CRISPR/Cas9-mediated NlInR2 mutants:Analyses of residual mRNA and truncated proteins

CRISPR/Cas9 technology is a powerful genome manipulation tool in insects.However,little is known about whether mRNA and protein of a target gene are completely cleared in homozygous mutants.This study generated homozygous mutants of the insulin receptor gene 2(NlInR2)in the brown planthopper(Nilaparvata lugens)using CRISPR/Cas9 genome editing.Both frameshift mutants,E5_D17 and E6_I7,differentiated towards long wings,but there were differences in wing morphology,with E5_D17 showing wing deformities.Subsequent investigations revealed the presence of residual expression of NlInR2 mRNA in both mutants,as well as the occurrence of spliceosomes featuring exon skipping splicing in E5_D17.Additionally,the E5_D17 exhibited the detection of N-terminally truncated NlInR2 protein.RNA interference experiments indicated that the knockdown of NlInR2 expression in the E5_D17 mutant line increased the proportion of wing deformities from 11.1 to 65.6%,suggesting that the residual NlInR2 mRNA of the E5_D17 mutant might have retained some genetic functions.Our results imply that systematic characterization of residual protein expression or function in CRISPR/Cas9-generated mutant lines is necessary for phenotypic interpretation.

Pancreatic agenesis and altered m6A methylation in the pancreas of PDX1-mutant cynomolgus macaques

As an essential transcriptional activator,PDX1 plays a crucial role in pancreatic development andβ-cell function.Mutations in the PDX1 gene may lead to type 4 maturityonset diabetes of the young(MODY4)and neonatal diabetes mellitus.However,the precise mechanisms underlying MODY4 remain elusive due to the paucity of clinical samples and pronounced differences in pancreatic architecture and genomic composition between humans and existing animal models.In this study,three PDX1-mutant cynomolgus macaques were generated using CRISPR/Cas9 technology,all of which succumbed shortly postpartum,exhibiting pancreatic agenesis.Notably,one tri-allelic PDX1-mutant cynomolgus macaque(designated as M4)developed a pancreas,whereas the two monoallelic PDX1-mutant cynomolgus macaques displayed no anatomical evidence of pancreatic formation.RNA sequencing of the M4 pancreas revealed substantial molecular changes in both endocrine and exocrine functions,indicating developmental delay and PDX1haploinsufficiency.A marked change in m6A methylation was identified in the M4 pancreas,confirmed through cultured PDX1-mutantisletorganoids.Notably,overexpression of the m6A modulator METTL3 restored function in heterozygous PDX1-mutant islet organoids.This study highlights a novel role of m6A methylation modification in the progression of MODY4 and provides valuable molecular insights for preclinical research.

Three new species of Discoglypha Warren(Lepidoptera:Geometridae)from China with a key to Chinese species

Three new species of the genus Discoglypha Warren,1896(Lepidoptera:Geometridae),D.maculata Xian&Han sp.nov.,D.obliquifascia Xian&Cheng sp.nov.and D.pupula Xian&Jiang sp.nov.,from China are described.The species D.punctimargo(Hampson,1895),D.aureifloris Warren,1896 and D.parvifloris Prout,1917 stat.nov.are newly recorded from China.A key to eight known species from China is presented.Examined specimens and distribution of the Chinese species of Discoglypha are recorded.External features and genitalia are depicted.

Soil Acidification in Response to Acid Deposition in Three Subtropical Forests of Subtropical China

Long-term changes in soil pH, the current status of soil acidification, and the response of bulk soil and soil water pH to experimental nitrogen addition under three subtropical forests were investigated in Dinghushan Biosphere Reserve of subtropical China. The results showed that the mineral soil pH at 0-20 cm depth declined significantly from 4.60-4.75 in 1980s to 3.84-4.02 in 2005. Nitrogen addition resulted in the decrease of pH in both bulk soil and soil water collected at 20-cm depth. The rapid decline of soil pH was attributed to long-term high atmospheric acid deposition (nitrogen and sulphur) therein. The forest at earlier succession stage with originally higher soil pH appeared to be more vulnerable to acid deposition than that at later succession stage with originally low soil pH.

