Morphological and Molecular Data Revealed One New Species of the Short-legged Toads Brachytarsophrys Tian and Hu,1983(Anura,Megophryidae)from Yunnan,China

A new species of the genus Brachytarsophrys,named Brachytarsophrys wenshanensis sp.nov.,has been identified in southeastern Yunnan,China.This new species can be readily distinguished from other known congeners by both morphological criterion and molecular analysis of three mitochondrial gene segments:16S,COI,and Cytb.This classification is based on the following morphological characters:(1)medium body size(SVL 83.8–85.1 mm in two adult males);(2)enormous head,with head width nearly 1.2 times the length;(3)tongue pyriform,feebly notched;(4)non-meeting heels;(5)male lacking nuptial pad;(6)tibiotarsal articulation reaching angle of mouth when hindlimbs are extended forward alongside the body;(7)absence of outer metatarsal tubercle,inner metatarsal tubercle elliptic and approximately equal to first toe;(8)rudimentary toe webbing,webbing formula:Ⅰ(2–)–(2^(++))Ⅱ(2^(–))–(3^(++))Ⅲ(2^(½))–(4)Ⅳ(4^(+))–(2^(⅔))V;(9)lateral fringes narrow;(10)dermal ridge or glandular fold on dorsum absent;(11)pectoral glands distinct and irregular,femoral gland small.Our work increases the number of species within the genus Brachytarsophrys to 9.

Involvement of Sox9a in chondrogenesis and gonadal development in teleost Nile tilapia(Oreochromis niloticus)

DEAR EDITOR,Sox9 is a member of the Sry-related high-mobility group box(Sox)transcription factor family in animals.In teleost fish,Sox9 undergoes duplication to generate two duplicates,namely Sox9a and Sox9b.However,the functions of these duplicates in the teleost Nile tilapia(Oreochromis niloticus)remain unclear.In this study,we characterized the roles of Nile tilapia Sox9a in chondrogenesis and gonadal development.In situ hybridization assays showed that Sox9a was mainly expressed in cartilage tissues and somatic cells surrounding germ cells of the gonads.CRISPR/Cas9-mediated homozygous mutation of the Sox9a gene resulted in craniofacial deformities and missed mandibles,as well as impaired the expression of Col2a1a that is involved in chondrogenesis.In addition,germ cell number and DNA replication in somatic cells in the gonads of both sexes were reduced following Sox9a mutation.Taken together,this study demonstrates that Sox9a is involved in cartilage development and germ cell proliferation in Nile tilapia.

Use of tetracycline hydrochloride and alizarin complexone for immersion marking black rockfish Sebastes schlegelii

We tested the utility of chemical marking techniques in the juvenile black rockfish Sebastes schlegelii. Juveniles(30–40 mm total length) were immersed in a range of tetracycline hydrochloride(TC) solutions at concentrations ranging from 300 to 500 mg/L, and alizarin complexone(ALC) solutions at concentrations ranging from 200 to 400 mg/L in filtered sea water(salinity of 30) for 24 h, respectively. Otoliths(sagittae, asteriscus), scales, fin rays(dorsal, pectoral, ventral, anal, and caudal fin rays), and fin spines(dorsal, ventral, and anal fin spines) were sampled and used to detect fl uorescent marks after a 60-day growth experiment. With the exception of 300 mg/L TC, acceptable marks were produced in the otoliths and fin spines by all concentrations of TC and ALC. In particular, we observed clearly visible marks in the sagittae, asteriscus, and fin spines under normal light at concentrations of 200–400 mg/L, 250–400 mg/L, and 250–400 mg/L ALC, respectively. Scales and fin rays had acceptable marks at much higher concentrations(≥350 mg/L TC, ≥250 mg/L ALC for scales and ≥350 mg/L TC, ≥300 mg/L ALC for fin rays). The best mark quality(i.e., acceptable marks were observed in all sampled structures after immersion marking) were obtained following immersion in TC at between 350–500 mg/L, and ALC between 300–400 mg/L. In addition, there was no significant difference in survival and growth of TC and ALC marked fish compared to their controls up to 60 days post-marking( P > 0.05).

