited in an autosomal and recessive mode, and genetically linked with the I4790K knockin mutation. Computational modeling suggests the I4790K mutation reduces the binding of diamides to PxRyR by disrupting key hydrogen bonding interactions within the binding cavity. The approximate frequencies of the 4790M, 4790K, and 4946E alleles were assessed in ten geographical field populations of P. xylostella collected in China in 2021. The levels of chlorantraniliprole resistance (2.3- to 1444-fold) in these populations were significantly correlated with the frequencies (0.017-0.917) of the 4790K allele, but not with either 4790M (0-0.183) or 4946E (0.017-0.450) alleles. This demonstrates that the PxRyR I4790K mutation is currently the major contributing factor to chlorantraniliprole resistance in P. xylostella field populations within China. Our findings provide in vivo functional evidence for the causality of the I4790K mutation in PxRyR with high levels of diamide resistance in P. xylostella, and suggest that tracking the frequency of the I4790K allele is crucial for optimizing the monitoring and management of diamide resistance in this crop pest.

Four new hypogean species of the genus Triplophysa (Osteichthyes, Cypriniformes, Nemacheilidae) from Guizhou Province, Southwest China, based on molecular and morphological data.

Recently described cave species of the genus Triplophysa have been discovered in southwestern China, suggesting that the diversity of the genus is severely underestimated and that there may be many undescribed species. In this work, four new species of the genus Triplophysa are described from southwestern Guizhou Province, China, namely Triplophysacehengensis Luo, Mao, Zhao, Xiao & Zhou, sp. nov. and Triplophysarongduensis Mao, Zhao, Yu, Xiao & Zhou, sp. nov. from Rongdu Town, Ceheng County, Guizhou, Triplophysapanzhouensis Yu, Luo, Lan, Xiao & Zhou, sp. nov. from Hongguo Town, Panzhou City, Guizhou, and Triplophysaanlongensis Song, Luo, Lan, Zhao, Xiao & Zhou, sp. nov. from Xinglong Town, Anlong County, Guizhou. These four new species can be distinguished from all recognized congeners by a combination of morphological characteristics and significant genetic divergences. The discovery of these species increases the number of known cave species within the genus Triplophysa to 39, making the genus the second most diverse group of cave fishes in China after the golden-line fish genus Sinocyclocheilus. Based on the non-monophyletic relationships of the different watershed systems in the phylogenetic tree, this study also discusses the use of cave species of the genus Triplophysa to determine the possible historical connectivity of river systems.

Correlation between clinicopathological characteristics and the clinical prognosis of patients with gastroenteropancreatic neuroendocrine tumors.

Gastroenteropancreatic (GEP) neuroendocrine tumors (NETs) are one of the most common types of NETs, accounting for 65-75% of all NETs. However, epidemiological characteristics of patients with GEP-NETs in China are still lacking. The present retrospective study aimed to investigate the local epidemiology of GEP-NETs and assess the prognostic factors in China. The data of 267 patients with GEP-NETs who were admitted to the First Affiliated Hospital of Bengbu Medical College (Bengbu, China) and the Affiliated Hospital of West Anhui Health Vocational College (Lu'an, China) were retrospectively reviewed. The clinical and pathological characteristics of the patients, as well as follow-up information, were collected, and the 5-year survival rate was calculated. Kaplan-Meier curves and log-rank analysis were used to analyze the prognostic factors. The stomach (100/267; 37.5%) was the most common site of GEP-NETs and the liver (25/39; 64.1%) was the most common metastatic site. A total of 166 (62.2%) and 219 (82.0%) patients had positive results for chromogranin A (CgA) and synaptophysin (Syn), respectively. The percentage of patients with tumor grade G1, G2 and G3 was 33.3, 21.0 and 45.7%, respectively. The 5-year overall survival rate was 79.7%, and the age, tumor site, distant metastasis and tumor grading upon diagnosis were all prognostic factors. In conclusion, the present case series investigated the epidemiology and prognostic factors of GEP-NETs in China. CgA and Syn could be used as diagnostic markers for NETs and the stomach was the most common primary tumor site. Lymph node metastasis, tumor site, distant metastasis and tumor grading were important prognostic factors.