Effects of back fat,growth rate,and age at first mating on Yorkshire and Landrace sow longevity in China

In the present study, genetic parameters for longevity and genetic correlations with back fat （BF）, average daily gain （ADG）, and first mating age （FMA） were estimated from 19 300 Yorkshire and 18 378 Landrace sows from 12 farms derived from one breeding system. General linear models （GLM） were employed to estimate genetic correlations with heritability （h2）. Longevity was defined by six different parameters （i.e., traits） and heritability ranged from h2=0.13 to 0.16 （P〈0.05） in both swine breeds. Genetic correlations between different longevity definitions were positive and high （r=0.79 to 0.99; P〈0.05）. BF exhibited low positive genetic correlations （r=0.12 to 0.15; P〈0.05） with longevity traits identified in both pig breeds which were dependent on trait definition, whereas ADG and FMA showed weak negative genetic correlations with longevity traits. The influence of BF, ADG, and FMA on longevity traits was examined by dividing the production and reproductive traits into several numerical groups; the influence of each longevity trait was subsequently analyzed among the different groups. The results indicated BF was the best trait to improve longevity among BF, ADG, and FMA, which showed positive effects for almost all longevity traits. For Yorkshire sows, gilts with over 18 mm BF were most desirable; Landrace with 10.6-13 mm BF were suitable for longevity. ADG was not favorable for gilt selection, because the correlation between growth rate and longevity very low. For FMA, our results indicated gilts mating before 230 days exhibited a longer life span. Therefore, we suggest farmers should select for thicker back fat and mate at an earlier age to improve sow longevity.

Distribution patterns of octylphenol and nonylphenol in the aquatic system at Mai Po Marshes Nature Reserve,a subtropical estuarine wetland in Hong Kong

Spatial and temporal distribution of octylphenol （OP） and nonylphenol （NP） in Mai Po Marshes, a subtropical estuarine wetland in Hong Kong, were investigated. Surface water samples were collected every month from 11 sites during the period of September- December 2004. Concentrations of OP and NP ranged from 11.3 to 348 ng/L and from 29 to 2591 ng/L, respectively. The high levels of NP and OP were found in November and December than in September and October. The levels of OP and NP have no significant spatial differences except September. Total organic matter in the sediments appeared to be an important factor in controlling the fate of these compounds in the aquatic environment.

Keeping the right time in space: importance of circadian clock and sleep for physiology and performance of astronauts

The circadian clock and sleep are essential for human physiology and behavior; deregulation of circadian rhythms impairs health and performance. Circadian clocks and sleep evolved to adapt to Earth's environment, which is characterized by a 24-hour light–dark cycle. Changes in gravity load, lighting and work schedules during spaceflight missions can impact circadian clocks and disrupt sleep, in turn jeopardizing the mood, cognition and performance of orbiting astronauts. In this review, we summarize our understanding of both the influence of the space environment on the circadian timing system and sleep and the impact of these changes on astronaut physiology and performance.

Spatial distribution patterns of Symplocos congeners in a subtropical evergreen broad-leaf forest of southern China

Studies on spatial distribution of congeneric species can supplement our understanding of species ecological processes. We analyzed population structure, spatial distribution, intra-and interspecific associations among six Symplocos tree species on a large(50 ha) plot in a subtropical, evergreen broad-leaf forest in southern China using spatial point pattern analysis methods. Our results suggested that the six Symplocos tree species were all clustered at small scales. The aggregation intensity of S. wikstroemiifolia with low abundance but relatively numerous large-diameter trees was much higher than that of the other five Symplocos species that occurred at high abundance but included few large-diameter trees. Spatial associations among the six congeners showed that 12 of 30 pairs were associated positively at small scales, and 13 of 30 pairs were unrelated. For species among different size classes, 79 of 120 pairs were not correlated, and 17 of 120 pairs were associated positively. These results showed insufficient evidence for interspecific competition and congeneric Symplocos species commonly coexist within subtropical plant communities. The spatial patterns of Symplocos species and their correlations changed with size(DBH) class and were simultaneously affected by spatial scales; the intensity of their aggregation and association decreased with increasing area of the sample plot.