Loss of behavioral stress response in blind cavefish reduces energy expenditure

The stress response is essential for animal self-defense and survival. However, species may exhibit stress response variation depending on their specific environmental and selection pressures. Blind cavefish dwell in cave environments, which differ markedly in stressors and resource availability compared to surface aquatic environments. However, whether blind cavefish exhibit differences in stress response as an adaptation to their cave environments remains unclear. Here, we investigated differences in stress response in six closely related Triplophysa species, including three blind cavefish (T. longibarbata, T. jiarongensis, and T. rosa) and three normal-sighted river fish (T. nasobarbatula, T. dongsaiensis, and T. bleekeri). Results showed that blind cavefish exhibited a range of distinct behavioral responses compared to sighted river fish, including greater levels of activity, shorter duration of freezing, absence of erratic movements or thrashing behavior, and opposite behavioral trends over time. Furthermore, the cavefish species demonstrated attenuated increases in metabolic rate in response to stressors related to novel environments. Cave-dwelling T. rosa also exhibited lower basal hypothalamic-pituitary-inter-renal (HPI) axis-related gene expression levels and stress hormone concentrations compared to river-dwelling T. bleekeri. These results suggest that blind cavefish may have lost their behavioral stress response, potentially mediated by a reduction in basal activity of the HPI axis, thus enabling the conservation of energy by reducing unnecessary expenditure in energy-limited caves.

Genome of striped eel catfish(Plotosus lineatus) provides insights into the adaptive evolution of amphidromous fish

DEAR EDITOR,Dramatic changes in habitat can have a range of effects on organisms(Bi & Zhang, 2021). Plotosus lineatus is a representative fish within Ostariophysi showing adaptation from freshwater habitats to the ocean. Here, we sequenced the genome of P. lineatus using long-read and Hi-C sequencing technology. Based on the high-quality chromosome-level genome, we explored the molecular basis of various P. lineatus features, including large genome with fewer chromosomes, novel dendritic organ(DO).

3DPhenoFish:Application for two-and three-dimensional fish morphological phenotype extraction from point cloud analysis

Fish morphological phenotypes are important resources in artificial breeding,functional gene mapping,and population-based studies in aquaculture and ecology.Traditional morphological measurement of phenotypes is rather expensive in terms of time and labor.More importantly,manual measurement is highly dependent on operational experience,which can lead to subjective phenotyping results.Here,we developed 3DPhenoFish software to extract fish morphological phenotypes from three-dimensional(3D)point cloud data.Algorithms for background elimination,coordinate normalization,image segmentation,key point recognition,and phenotype extraction were developed and integrated into an intuitive user interface.Furthermore,18 key points and traditional 2D morphological traits,along with 3D phenotypes,including area and volume,can be automatically obtained in a visualized manner.Intuitive fine-tuning of key points and customized definitions of phenotypes are also allowed in the software.Using 3DPhenoFish,we performed high-throughput phenotyping for four endemic Schizothoracinae species,including Schizopygopsis younghusbandi,Oxygymnocypris stewartii,Ptychobarbus dipogon,and Schizothorax oconnori.Results indicated that the morphological phenotypes from 3DPhenoFish exhibited high linear correlation(>0.94)with manual measurements and offered informative traits to discriminate samples of different species and even for different populations of the same species.In summary,we developed an efficient,accurate,and customizable tool,3DPhenoFish,to extract morphological phenotypes from point cloud data,which should help overcome traditional challenges in manual measurements.3DPhenoFish can be used for research on morphological phenotypes in fish,including functional gene mapping,artificial selection,and conservation studies.3DPhenoFish is an open-source software and can be downloaded for free at https://github.com/lyh24k/3DPhenoFish/tree/master.

