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.

Origin and evolution of a new tetraploid mangrove species in an intertidal zone

Polyploidy is a major factor in the evolution of plants,yet we know little about the origin and evolution of polyploidy in intertidal species.This study aimed to identify the evolutionary transitions in three truemangrove species of the genus Acanthus distributed in the Indo-West Pacific region.For this purpose,we took an integrative approach that combined data on morphology,cytology,climatic niche,phylogeny,and biogeography of 493 samples from 42 geographic sites.Our results show that the Acanthus ilicifolius lineage distributed east of the Thai-Malay Peninsula possesses a tetraploid karyotype,which is morphologically distinct from that of the lineage on the west side.The haplotype networks and phylogenetic trees for the chloroplast genome and eight nuclear genes reveal that the tetraploid species has two sub-genomes,one each from A.ilicifolius and A.ebracteatus,the paternal and maternal parents,respectively.Population structure analysis also supports the hybrid speciation history of the new tetraploid species.The two sub-genomes of the tetraploid species diverged from their diploid progenitors during the Pleistocene.Environmental niche models revealed that the tetraploid species not only occupied the near-entire niche space of the diploids,but also expanded into novel environments.Our findings suggest that A.ilicifolius species distributed on the east side of the Thai-Malay Peninsula should be regarded as a new species,A.tetraploideus,which originated from hybridization between A.ilicifolius and A.ebracteatus,followed by chromosome doubling.This is the first report of a true-mangrove allopolyploid species that can reproduce sexually and clonally reproduction,which explains the long-term adaptive potential of the species.

Genetic mechanism of body size variation in groupers:Insights from phylotranscriptomics

Animal body size variation is of particular interest in evolutionary biology,but the genetic basis remains largely unknown.Previous studies have shown the presence of two parallel evolutionary genetic clusters within the fish genus Epinephelus with evident divergence in body size,providing an excellent opportunity to investigate the genetic basis of body size variation in vertebrates.Herein,we performed phylotranscriptomic analysis and reconstructed the phylogeny of 13 epinephelids originating from the South China Sea.Two genetic clades with an estimated divergence time of approximately 15.4 million years ago were correlated with large and small body size,respectively.A total of 180 rapidly evolving genes and two positively selected genes were identified between the two groups.Functional enrichment analyses of these candidate genes revealed distinct enrichment categories between the two groups.These pathways and genes may play important roles in body size variation in groupers through complex regulatory networks.Based on our results,we speculate that the ancestors of the two divergent groups of groupers may have adapted to different environments through habitat selection,leading to genetic variations in metabolic patterns,organ development,and lifespan,resulting in body size divergence between the two locally adapted populations.These findings provide important insights into the genetic mechanisms underlying body size variation in groupers and species differentiation.

Diverse foraging strategies of breeding Swinhoe's Storm-petrel in the productive marginal sea of the Northwest Pacific

Understanding the foraging behavior is essential for investigating seabird ecology and conservation,as well as monitoring the well-being of the marine environment.Breeding seabirds adopt diverse foraging strategies to maximize energy gains and cope with the intensified challenges of parenting and self-maintenance.Such tradeoff may stem from the heterogeneity of food resources and the constraints of central place foraging.Nevertheless,abundant marine productivity could alleviate the energy limitation for seabirds,resulting in a consistent foraging approach.Here,we investigated the foraging strategy during the breeding season of a cryptic small-sized seabird,Swinhoe’s Storm-petrel(Hydrobates monorhis),in the Yellow Sea,a productive marginal sea of the Northwest Pacific.Using GPS tracking,we evaluated habitat preference,quantified the foraging strategy,and tested if environmental conditions and individual traits influence foraging trips.We found that Swinhoe’s Storm-petrels preferred nearshore areas with shallow water and engaged in primarily short foraging trips.Distinctive southeastward and southwestward strategies emerged when combining trip metrics,including foraging direction,duration,and maximum distance.The bathymetry,proximity to the coastline,and sea surface temperature differed in two foraging strategies.Foraging strategies exhibited flexibility between individuals,potentially explained by wing morphology,in which longer-winged birds are more likely to embark on longer-distance foraging trips.These findings highlight the impact of environmental factors and individual traits on seabirds’foraging decisions in productive marginal sea ecosystems.Our study also provides valuable insights into the foraging ecology of this Asian endemic storm-petrel.