Pancreatic triglyceride lipase is involved in the virulence of the brown planthopper to rice plants

The brown planthopper(BPH),Nilaparvata lugens,an important rice insect pest,can enhance its virulence to BPH-resistant rice within as short a span as several generations.Here,we cloned a pancreatic triglyceride lipase(PTL)gene(NlPTL)in N.lugens,and found that its mRNA level was higher in the high virulence population(fed on variety Rathu Heenati,P-RH)than in the low virulence population(fed on variety Taichung Native 1,P-TN1).Knocking down NlPTL caused BPH individuals to spend more time in non-penetration and the pathway phases and less time feeding on the phloem of rice plants;these changes consequently decreased food intake,lipid content,survival rate,and fecundity in the insects.These findings reveal for the first time that PTL in BPH is involved in its virulence to rice plants.

A comparative study of mangrove floras in China and India

Species compositions in the mangrove habitats of China and India, especially on Hainan Island （China） and on the Sun- darbans and the Andaman ＆ Nicobar Islands （India）, are of interest, showing several similarities and differences. The total mangrove areas of India cover about 6419 km^2 with 43 species （23 major and 20 minor species）, while the mangrove areas of China cover about 150 km^2 with 26 speices （19 major and 7 minor speices）. We carried out a comparison of mangrove floras in the Indian Sundarbans, Andaman ＆ Nicobar Islands and in Hainan Island. The mangrove areas of the Indian Sundarbans comprise about 66.5% of the total Indian mangroves, presently covering an area of 4267 km^2 with 35 species from 22 genera and 16 families. There are 34 species from 17 genera and l 3 families in the Andaman ＆ Nicobar Islands. Hainan Island is considered a distribution center of mangroves; it has the largest number of mangrove species in China, i.e., 25 species from 15 genera and 12 families, accounting for about 96% of the total number of mangrove species in China. The mangroves of Hainan Island and the Andaman ＆ Nicobar Islands belong to island types, while those of Sundarbans belong to inter-tidal deltaic types. Differences in soil type, rainfall, temperature, tidal fluctuation and other climatic features possibly cause variations in floristic composition of these regions.

Comparative analysis of the ecological fitness and transcriptome between two genotypes of the brown planthopper Nilaparvata lugens

Insect quantity is an important index for determining the degree of damage to plants.The brown planthopper(BPH),Nilaparvata lugens(Stal),is one of the most important rice pests in Asia.Here,we used two genotypes of BPH,a highfecundity genotype(HFG)and a low-fecundity genotype(LFG),to measure their ecological fitness and to explore possible causes for genotype differentiation by transcriptome sequencing.Our results showed that there were significant differences in body weight,egg hatching rate and honeydew secretion between the HFG and LFG,particularly,the number of eggs laid per female was more significant.Transcriptional analysis showed that a total of 1966 differentially expressed genes(DEGs)were mainly involved in energy metabolism,carbohydrate and lipid syn thesis,in sect horm one syn thesis and fecun dity-related pathways.Moreover,we found that the phosphorylation levels of AMPK,AKT and FoxO and the synthesis of ecdysone were different between the two genotypes of BPH.These findings may partially explain the differenee in the ecological fitness between HFG and LFG.This study is the first to compare the differences between two genotypes of BPH and to provide transcriptomic evidenee to further understand the fecundity of BPH.

Screening of Brown Planthopper Resistant miRNAs in Rice and Their Roles in Regulation of Brown Planthopper Fecundity

MicroRNAs(miRNAs)can participate in plant-insect interactions,which regulate plant defense networks.In this study,we analyzed the miRNA expression profiles of six rice varieties before and after brown planthopper(BPH)-feeding.We identified 45 differentially expressed miRNAs between BPH-susceptible and BPH-resistant rice varieties and 144 miRNAs that responded to BPH-feeding.Thus,miRNAs may be involved in multiple pathways regulating rice defense response against BPH.In addition,we found that the genetic history of rice varieties determined the regulation mode of the miRNA and affected the amounts,types,changing trends and response periods of miRNAs in response to BPH-feeding.To conclude,we scanned seven potential cross-kingdom miRNAs,of which miR5795 may target the vitellogenin gene in BPH,causing a 16.07%reduction in BPH oviposition.The results provide new miRNA information of rice-BPH interactions and BPH-resistant rice variety breeding.