Genome and population evolution and environmental adaptation of Glyptosternon maculatum on the Qinghai-Tibet Plateau

Persistent uplift means the Qinghai-Tibet Plateau(QTP)is an ideal natural laboratory to investigate genome evolution and adaptation within highland environments.However,how paleogeographic and paleoclimatic events influence the genome and population of endemic fish species remains unclear.Glyptosternon maculatum is an ancient endemic fish found on the QTP and the only critically endangered species in the Sisoridae family.Here,we found that major transposons in the G.maculatum genome showed episodic bursts,consistent with contemporaneous geological and climatic events during the QTP formation.Notably,histone genes showed significant expansion in the G.maculatum genome,which may be mediated by long interspersed nuclear elements(LINE)repetitive element duplications.Population analysis showed that ancestral G.maculatum populations experienced two significant depressions 2.6 million years ago(Mya)and 10000 years ago,exhibiting excellent synchronization with Quaternary glaciation and the Younger Dryas,respectively.Thus,we propose that paleogeography and paleoclimate were dominating driving forces for population dynamics in endemic fish on the QTP.Tectonic movements and temperature fluctuation likely destroyed the habitat and disrupted the drainage connectivity among populations.These factors may have caused severe bottlenecks and limited migration among ancestral G.maculatum populations,resulting in the low genetic diversity and endangered status of the species today.

Comparative multi-locus assessment of modern Asian newts(Cynops, Paramesotriton, and Pachytriton:Salamandridae) in southern China suggests a shared biogeographic history

Evolutionary biologists are always interested in deciphering the geographic context of diversification,therefore they introduced the concept of comparative phylogeography, which helps to identify common mechanisms that contribute to shared genetic structures among organisms from the same region.Here, we used multi-locus genetic data along with environmental data to investigate shared phylogeographic patterns among three Asianendemic newt genera, Cynops, Paramesotriton and Pachytriton, which occurred in montane/submontane streams or ponds in southern China. Our 222samples from 78 localities covered the entire range of the three genera and represented the largest dataset of this group to date. We reconstructed matrilineal genealogies from two protein-coding,mitochondrial genes, and gene network from two nuclear genes. We also estimated divergence times of major cladogenetic events and used occurrence data to evaluate niche difference and similarity between lineages. Our results revealed a common basal split in all three genera that corresponds to the separation of two geographic terrains of southern China.Those ancient divergence occurred during middle to late Miocene and likely correlate with paleoclimatic fluctuations caused by the uplift of the Qinghai-Xizang (Tibet) Plateau (QTP).Particularly,the strengthening and weakening of Asian summer monsoons during the Miocene may have profoundly impacted southern China and led to repeatedly vicariance in those newts.However,despite differences in realized niches between lineages,there is no evidence for divergence of fundamental niches.Preservation of old newt matriline lineages in mountains of southern China suggests that the region acts as both museums and cradles of speciation.Based on those results,we advocate a multi-pronged protection strategy for newts in the three genera.

Chromosome-level genome assembly of the Chinese longsnout catfish Leiocassis longirostris

The Chinese longsnout catfish(Leiocassis longirostris Günther)is one of the most economically important freshwater fish in China.As wild populations have declined sharply in recent years,it is also a valuable model for research on sexual dimorphism,comparative biology,and conservation.However,the current lack of high-quality chromosome-level genome information for the species hinders the advancement of comparative genomic analysis and evolutionary studies.Therefore,we constructed the first high-quality chromosomelevel reference genome for L.longirostris.The total genome was 703.19 Mb,with 389 contigs and contig N50 length of 4.29 Mb.Using high-throughput chromosome conformation capture(Hi-C)data,the genome sequences(685.53 Mb)were scaffolded into 26 chromosomes ranging from 17.36 to 43.97 Mb,resulting in a chromosomal anchoring rate for the genome of 97.44%.In total,23708 protein-coding genes were identified in the genome.Phylogenetic analysis indicated that L.longirostris and its closest related species P.fulvidraco diverged approximately 26.6 million years ago.This highquality reference genome of L.longirostris should pave the way for future genomic comparisons and evolutionary research.