Susceptibility and diagnostic concentration for Bacillus thuringiensis toxins and newer chemical insecticides in Spodoptera frugiperda (Lepidoptera: Noctuidae) from China.

The fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), is a cosmopolitan pest that exploits more than 350 host plants, including economically important crops such as corn, cotton and rice. Control of S. frugiperda largely relies on transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) and spraying synthetic insecticides. Here, we established the susceptibility and diagnostic concentration for 2 Bt toxins and 5 newer insecticides in invasive populations of S. frugiperda from southeastern China. Concentrations causing 50% mortality (LC50) in ten field populations sampled in 2022 ranged from 2.13 to 19.29 and 22.43 to 71.12 ng/cm2 for Cry1Fa and Vip3Aa, and 0.83 to 5.30, 2.83 to 9.94, 0.04 to 0.23, 4.59 to 8.40, and 1.49 to 6.79 mg/liter for chlorantraniliprole, chlorfenapyr, emamectin benzoate, indoxacarb, and spinosad, respectively. Relative to the susceptible strain YJ-19, the largest resistance ratio in the field populations was 5.1, 1.6, 6.2, 3.9, 4.6, 2.2, and 3.6 for Cry1Fa, Vip3Aa, chlorantraniliprole, chlorfenapyr, emamectin benzoate, indoxacarb, and spinosad, respectively, indicating that the field populations were generally susceptible to these Bt toxins and insecticides. Based on the pooled response of the field populations, the diagnostic concentration for resistance monitoring, estimated as ca. twice the LC99, was 400 and 1,500 ng/cm2 for Cry1Fa and Vip3Aa, and 2, 40, 60, 60, and 100 mg/liter for emamectin benzoate, chlorantraniliprole, chlorfenapyr, spinosad, and indoxacarb, respectively. These results provide useful information for monitoring resistance to key Bt toxins and insecticides for the control of S. frugiperda in China.

Cross-resistance and genetics of field-evolved resistance to chlorfenapyr in Plutella xylostella.

Chlorfenapyr is a broad-spectrum halogenated pyrrole insecticide with a unique mode of action. Due to the misuse and overuse of this chemical, resistance has been reported in several arthropods, including Plutella xylostella, which is one of the most destructive insect pests afflicting crucifers worldwide. A better understanding of the cross-resistance and genetics of field-evolved chlorfenapyr resistance could effectively guide resistance management practices. Here, the chlorfenapyr resistance of a field-derived population of P. xylostella was introgressed into the susceptible IPP-S strain using a selection-assisted multigenerational backcrossing approach. The constructed near-isogenic strain, TH-BC5 F2 , shared 98.4% genetic background with the recurrent parent IPP-S strain. The TH-BC5 F2 strain showed 275-fold resistance to chlorfenapyr, but no significant cross-resistance to spinosad, abamectin, chlorpyrifos, ß-cypermethrin, indoxacarb, chlorantraniliprole, or broflanilide (no more than 4.2-fold). Genetic analysis revealed that resistance was autosomal, incompletely dominant, and conferred by 1 major gene or a few tightly linked loci. The synergism of metabolic inhibitors (PBO, DEM, and DEF) to chlorfenapyr was very weak (<1.7-fold), and the metabolic enzyme activities in the TH-BC5 F2 strain were not significantly elevated compared with the IPP-S strain. The results enhances our understanding of the genetic traits of chlorfenapyr resistance, and provides essential information for improving resistance management strategies.

New insights into the germline genes and CDR3 repertoire of the TCRß chain in Chiroptera.