Effects of sesame nectar on longevity and fecundity of seven Lepidoptera and survival of four parasitoid species commonly found in agricultural ecosystems

Ecological engineering involves the use of plants to promote establishment, survival and efficiency of natural enemies in agricultural systems. Some plant species may be hosts or provide resources to some pest species. We assessed the risks and benefits of sesame （Sesamum indicum L.）, as a nectar source for seven economically important Lepidopteran pest and four parasitoid species in a range of vegetable crop systems. Our results showed that the mean Iongevities of arthropod parasitoids Pteromalus puparum （L.）, Encarsia sophia （Girault ＆ Dodd） and male Microplitis tuberculifer （Wesmael） were significantly extended when fed on sesame flowers compared to the water control. Sesame flowers had no effect on adult Iongevities and fecundities of six out of the seven Lepidoptera pest species tested except Plutella xyllostella （L.） females laid more eggs when fed on sesame flowers. It is likely that the increased fecundity is due to accessibility to nectar at the bottom of corolla because of their smaller body sizes. Our findings provide a first step towards better understanding of the risks and benefits of using sesame to implement ecological engineering for the management of vegetable pests.

Mitochondrial DNA diversity and origin of indigenous pigs in South China and their contribution to western modern pig breeds

Indigenous pigs in South China are valuable genetic resources with many specific and unique characters, which have played an important role in the establishment of some western modern pig breeds. However, the origin and genetic diversity of indigenous pigs in South China have not been fully understood. In the present study, we sequenced 534 novel mitochondrial DNA (mtDNA) D-loop and assembled 54 complete mitogenome sequences for all 17 indigenous pig breeds from Fujian, Guangdong, Guangxi and Hainan in South China. These data were analyzed together with previously published homologous sequences relevant to this study. We found that all 13 coding genes of the mitogenomes were under purifying selection, but ND1 had the most variable sites and CYTB contained the most non-synonymous SNPs. Phylogenetic analysis showed that all indigenous pigs in South China were clustered into the D haplogroup with D1a1, D1b, D1c and D1e sub-haplogroups found to be dominant. Haplotype and nucleotide diversities of D-loop sequences ranged from 0.427 to 0.899 and from 0.00342 to 0.00695, respectively, among which all pigs in Guangdong had the lowest diversity. The estimates of pairwise FST, gene flow (Nm) and genetic distance (Da) indicated that most of these indigenous pig breeds differentiated from each other significantly (P<0.05). Among the western modern breeds, Berkshire and Yorkshire had significant Asian matrilineal footprints from indigenous pigs in South China, especially the Spotted pigs distributed in Guangdong and Guangxi. The neutrality test (Fu’s FS) indicated that indigenous pigs from Fujian and Guangxi had gone through recent population expansion events (P<0.05). It is concluded that indigenous pigs in South China were most likely derived from the Mekong region and the middle and downstream regions of Yangtze River through Guangxi and Fujian. Our findings provide a complete and in-depth insight on the origin and distribution pattern of maternal genetic diversity of indigenous pigs in South China.

Protective immunity of orange-spotted grouper(Epinephelus coioids) against a nervous necrosis virus isolated from China,and determination of the complete sequences of the virus

On the basis of the sequence and analysis of genome from the orange-spotted nervous necrosis virus（ OGNNV）, China strain, a pair of special primers were designed according to the nucleotide sequences of RNA2 from OGNNV. The major capsid protein （ MCP）gene of OGNNV was cloned by means of reverse-transcriptase polymerase chain reaction （RT-PCR） and ligated into the pET32a expression plasmid. The MCP gene of OGNNV was 1 017 bases, encoded a protein of 338 amino acid with a molecular mass of 37.1 kDa. Recombinant protein with a molecular mass of 57.4 kDa was expressed in E. coli BL21 （DE3）. Vaccine was prepared from the recombinant protein expressed in recombinant cells. The juvenile orange-spotted groupers （8 cm in average length） were immunized by intraperitoneal injection. Group A was challenged with infected tissue filtrates 25 d post-vaccination. The mortality in the vaccined group （ A1,30% ） was a little higher than the unvaccined group （ B2, 27.8% ）. Group B was challenged after three vaccine injections. The mortality in the vaccined group （B1, 16.7% ） was lower than the unvaccined group （132, 27.8% ）, And the relative percentage survival （RPS） value of vaccined group, compared with the unvaccined group, was 40%. The anti-recombinant protein sera with a 1 ： 100 dilution were mixed with double volume of infected tissue filtrates and incubated at 4 ℃ for 12 h and then intramuscularly injected into the juvenile orange-spotted grouper. Treatment of infected tissue filtrates with anti-recombinant protein serum resulted in a significantly lower mortality of fish （ Group C1, mortality of 18.18% ）, compared with the fish （ Group C2, mortality of 40% ） which received infected tissue filtrates treated with control serum. Results implied the potential use of the capsid protein in immunization against OGNNV.