First Record of the Genus Wijayarana (Anura, Ranidae) from Yunnan, China

Dear Editor,e family Ranidae is one of the largest and most families of amphibians(Che et al.,2007),with 64 recognized genera and429 species(Frost,2022).However,the systematics and natural history of some ranid groups have not been well-studied.One such example is the taxonomic relationship between Sylvirana,H ylarana and other closely-related taxa(Che et al.,2007;Pyron and Wiens,2011;Oliver et al.,2015).China has a large diversity of ranid frogs,with 146 speciesbelonging to 7 genera,and48 species belonging to 7 genera in the southern province of Yunnan(AmphibiaChina,2022).

Taxonomic revision of genus Rohanixalus(Anura:Rhacophoridae)in China with description of one new species

DEAR EDITOR,Based upon morphological and molecular evidence,the authors revised the genus Rohanixalus Biju,Garg,Gokulakrishnan,Chandrakasan,Thammachoti,Ren,Gopika,Bisht,Hamidy and Shouche,2020(Anura:Rhacophoridae)in China through describing one new species,adding one species to the fauna(R.shyamrupus)and supplementing data on one species(Rohanixalus hansenae;Supplementary Materials).

Interspecific differences and ecological correlations between scale number and skin structure in freshwater fishes

Fish skin is mainly composed of the epidermis,dermis,and its derivative scales.There is a wide diversity in scale number in fishes,but the diversity of skin structure lacks systematic histological comparison.This research aimed to improve our understanding of the functional relationship between the scale number and the skin structure in freshwater fishes and to determine which ecological factors affect the scale number and skin structure.First,we presented a method to quantify skin structure in fish and histologically quantified the skin structure of 54 freshwater fishes.Second,we collected the scale number and habitat information of 509 Cyprinidae fishes in China and explored which ecological factors were related to their scale number.Third,common carp and scaleless carp were used as models to study the effects of scale loss on swimming.We found a strong negative correlation between scale thickness and scale number.The main factor affecting the skin structure of fishes was the species’water column position,and the skin of benthic fishes was the most well-developed(thicker skin layers(dermis,epidermis)or more/larger goblet cells and club cells).The scale number was related to two factors,namely,temperature and water column position,and cold,benthic and pelagic adaptation may have contributed to increased scale numbers.Only in benthic fishes,the more well-developed their skin,the more scales.In common carp,scale loss did not affect its swimming performance.In summary,we suggest that there is a rich diversity of skin structure in freshwater fishes,and the scales of fish with well-developed skin tend to degenerate(greater number/smaller size/thinner,or even disappear),but the skin of fish with degenerated scales is not necessarily well developed.

A New Species of Cyrtodactylus(Squamata:Gekkonidae)from the Karst Forests of Daweishan National Nature Reserve,Yunnan,China

Cyrtodactylus geckos are one of the most speciose and diverse groups of extant lizards known,distributed throughout the Asian and Pacific realms.Using molecular phylogenetic methods and supporting morphological data,we describe a new species of Cyrtodactylus in Daweishan National Nature Reserve,Yunnan Province,China.Cyrtodactylus hekouensis sp.nov.can be morphologically distinguished from its nearby congeners by the following characters:maximum SVL 92.3 mm and TL 98.5 mm;11-12 supralabials;11-12 infralabials;36-57 scale rows between the fifth supralabials;10-13 dorsal tubercles rows;3 postnasals on blunt and smooth front snout;precloacalfemoral pores in a continuous series of 33-39(females with pitted scales)located under vent/cloaca and thighs in both sexes;precloacal groove absent;3/3 postcloacal tubercles;subdigital lamellae under the fourth finger 21 or 22,under the fourth toe 20-23;smooth midbody with smooth venter and tuberculate dorsal scale rows,tubercles from head to tail base;dorsal transverse patterns are generally large,bilaterally symmetrical.The results of the phylogenetic analysis recover specimens of this new species as sister to a clade containing C.wayakonei and C.martini.Uncorrected pairwise intraspecific distances were<1%,and distances between our new species and other Cyrtodactylus species from nearby countries ranged from 14.2%to 26.8%.