Introduction: Bats are recognized as natural reservoirs for many viruses, and their unique immune system enables them to coexist with these viruses without frequently exhibiting disease symptoms. However, the current understanding of the bat adaptive immune system is limited due to the lack of a database or tool capable of processing T-cell receptor (TCR) sequences for bats. Methods: We performed germline gene annotation in three bat species using homologous genes and RSSs (Recombinational Signal Sequences) scanning method. Then we used the conserved C gene to construct the TCRß chain receptor library of the Intermediate Horseshoe Bat. Bats' TCRß data will be analyzed using MiXCR and constructed reference library. Results: Regarding the annotation results, we found that the Pale Spear-nosed Bat has 37 members in the TRBV12 family, which is more than the total number of TRBV genes in the Greater Horseshoe Bat. The average number of unique TCRß chain receptor sequences in each Intermediate Horseshoe Bat sample reached 24,904. Discussion: The distinct variations in the distribution of TRBV genes among the three types of bats could have a direct impact on the diversity of the TCR repertoire, as evidenced by the presence of conserved amino acids that indicate the T-cell recognition of antigens in bats is MHC-restricted. The bats' TCRß repertoire is formed through the rearrangement of the V-D-J-C genes, with D-J/V-D deletions and insertions resulting in high diversity.

Equivalent intensity but differential dominance of sodium channel blocker insecticide resistance conferred by F1845Y and V1848I mutations of the voltage-gated sodium channel in Plutella xylostella.

Two point mutations (F1845Y and V1848I) in the voltage-gated sodium channel gene of Plutella xylostella are involved in the target-site resistance to sodium channel blocker insecticides (SCBIs). The contribution of the individual mutations to the SCBI resistance and the associated inheritance modes is as yet unclear. Through 2 rounds of single-pair crossing and marker-assisted selection, 2 P. xylostella strains (1845Y and 1848I) bearing homozygous F1845Y or V1848I mutant alleles were successfully established from a field-collected population, and the contribution of each mutation to SCBI resistance, as well as associated inheritance patterns, was determined. When compared with the susceptible SZPS strain, each of the mutations individually conferred equally high-level resistance to indoxacarb (378 and 313 fold) and metaflumizone (734 and 674 fold), respectively. However, dominance levels of resistance to SCBIs were significantly different between the 2 resistant strains. Resistance of the 1845Y strain to indoxacarb and metaflumizone was inherited as an autosomal and incompletely dominant trait (D values ranged from 0.43 to 0.76). In contrast, that of the 1848I strain followed an autosomal but incompletely recessive to semidominant mode (D values: -0.24 to 0.09). Our findings enriched the current understanding of inheritance and mechanisms of SCBI resistance in P. xylostella, and will help develop resistance management programs for P. xylostella and other economic pests.

Baseline susceptibility of Helicoverpa armigera, Plutella xylostella, and Spodoptera frugiperda to the meta-diamide insecticide broflanilide.

Broflanilide is a novel meta-diamide insecticide that acts as a γ-aminobutyric acid-gated chloride channel allosteric modulator. With its unique mode of action, broflanilide has no known cross-resistance with existing insecticides and is expected to be an effective tool for the management of insecticide resistance. Establishing the baseline susceptibility to this insecticide is an essential step for developing and implementing effective resistance management strategies. Here we evaluated the baseline susceptibility to broflanilide for 3 cosmopolitan lepidopteran pest species, Helicoverpa armigera, Plutella xylostella, and Spodoptera frugiperda. Broflanilide exhibited high activity against populations sampled in the major distribution range of these pests in China, with median lethal concentrations (LC50 ) ranging between 0.209 and 0.684, 0.076 and 0.336, and 0.075 and 0.219 mg/L for H. armigera, P. xylostella, and S. frugiperda, respectively. Among-population variability in susceptibility to broflanilide was moderate for H. armigera (3.3-fold), P. xylostella (4.4-fold), and S. frugiperda (2.9-fold). The recommended diagnostic concentrations for H. armigera, P. xylostella, and S. frugiperda were 8, 4, and 2 mg/L, respectively. Little or no cross-resistance to broflanilide was detected in 3 diamide-resistant strains of P. xylostella and 1 spinosyns-resistant strain of S. frugiperda. Our results provide critical information for the development of effective resistance management programs to sustain efficacy of broflanilide against these key lepidopteran pests.