The combination of NlMIP and Gαi/q coupled-receptor NlA10promotes abdominal vibration production in female Nilaparvata lugens(Stal)

For various sexually mature insects,including the brown planthopper(BPH,Nilaparvata lugens),the abdominal vibration(AV)signal is the initiation of the mating process,and itiscritical to the successofmating.Currently,there are few studies on the genetic and molecular mechanisms of AV regulation.Ourprevious AV-related transcriptomic study in female BPH identified myoinhibitory peptide(NlMIP)as a gene that potentially affects AV statusinfemales,buthow NlMIP affects AV status remains unknown.In this study,we confirmed that NlMIP regulates AV production and mating behavior in female BPH.When the RNAi knockdown efficiency of NlMIP was 59.00%,the probability of females producing AV and the mating rate in 1 h decreased by 38.89 and 61.11%,respectively.In addition,six mature peptides of NlMIP were synthesized and they were able to regulate AV production and mating behavior in females,with NlMIP2having the strongest effect.The A-family neuropeptide GPCR 10(NlA10)was found to be a potential receptor for NlMIP based on a phylogenetic tree analysis and the fact that NlMIP mature peptides effectively activated NlA10.After NlA10was knocked down,the probability of females producing AV and the mating rate in 1 h had reductions of 28.89 and 43.33%,respectively.When activated by NlMIP2,NlA10 coupled the Gαi/q signalling pathways,thereby inhibiting the downstream AC/cAMP/PKA,activating the PLC/Ca^(2+)/PKC signalling pathways and then activating MEK1/2 in a cascade to mediate the phosphorylation of ERK1/2,and finally regulating the AV of females.These results provide a basis for the prevention and control of the brown planthopper pest by disrupting female AV.

An integrated analysis of mRNA-miRNA transcriptome data revealed hub regulatory networks in three genitourinary cancers

Bladder,kidney,prostate and testicular carcinoma are the top four genitourinary cancers in China.Here we analyzed mRNA and miRNA expression profiles of carcinomas of the bladder(TCC),kidney(ccRCC)and testis(TGCT)to uncover their specific regulatory mechanisms.The gene expression profiles of GSE31617 were downloaded from GEO database,which contained 27 samples,including 10 TCC,7 TGCT and 10 ccRCC.Specific up-and downregulated differentially expressed genes(DEGs)and differentially expressed microRNAs(DEmiRNAs)of each cancer were selected and target genes of DEmiRNAs were predicted.Gene interaction network of the shared genes and target genes of DEmiRNAs of each cancer was predicted by STRING and constructed by Cytoscape.In each cancer,we build regulatory networks of hub genes selected and conducted GO analysis of enriched genes.Furthermore,we chose four hub genes(SALL4,RHEB,CDC42 and TNN)for survival analysis in OncoLnc database,and they all had effects on the survival rate of another genitourinary cancer-kidney renal clear cell carcinoma(KIRC).In conclusion,the present study indicated that the identified hub genes promote our understanding of molecular mechanisms underlying the development of three genitourinary cancers,and might be used as molecular targets and diagnostic biomarkers for the treatment of them.