Single-cell analyses identify distinct and intermediate states of zebrafish pancreatic islet development

Pancreatic endocrine islets are vital for glucose homeostasis. However, the islet developmental trajectory and its regulatory network are not well understood. To define the features of these specification and differentiation processes, we isolated individual islet cells from TgBAC(neurod1:EGFP) transgenic zebrafish and analyzed islet developmental dynamics across four different embryonic stages using a single-cell RNA-seq strategy. We identified proliferative endocrine progenitors, which could be further categorized by different cell cycle phases with the G1/S subpopulation displaying a distinct differentiation potential. We identified endocrine precursors, a heterogeneous intermediate-state population consisting of lineage-primed alpha, beta and delta cells that were characterized by the expression of lineage-specific transcription factors and relatively low expression of terminally differentiation markers. The terminally differentiated alpha, beta, and delta cells displayed stage-dependent differentiation states, which were related to their functional maturation. Our data unveiled distinct states, events and molecular features during the islet developmental transition, and provided resources to comprehensively understand the lineage hierarchy of islet development at the single-cell level.

Vertebral deformities in hatchery-reared and wild-caught juvenile Japanese flounder,Paralichthys olivaceus

The present study compared vertebral deformities of hatchery-reared and wild-caught juvenile Japanese flounder,Paralichthys olivaceus.A total of 362 hatchery-reared flounder(total length 122.5-155.8 mm) were collected from three commercial hatcheries located in Yantai,East China,and 89 wild fish(total length 124.7-161.3 mm) were caught off Yangma Island near Yantai City(37°27′N,121°36′E).All the fish were dissected,photographed,and images of the axial skeleton were examined for vertebral deformities.Compared with wild-caught flounder in which no deformed vertebrae were detected,48(13.3%) hatcheryreared fish had deformed vertebrae.The deformities were classified as compression,compression-ankylosis,and dislocation-ankylosis.The vertebral deformities were mainly localized between post-cranial vertebra 1and 3,with vertebrae number 1 as the most commonly deformed.The causative factors leading to vertebral deformities in reared Japanese flounder may be related to unfavorable temperature conditions,inflammation,damage,or rupture to the intervertebral ligaments under rearing conditions.Furthermore,no significant difference in the total number of vertebral bodies was observed between wild-caught(38.8±0.4) and hatchery-reared flounder(38.l±0.9)(P>0.05).However,the number of vertebral bodies of hatchery-reared and wild-caught flounder ranged from 35 to 39 and from 38 to 39,respectively.

A New Cyrtodactylus Species(Reptila:Gekkonidae)from Nan Province,Northern Thailand

Here,a new species of bent-toed gecko,Cyrtodactylus phukhaensis sp.nov.,is described from Doi Phu Kha,Nan province,Thailand based on molecular and morphological evidence.A phylogeny based on NADH dehydrogenase subunit 2(ND2)and its flanking tRNAs places the new species in the chauquangensis group as a sister taxon to Cyrtodact ylus wayakonei.The new species can be differentiated from other members of the chauquangensis group by having a unique combina tion of 7 or 8 suprala bials,23-28 longitudinal rows of dorsal tubercles,8-10 infrala bials,9 femoral pores,7 precloacal pores,40-47 ventral scales,and a lack of bands crossingthe temporal area.In addition,results from a chromosome study of C.phukhaensis sp.nov.showed that the new species has a diploid chromosome number of 40 with a fundamental number of 46.The formula of the karyotype was as follows:2n(40)=2m+4sm+34t.Our findings suggest that further studies of Cyrtodactylus biodiversity in northern Thailand are needed.

Ccdc57 is required for straightening the body axis by regulating ciliary motility in the brain ventricle of zebrafish