Variation of the toxicity caused by key contaminants in industrial wastewater along the treatment train of Fenton-activated sludge-advanced oxidation processes.

Industrial wastewater contains a mixture of refractory and hazardous pollutants that have comprehensive toxic effects. We investigated the treatment of a long-chain industrial wastewater treatment train containing Fenton, biological anoxic/oxic (AO), and heterogeneous ozone catalytic oxidation (HOCO) processes, and evaluated their detoxification effect based on the analysis of the genic toxicity of some key contaminants. The results showed that although the effluent met the discharge standard in terms of traditional quality parameters, the long-chain treatment process could not effectively detoxify the industrial wastewater. The analysis results of summer samples showed that the Fenton process increased the total toxicity and genotoxicity of the organics, concerned metals, and non-volatile pollutants, whereas the A/O process increased the toxicity of the organics and non-volatile pollutants, and the HOCO process led to higher toxicity caused by metals and non-volatile pollutants. The outputs of the winter samples indicated that the Fenton process reduced the total toxicity and genotoxicity caused by non-volatile pollutants but increased that of the organics and concerned metals. The effect of the A/O process on the effluent toxicity in winter was the same as that in summer, whereas the HOCO process increased the total toxicity and genotoxicity of the metals in winter samples. Correlation analysis showed that various toxicity stresses were significantly correlated with the variation of these key pollutants in wastewater. Our results could provide a reference for the optimization of industrial wastewater treatment plants (IWTPs) by selecting more suitable treatment procedures to reduce the toxicity of different contaminants.

CRISPR-mediated knockout of nicotinic acetylcholine receptor (nAChR) α6 subunit confers high levels of resistance to spinosyns in Spodoptera frugiperda.

Spinosyn insecticides (spinosad and spinetoram) have been widely used to control a number of agricultural pests including the fall armyworm, Spodoptera frugiperda. Mutations of the nicotinic acetylcholine receptor α6 subunit (nAChRα6) have been reported to confer high levels of resistance to spinosyns in several insect pests. Here we used CRISPR-mediated gene knockout to determine the involvement of S. frugiperda nAChRα6 (Sfα6) in spinosyns susceptibility. A Sfα6 knockout strain of S. frugiperda (Sfα6-KO) was established using dual single guide RNA (sgRNA) directed large fragment deletion with the CRISPR/Cas9 system. Sfα6-KO showed high levels of resistance to spinosad (307-fold) and spinetoram (517-fold) compared with the progenitor strain YJ-19, while no resistance was observed to emamectin benzoate, indoxacarb, chlorfenapyr, chlorantraniliprole and broflanilide. Genetic analyses confirmed that spinosad resistance in Sfα6-KO was autosomal, incompletely recessive and tightly linked to the edited deletion mutation of Sfα6. Our results provided in vivo functional evidence for Sfα6 as the major target of spinosyns against S. frugiperda, and demonstrated that disruption of Sfα6 causes high level resistance to spinosyns. Although no mutations of Sfα6 have yet been reported in any field populations of S. frugiperda, it is critical to develop F1 screens and/or DNA-based methods to detect and monitor the mutant allele frequencies of Sfα6 across global populations of S. frugiperda.

Phylogenetic analysis of combined mitochondrial genome and 32 nuclear genes provides key insights into molecular systematics and historical biogeography of Asian warty newts of the genus Paramesotriton (Caudata: Salamandridae).