Sexual competition and kin recognition co-shape the traits of neighboring dioecious Diospyros morrisiana seedlings

Plants respond differently to the identity of their neighbors,such as their sex and kinship,showing plasticity in their traits.However,how the functional traits of dioecious trees are shaped by the recognition of neighbors with different sex and kinship remains unknown.In this study,we set up an experiment with different kin/nonkin and inter/intrasexual combinations for a dioecious tree species,Diospyros morrisiana.The results showed that plants grew better with nonkin and intrasexual neighbors than with kin and intersexual neighbors.Kin combinations had significantly shorter root length in the resource-overlapping zone than nonkin combinations,suggesting that kin tended to reduce competition by adjusting their root distribution,especially among female siblings.Our study suggested that the seedling growth of D.morrisiana was affected by both the relatedness and sexual identity of neighboring plants.Further analysis by gas chromatography-mass spectrometry showed that the root exudate composition of female seedlings differed from that of male seedlings.Root exudates may play important roles in sex competition in dioecious plants.This study indicates that sex-specific competition and kin recognition interact and co-shape the traits of D.morrisiana seedlings,while intrasexual and nonkin neighbors facilitate the growth of seedlings.Our study implies that kin-and sex-related interactions depend on different mechanisms,kin selection,and niche partitioning,respectively.These results are critical for understanding how species coexist and how traits are shaped in nature.

Single-base editing in IGF2 improves meat production and intramuscular fat deposition in Liang Guang Small Spotted pigs

Background Chinese indigenous pigs are popular with consumers for their juiciness,flavour and meat quality,but they have lower meat production.Insulin-like growth factor 2(IGF2) is a maternally imprinted growth factor that promotes skeletal muscle growth by regulating cell proliferation and differentiation.A single nucleotide polymorphism(SNP) within intron 3 of porcine IGF2 disrupts a binding site for the repressor,zinc finger BED-type containing 6(ZBED6),leading to up-regulation of IGF2 and causing major effects on muscle growth,heart size,and backfat thickness.This favorable mutation is common in Western commercial pig populations,but absent in most Chinese indigenous pig breeds.To improve meat production of Chinese indigenous pigs,we used cytosine base editor 3(CBE3)to introduce IGF2 intron3-C3071T mutation into porcine embryonic fibroblasts(PEFs) isolated from a male Liang Guang Small Spotted pig(LGSS),and single-cell clones harboring the desired mutation were selected for somatic cell nuclear transfer(SCNT) to generate the founder line of IGF2^(T/T) pigs.Results We found the heterozygous progeny IGF2^(C/T) pigs exhibited enhanced expression of IGF2,increased lean meat by 18%-36%,enlarged loin muscle area by 3%-17%,improved intramuscular fat(IMF) content by 18%-39%,marbling score by 0.75-1,meat color score by 0.53-1.25,and reduced backfat thickness by 5%-16%.The enhanced accumulation of intramuscular fat in IGF2^(C/T) pigs was identified to be regulated by the PI3K-AKT/AMPK pathway,which activated SREBP1 to promote adipogenesis.Conclusions We demonstrated the introduction of IGF2-intron3-C3071T in Chinese LGSS can improve both meat production and quality,and first identified the regulation of IMF deposition by IGF2 through SREBP1 via the PI3KAKT/AMPK signaling pathways.Our study provides a further understanding of the biological functions of IGF2and an example for improving porcine economic traits through precise base editing.

Current therapies for osteoarthritis and prospects of CRISPR-based genome,epigenome,and RNA editing in osteoarthritis treatment

Osteoarthritis(OA)is one of the most common degenerative joint diseases worldwide,causing pain,disability,and decreased quality of life.The balance between regeneration and inflammation-induced degradation results in multiple etiologies and complex pathogenesis of OA.Currently,there is a lack of effective therapeutic strategies for OA treatment.With the development of CRISPR-based genome,epigenome,and RNA editing tools,OA treatment has been improved by targeting genetic risk factors,activating chondrogenic elements,and modulating inflammatory regulators.Supported by cell therapy and in vivo delivery vectors,genome,epigenome,and RNA editing tools may provide a promising approach for personalized OA therapy.This review summarizes CRISPR-based genome,epigenome,and RNA editing tools that can be applied to the treatment of OA and provides insights into the development of CRISPR-based therapeutics for OA treatment.Moreover,in-depth evaluations of the efficacy and safety of these tools in human OA treatment are needed.