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.

Generic classification of Asian horned toads(Anura:Megophryidae: Megophryinae) and monograph of Chinese species

The subfamily Megophryinae,as a representative batrachian group of the Oriental Realm and one of the most diverse groups of amphibians,has attracted considerable attention due to continued conjecture regarding its generic classification and failure to reach a satisfactory consensus.China boasts the richest diversity of Asian horned toads,containing some two thirds of the total species cataloged.However,most species have a complicated taxonomic history,resulting in multiple misidentifications.As such,an overall clarification of historical records and regional checklists is required.In the current investigation,we established the phylogeny of the Asian horned toads and performed detailed examinations with redefinitions of several important morphological traits.Based on the phylogenetic relationships and morphological differences,we propose a new ten-genus classification for the Asian horned toad subfamily Megophryinae:i.e.,Brachytarsophrys,Atympanophrys,Grillitschia,Sarawakiphrys gen.nov.,Jingophrys gen.nov.,Xenophrys,Megophrys,Pelobatrachus,Ophryophryne,and Boulenophrys.Revisions on the diagnosability,distribution,and content of each genus are provided.Furthermore,we present a careful review of the taxonomic history of Asian horned toad species from China and provide a monograph of congeners,including six species of Brachytarsophrys,four species of Atympanophrys,five species of Jingophrys gen.nov.,10 species of Xenophrys,two species of Ophryophryne,and 60 species of Boulenophrys.Finally,we discuss the importance of traditional morphological traits based on multiple populations in taxonomic work as well as taxonomic inflation caused by the genetic species delimitation.

Late Oligocene fossil acorns and nuts of Quercus section Cyclobalanopsis from the Nanning Basin,Guangxi,South China

Quercus is the largest genus within the Fagaceae and has a rich fossil record.Most of the fossil material is attributed to the subgenus Quercus based on leaves,pollen or rarely acorns and nuts.Fossil records of Q.section Cyclobalanopsis characterized by ring-cupped acorns are relatively few and especially those described based on nuts are scant.In this study,we described four new species of Quercus section Cyclobalanopsis based on mummified acorns and nuts:Q.paleodisciformis X.Y.Liu et J.H.Jin sp.nov.,Q.paleohui X.Y.Liu et J.H.Jin sp.nov.,Q.nanningensis X.Y.Liu et J.H.Jin sp.nov.and Q.yongningensis X.Y.Liu et J.H.Jin sp.nov.These species closely resemble the extant species Q.disciformis,Q.hui,Q.kerrii,and Q.dinghuensis.The occurrence of Q.section Cyclobalanopsis in the Oligocene stratum of Guangxi,South China,suggests that the section has diversified within its extant distribution center since the Oligocene.By combining records from other areas,we propose that the section first appeared in the middle Eocene of East Asia(Sino-Japan),has diversified in situ with a few elements scattering into West Asia and southern Europe since the Oligocene and Pliocene,respectively,and finally became restricted in East Asia since the Pleistocene.This indicates that the section originated and diversified in East Asia,before spreading into West Asia no later than the Oligocene and into southern Europe by the Pliocene.Subsequently it disappeared from South Europe and West Asia due to the appearance of the(summer dry)Mediterranean climate and widespread cooling during the Pleistocene.