The Paramesotriton Chang, 1935 genus of Asian warty newts is the second most diverse genus in the family Salamandridae, currently containing 14 recognized species from northern Vietnam to southwest-central and southern China. Although species of this genus have been included in previous phylogenetic studies, the origin and interspecific relationships of the genus are still not fully resolved, especially at key nodes in the phylogeny. In this study, we sequenced mitochondrial genomes and 32 nuclear genes from 27 samples belonging to 14 species to reconstruct the interspecific phylogenetic relationships within Paramesotriton and explore its historical biogeography in southern China. Both Bayesian inference and maximum-likelihood analyses highly supported the monophyly of Paramesotriton and its two recognized species groups ( P. caudopunctatus and P. chinensis groups) and further identified five hypothetical phylogenetic cryptic species. Biogeographic analyses indicated that Paramesotriton originated in southwestern China (Yunnan-Guizhou Plateau/South China) during the late Oligocene. The time of origin of Paramesotriton corresponded to the second uplift of the Himalayan/Qinghai-Xizang (Tibetan) Plateau (QTP), rapid lateral extrusion of Indochina, and formation of karst landscapes in southwestern China. Principal component analysis (PCA), independent sample t-tests, and niche differentiation using bioclimatic variables based on locations of occurrence suggested that Paramesotriton habitat conditions in the three current regions (West, South, and East) differ significantly, with different levels of climatic niche differentiation. Species distribution model (SDM) predictions indicated that the most suitable distribution areas for the P. caudopunctatus and P. chinensis species groups are western and southern/eastern areas of southern China. This study increases our knowledge of the taxonomy, biodiversity, origin, and suitable distribution areas of the genus Paramesotriton based on phylogenetic, biogeographic, and species distribution models.

The complete mitochondrial genome of the Great evening bat Ia io (Chiroptera: Vespertiilionidae) from karst area, Southwestern China.

In this study, the complete mitochondrial genome of Ia io from Guizhou Province, China. The genome was a circular mitochondrial genome of 16689 bp in length, containing 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNA), 2 ribosomal RNA genes (rRNA), and a control region. The average base composition is 32.76% A, 24.59% C, 14.49% G, and 28.16% T. The first complete mitochondrial genome of I. io provides fundamental data for future systematic taxonomic studies of the genus Ia.

Effects of Lateral Patellar Retinaculum Release for Recurrent Patella Dislocation: A Prospective Study.

PURPOSE: Recurrent patellar dislocation (RPD) is the most common complication of patellar instability and the medial patellofemoral ligament (MPFL) reconstruction has become its reference treatment. Lateral patellar retinaculum (LPR) release used to be performed in association with MPFL reconstruction. The aim of this study was to investigate the added values of MPFL reconstruction plus LPR release for RPD. METHODS: After Institutional Review Board approval, RPD patients from October 2014 to April 2019 were randomly assigned into two groups (isolated MPFL reconstruction [Group I] and MPFL reconstruction plus LPR release [Group II]) and prospectively assessed until 12 months after surgery. Knee joints with flexion of 20° were scanned by a 64-row CT scanner. Congruence angle (CA), patella tilt angle (PTA), lateral patellofemoral angle (LPFA), tibial tuberosity-trochlear groove distance and patellar tilt with the quadriceps relaxed and contracted were measured. Knee function was assessed by Lysholm knee score and International Knee Documentation Committee (IKDC) score. Patients were followed up for at least 12 months. RESULTS: A total of 87 RPD patients (45 for Group I and 42 for Group II) were selected in this study. Preoperative clinical characteristics were not significantly different across groups. No serious complications were noted in either group. It was statistically insignificant between the two group patients in terms of postoperative patella associated measurements (P > 0.05 for all). The Lysholm score and IKDC score of Group I (84.5 ± 7.1 and 87.9 ± 7.2) were significantly less than that of Group II (89.7 ± 8.7 and 93.1 ± 7.7), which indicated the better knee function of Group II. CONCLUSION: LPR release plus MPFL reconstruction provides additional benefits compared with isolated MPFL reconstruction in knee function. A combination of surgical treatments for RPD should be recommended.

Cadherin Protein Is Involved in the Action of Bacillus thuringiensis Cry1Ac Toxin in Ostrinia furnacalis.