Genetic relatedness and association mapping of horticulturally valuable traits for the Ceiba plants using ddRAD sequencing

Ceiba species have high ornamental value and are widely cultivated in tropical regions.However,genetic background of cultivated Ceiba plants remains unclear.To understand the genetic relatedness of cultivated Ceiba plants and genetic basis of key horticultural traits,here we explored the genetic relatedness of 153 accessions of Ceiba plants cultivated in Southern China and identified SNPs associated with five horticultural traits,based on 11704 SNPs derived from double-digest restriction-site associated DNA sequencing(ddRAD-seq).Clustering analysis revealed that these accessions were composed of three groups:C.speciosa group,C.insignis group,and hybrid group.The GWAS identified two,four,two,three,and four SNPs related to petal color,petal striation number,flowering time,trunk shape,and prickles on the trunk and branches,respectively.One to two candidate genes were found near the SNPs strongly associated with these traits.This study revealed the genetic relatedness in the Ceiba plants cultivated in Southern China and presented the first GWAS analysis for five horticultural traits for them,laying a foundation for phenotype-related marker selection and molecular breeding.

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.

Two New Species of Fire-Bellied Newts (Caudata, Salamandridae, Cynops) from Southeastern China

The fire-bellied newt genus Cynops contains ten known species distributed in China and Japan in East Asia.In this work,two new Cynops species are described,namely Cynops jiaoren sp.nov.distributed in northern Guangdong and Cynops maguae sp.nov.distributed in eastern Jiangxi.The two new species can be distinctly distinguished from their congeners by the independent phylogenetic placements and a combination of morphological characteristics.The discovery of these two new species in the Southeast Chinese Hilly Area,where half of congeners occur,increases the known diversity of Cynops,and indicates unresolved relationships among the species in southeastern China.Further discussions on the taxonomic status of Cynops cyanurus are also provided.

Redefinition of the Odorrana versabilis Group,with a New Species from China(Anura,Ranidae,Odorrana)

Taxonomic studies of the Odorrana versabilis group have been consistently disorganized,and they have often been incorrectly associated with an abandoned subgenus or genus Bamburana in existing literature.In this study,we conducted an integrative taxonomic analysis using molecular phylogenetic analysis of two mitochondrial gene fragments(16S rRNA and CO1)and morphological examination of 41 specimens collected during herpetological surveys in southern China.Our results confirmed the monophyly of O.versabilis group which exhibited distinct morphological traits.We also identified a cryptic lineage sister with O.exiliversabilis.Additionally,our study revealed that O.trankieni was nested within O.nasuta.Consequently,we redefined the morphological characteristics of the O.versabilis group,described a new species in this group,O.confusa sp.nov.,and proposed that O.trankieni be considered a junior synonym of O.nasuta.One of the most significant findings of this study was the observation of distinct morphological changes in adult males across all species within the O.versabilis group between the non-breeding and breeding seasons.This study contributes to research on various aspects of Asian amphibian fauna,including taxonomy,ecology,evolution,and physiology.

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.

Different mechanisms underlie similar species-area relationships in two tropical archipelagoes

Despite much research in the field of island biogeography,mechanisms regulating insular diversity remain elusive.Here,we aim to explore mechanisms underlying plant species-area relationships in two tropical archipelagoes in the South China Sea.We found positive plant species-area relationships for both coral and continental archipelagoes.However,our results showed that different mechanisms contributed to similar plant species-area relationships between the two archipelagoes.For coral islands,soil nutrients and spatial distance among communities played major roles in shaping plant community structure and species diversity.By contrast,the direct effect of island area,and to a lesser extent,soil nutrients determined plant species richness on continental islands.Intriguingly,increasing soil nutrients availability(N,P,K)had opposite effects on plant diversity between the two archipelagoes.In summary,the habitat quality effect and dispersal limitation are important for regulating plant diversity on coral islands,whereas the passive sampling effect,and to a lesser extent,the habitat quality effect are important for regulating plant diversity on continental islands.More generally,our findings indicate that island plant species-area relationships are outcomes of the interplay of both niche and neutral processes,but the driving mechanisms behind these relationships depends on the type of islands.