Transgenic crops expressing Bacillus thuringiensis (Bt) insecticidal proteins have been extensively planted for insect pest control, but the evolution of Bt resistance in target pests threatens the sustainability of this approach. Mutations of cadherin in the midgut brush border membrane was associated with Cry1Ac resistance in several lepidoptera species, including the Asian corn borer, Ostrinia furnacalis, a major pest of maize in Asian-Western Pacific countries. However, the causality of O. furnacalis cadherin (OfCad) with Cry1Ac resistance remains to be clarified. In this study, in vitro and in vivo approaches were employed to examine the involvement of OfCad in mediating Cry1Ac toxicity. Sf9 cells transfected with OfCad showed significant immunofluorescent binding with Cry1Ac toxin and exhibited a concentration-dependent mortality effect when exposed to Cry1Ac. The OfCad knockout strain OfCad-KO, bearing homozygous 15.4 kb deletion of the OfCad gene generated by CRISPR/Cas9 mutagenesis, exhibited moderate-level resistance to Cry1Ac (14-fold) and low-level resistance to Cry1Aa (4.6-fold), but no significant changes in susceptibility to Cry1Ab and Cry1Fa, compared with the original NJ-S strain. The Cry1Ac resistance phenotype was inherited as autosomal, recessive mode, and significantly linked with the OfCad knockout in the OfCad-KO strain. These results demonstrate that the OfCad protein is a functional receptor for Cry1Ac, and disruption of OfCad confers a moderate Cry1Ac resistance in O. furnacalis. This study provides new insights into the mode of action of the Cry1Ac toxin and useful information for designing resistance monitoring and management strategies for O. furnacalis.

Varying contributions of three ryanodine receptor point mutations to diamide insecticide resistance in Plutella xylostella.

BACKGROUND: Although decoding the molecular mechanisms underlying insecticide resistance has often proven difficult, recent progress has revealed that specific mutations in the ryanodine receptor (RyR) of the diamondback moth, Plutella xylostella, can confer resistance to diamide insecticides. The extent to which specific RyR mutations contribute to the diamide resistance phenotype, the associated genetic traits and fitness costs remain limited. RESULTS: Three field-evolved PxRyR mutations (G4946E, I4790 M, and I4790 K) were respectively introgressed into a common susceptible background strain (IPP-S) of P. xylostella with marker-assisted backcrossing. The mutations alone can result in moderate to high levels of resistance to five commercial diamides (flubendiamide, chlorantraniliprole, cyantraniliprole, tetraniliprole, and cyclaniliprole), and the resistance intensity mediated by the three mutations was hierarchical in order of I4790 K (1199- to >2778-fold) > G4946E (39- to 739-fold) > I4790 M (16- to 57-fold). Flubendiamide resistance was autosomal and incompletely recessive, and was significantly linked with the introgressed mutations in the three constructed strains. In addition, the resistance levels to flubendiamide of hybrid progeny from any two resistant strains fell in between the status of their parents. Furthermore, by comparing the net replacement rate, the fitness of 4946E, 4790 M and 4790 K strains were 0.77, 0.93 and 0.92 relative to the IPP-S strain, respectively. CONCLUSION: Three independent PxRyR mutations confer varying degrees of resistance to diamides in P. xylostella. Among the three mutations, I4790 K confers highest levels of resistance (> 1000-fold) to all five commercial diamides. The findings can guide resistance management practices for diamides in P. xylostella and other arthropods.

Study on the characteristics of dissolution and acid production in waste activated sludge: Focusing on the pretreatment of thermal-alkali with rhamnolipid.