Biotransfer of heavy metals along a soil-plant-insect-chicken food chain:Field study

The accumulation and transfer of Pb,Zn,Cu,and Cd along a soil-plant-insect-chicken food chain at contaminated sites were investigated.The study site nearing the Pb/Zn mine had been contaminated by heavy metals severely.Cadmium and Pb concentrations steadily declined with increasing trophic level（p 〈 0.01）,but concentrations of Zn and Cu slightly increased from plant to insect larva（p 〉 0.05）.The concentrations of heavy metals were the highest in chicken muscle,with lower values in liver and blood.The bioaccumulation of Pb was observed in chicken livers.The eliminations of Pb,Zn,Cu,and Cd via insect and chicken feces avoid metal bioaccumulation in insect and chicken body.These results suggest that the accumulation of heavy metals in specific animal organ of tissues could not be neglected,although transfer of metals to chicken from plant and insect was limited.

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.

Removal of metals by sorghum plants from contaminated land

The growth of high biomass crops facilitated by optimal of agronomic practices has been considered as an alternative to phytoremediation of soils contaminated by heavy metals. A field trial was carded out to evaluate the phytoextraction efficiency of heavy metals by three varieties of sweet sorghum （Sorghum biocolor L.）, a high biomass energy plant. Ethylene diamine tetraacetate （EDTA）, ammonium nitrate （NH4NO3） and ammonium sulphate （（NH4）2SO4） were tested for their abilities to enhance the removal of heavy metals Pb, Cd, Zn, and Cu by sweet sorghum from a contaminated agricultural soil. Sorghum plants always achieved the greatest removal of Pb by leaves and the greatest removal of Cd, Zn and Cu by stems. There was no significant difference among the Keller, Rio and Mray varieties of sweet sorghums in accumulating heavy metals. EDTA treatment was more efficient than ammonium nitrate and ammonium sulphate in promoting Pb accumulation in sweet sorghum from the contaminated agricultural soil. The application of ammonium nitrate and ammonium sulphate increased the accumulation of both Zn and Cd in roots of sorghum plants. Results from this study suggest that cropping of sorghum plants facilitated by agronomic practices may be a sustainable technique for partial decontamination of heavy metal contaminated soils.

Whole-genome sequencing of leopard coral grouper(Plectropomus leopardus) and exploration of regulation mechanism of skin color and adaptive evolution

Leopard coral groupers belong to the Plectropomus genus of the Epinephelidae family and are important fish for coral reef ecosystems and the marine aquaculture industry. To promote future research of this species, a high-quality chromosome-level genome was assembled using PacBio sequencing and Hi-C technology. A 787.06 Mb genome was assembled, with 99.7%(784.57 Mb) of bases anchored to 24 chromosomes. The leopard coral grouper genome size was smaller than that of other groupers, which may be related to its ancient status among grouper species. A total of 22 317 proteincoding genes were predicted. This high-quality genome of the leopard coral grouper is the first genomic resource for Plectropomus and should provide a pivotal genetic foundation for further research. Phylogenetic analysis of the leopard coral grouper and 12 other fish species showed that this fish is closely related to the brown-marbled grouper.Expanded genes in the leopard coral grouper genome were mainly associated with immune response and movement ability, which may be related to the adaptive evolution of this species to its habitat. In addition, we also identified differentially expressed genes(DEGs) associated with carotenoid metabolism between red and brown-colored leopard coral groupers. These genes may play roles in skin color decision by regulating carotenoid content in these groupers.

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.

Marker-assisted selection of YY supermales from a genetically improved farmed tilapia-derived strain

DEAR EDITOR, Genetically improved farmed tilapia (GIFT)and GIFT-derived strains account for the majority of farmed tilapia worldwide.As male tilapias grow much faster than females,they are often considered more desirable in the aquacultural industry.Sex reversal of females to males using the male sex hormone 17-α-methyltestosterone (MT)is generally used to induce phenotypic males during large-scale production of all male fingerlings.However,the widespread use of large quantities of sex reversal hormone in hatcheries may pose a health risk to workers and ecological threats to surrounding environments. Breeding procedures to produce genetically all-male tilapia with limited or no use of sex hormones are therefore urgently needed.

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.