This paper studied the effect of thermal-alkali with rhamnolipid coupling pretreatment waste activated sludge (WAS) on the dissolution and acid production of organic matter. The results showed that when the dosage of rhamnolipid (RL) was 40 mg/g vs, the dissolution rate of soluble Chemical oxygen demand (SCOD) and soluble carbohydrate (SC) was the highest, and the concentration of soluble protein (SP), biopolymer and neutral low molecular substances was the highest. Three-dimensional fluorescence parallel factor analysis found that the addition of rhamnolipid promoted the formation of fulvic acids. When the dosage of rhamnolipid was 60 mg/g vs, the highest peak concentration of volatile fatty acids (VFAs) reached 3.5 days. The type of fermentation acid was butyric acid. The higher cracking rate and higher acid production rate showed that thermal-alkali with rhamnolipid had better acid production performance than thermal-alkali pretreatment sludge, but the amount of rhamnolipid affected the fermentation type.

Whole-genome sequencing to detect mutations associated with resistance to insecticides and Bt proteins in Spodoptera frugiperda.

The fall armyworm (FAW), Spodoptera frugiperda, is a major pest native to the Americas that has recently invaded the Old World. Point mutations in the target-site proteins acetylcholinesterase-1 (ace-1), voltage-gated sodium channel (VGSC) and ryanodine receptor (RyR) have been identified in S. frugiperda as major resistance mechanisms to organophosphate, pyrethroid and diamide insecticides respectively. Mutations in the adenosine triphosphate-binding cassette transporter C2 gene (ABCC2) have also been identified to confer resistance to Cry1F protein. In this study, we applied a whole-genome sequencing (WGS) approach to identify point mutations in the target-site genes in 150 FAW individuals collected from China, Malawi, Uganda and Brazil. This approach revealed three amino acid substitutions (A201S, G227A and F290V) of S. frugiperda ace-1, which are known to be associated with organophosphate resistance. The Brazilian population had all three ace-1 point mutations and the 227A allele (mean frequency = 0.54) was the most common. Populations from China, Malawi and Uganda harbored two of the three ace-1 point mutations (A201S and F290V) with the 290V allele (0.47-0.58) as the dominant allele. Point mutations in VGSC (T929I, L932F and L1014F) and RyR (I4790M and G4946E) were not detected in any of the 150 individuals. A novel 12-bp insertion mutation in exon 15 of the ABCC2 gene was identified in some of the Brazilian individuals but absent in the invasive populations. Our results not only demonstrate robustness of the WGS-based genomic approach for detection of resistance mutations, but also provide insights for improvement of resistance management tactics in S. frugiperda.

Complete mitochondrial genome of Pseudachorutes palmiensis (Collembola: Neanuridae).

We assembled one mitochondrial genome of Pseudachorutes palmiensis from Illumina sequencing data. The circularized mitochondrial assembly is 17,110 bp in length, including 13 protein-coding genes, 22 transfer RNA genes, and two ribosomal RNA genes, which showed the typical insect mitochondrial gene composition, but had different order with most springtails. The overall base composition is 33.5% for A, 31.7% for T, 22.2% for C, and 12.5% for G. A phylogeny of 10 collembolans showed P. palmiensi was clustered within Neanuridae.

CRISPR/Cas9 mediated ryanodine receptor I4790M knockin confers unequal resistance to diamides in Plutella xylostella.

The diamondback moth Plutella xylostella is a major destructive pest of Brassica worldwide. P. xylostella has evolved resistance to nearly all commercial insecticides used for its control, including the most recent chemical class, diamide insecticides. Several studies show that the G4946E and I4790M mutations of ryanodine receptor (RyR) are strongly associated with diamide resistance in insects. While the pivotal functional role of G4946E in conferring diamide resistance phenotype has confirmed by several studies in different species, no direct evidence has unambiguously confirmed the functional significance of the single I4790M mutation in diamide resistance. Here, we successfully constructed a knockin homozygous strain (I4790M-KI) of P. xylostella using CRISPR/Cas9 coupled with homology directed repair approach to introduce I4790M into RyR. When compared with the background susceptible IPP-S strain, the manipulated I4790M-KI strain exhibited moderate resistance to the phthalic acid diamide flubendiamide (40.5-fold) and low resistance to anthranilic diamides chlorantraniliprole (6.0-fold) and cyantraniliprole (7.7-fold), with no changes to the toxicities of indoxacarb and ß-cypermethrin. Furthermore, the acquired flubendiamide resistance was inherited in an autosomally recessive mode and significantly linked with the I4790M mutation of RyR in this I4790M-KI strain. Our findings provide in vivo functional evidence for the causality of I4790M mutation of PxRyR with moderate levels of resistance to flubendiamide in P. xylostella, and support the hypothesis that the diamide classes have different interactions with RyRs.

First Report of Purple Spot on Dandelion in China Caused by Alternaria tenuissima.

Dandelion (Taraxacum mongolicum) is a perennial herb of the family Asteraceae, with a high edible and medicinal value and widely grown at medium and low altitudes in China. In July 2019, purple spot of dandelion was found in a field near Harbin City, Heilongjiang province, China. The disease incidence regionally reached 95% in fields with yield losses between 10 and 20%, seriously reducing the economic and food value of dandelion. Multiple, irregular brown spots were first observed on the leaves of this plant- that later developed into circular or near-circular purple spots with raised centers, or purple lesions along the veins. When the leaf spots coalesced, the value of the commodity was lost. To isolate the pathogen, 5 × 5 mm pieces of leaf tissue from the margins of lesions were surface disinfected in 75% alcohol, rinsed in distilled water, and incubated on potato dextrose agar (PDA) plates at 28℃ until sporulation. Using single-spore isolation, a pure culture (YY-1) was obtained with abundant grayish white hyphae that later turned olive green. The underside of the colonies were brown. Conidia were typically obclavate, had a short beak with 1 to 6, but usually 3, transverse septa, and up to 3 longitudinal septa. The transverse septum was thicker and the wall of the conidium appeared brick-like. Conidia were pale brown, catenulate, and measured from 25 to 42 µm long by 6 to 10 µm wide. YY-1 was identified as Alternaria sp. based on morphological characteristics (Simmons 2007). Molecular identification was performed to detect the fungal species, and included the Internal Transcribed Spacer (ITS), translation elongation factor 1-alpha (EF1), actin gene (ACT), plasma membrane ATPase gene (ATP), and the calmodulin gene (CAL), which were respectively amplified with primers ITS4/ITS5 (Guo et al. 2012), EF1-728F/EF1-986R (Carbone and Kohn 1999), ACT-512F/ACT-783R (Carbone and Kohn 1999), and ATPDF1/ATPDR1 and CALDF1/CALDR1 (Lawrence 2013) (GenBank Accession Nos. MN746334, MT627208, MT627209, MT558864, MT558865). The species of Alternaria could not be confirmed by sequencing the above genes, as described previously (Zheng et al. 2015). Hence, a partial coding sequence of the histone 3 gene (GenBank Accession No. MN744235) was amplified using primers H3-1a/H3-1b (Zheng et al. 2015) and it shared 98.09% sequence identity with A. tenuissima (KP267543). The ITS sequence (MN746334) was 99.81% similar to the reference sequences of A. tenuissima (KT223327) in GenBank. A Maximum-likelihood tree was then reconstructed based on the ITS, CAL, and ATP sequences by MEGA7, which showed that YY-1 was most closely related to A. tenuissima. Therefore, YY-1 was identified as A. tenuissima based on its morphological and molecular characteristics. To perform Koch's postulates, 20 healthy leaves from greenhouse-grown dandelion were inoculated with 5-µL drops of a conidial suspension (1 x 105 conidia/ml) of isolate YY-1. Sterile water was used as a control. The inoculated plants were placed in a growth chamber at 28℃ and 80 to 90% relative humidity. After 10 days, similar symptoms were observed on plants inoculated with YY-1, while control plants did not produce symptoms. The pathogen was reisolated from the inoculated leaves and identified by morphological and molecular methods as A. tenuissima. To our knowledge, this is the first report of A. tenuissima causing purple spot on T. mongolicum in China.