Recently,cilia defects have been proposed to contribute to scoliosis.Here,we demonstrate that coiled-coil domain-containing 57(Ccdc57)plays an essential role in straightening the body axis of zebrafish by regulating ciliary beating in the brain ventricle(BV).Zygotic ccdc57(Zccdc57)mutant zebrafish developes scoliosis without significant changes in their bone density and calcification,and the maternal-zygotic ccdc57(MZccdc57)mutant embryos display curved bodies since the long-pec stage.The expression of ccdc57 is enriched in ciliated tissues and immunofluorescence analysis reveals colocalization of Ccdc57-HA with acetylated a-tubulin,implicating it in having a role in ciliary function.Further examination reveals that it is the coordinated cilia beating of multiple cilia bundles(MCB)in the MZccdc57 mutant embryos that is affected at 48 hours post fertilization,when the compromised cerebrospinal fluid flow and curved body axis have already occurred.Either ccdc57 m RNA injection or epinephrine treatment reverses the spinal curvature in MZccdc57 mutant larvae from ventrally curly to straight or even dorsally curly and significantly upregulates urotensin signaling.This study reveals the role of ccdc57 in maintaining coordinated cilia beating of MCB in the BV.

Alterations in immune cell heterogeneities in the brain of aged zebrafish using single-cell resolution

Immunocytes,including the microglia,are crucial in the neurodegenerative process in old people.However,the understanding regarding microglia heterogeneity and other involved immunocytes remains elusive.We analyzed 26,456 immunocytes from 12-and 26-month-old zebrafish brains at single-cell resolution.Microglia and T lymphocytes were detected in the brain at both time points.Two types of microglia were annotated,namely,ac+microglia and xr+microglia,which were clustered into subsets 1,2,3,4,5,and subsets 6,7,8,9,respectively.Diversified microglia predominated the adult brains and cooperated with T cells to perform the functions of immune response and neuronal nutrition.We validated the specific microglia markers.The novel transgenic lines,Tg(lgals3bpb:e GFP)and Tg(apoc1:e GFP),were created,which faithfully labeled ac+microglia and served as valuable labeling tools.However,the microglia population reduced while T cells of six subtypes intriguingly increased to serve as the primary immune cells in aged brains.Unlike in 12-month-old brains,T cells,together with microglia,exhibited a coordinated signature of inflammation in the 26-month-old brains.Our findings revealed the immunocytes atlas in aged zebrafish brains.It implied the involvement of microglia and T cells in the progression of neurodegeneration in aging.

Ribosome biogenesis protein Urb1 acts downstream of mTOR complex 1 to modulate digestive organ development in zebrafish

Ribosome biogenesis is essential for the cell growth and division. Disruptions in ribosome biogenesis result in developmental defects and a group of diseases, known as ribosomopathies. Here, we report a mutation in zebrafish urb1, which encodes an essential ribosome biogenesis protein. The urb1 cq31 mutant exhibits hypoplastic digestive organs, which is caused by impaired cell proliferation with the differentiation of digestive organ progenitors unaffected. Knockdown of mtor or raptor leads to similar hypoplastic phenotypes and reduced expression of urb1 in the digestive organs. Overexpression of Urb1 results in overgrowth of digestive organs, and can efficiently rescue the hypoplastic liver and pancreas in the mtor and raptor morphants. Reduced syntheses of free ribosomal subunits and impaired assembly of polysomes are observed in the urb1 mutant as well as in the mtor and raptor morphants, which can be rescued by the Urb1 overexpression. These data demonstrate that Urb1 plays an important role in governing ribosome biogenesis and protein synthesis downstream of mammalian/mechanistic target of rapamycin complex 1（mTORC1）, thus regulating the development of digestive organs. Our study indicates the requirement of hyperactive protein synthesis for the digestive organ development.

Using zebrafish as the model organism to understand organ regeneration

The limited regenerative capacity of several organs, such as central nervous system(CNS), heart and limb in mammals makes related major diseases quite difficult to recover. Therefore, dissection of the cellular and molecular mechanisms underlying organ regeneration is of great scientific and clinical interests. Tremendous progression has already been made after extensive investigations using several model organisms for decades. Unfortunately, distance to the final achievement of the goal still remains. Recently, zebrafish became a popular model organism for the deep understanding of regeneration based on its powerful regenerative capacity, in particular the organs that are limitedly regenerated in mammals. Additionally, zebrafish are endowed with other advantages good for the study of organ regeneration. This review summarizes the recent progress in the study of zebrafish organ regeneration, in particular regeneration of fin, heart, CNS, and liver as the representatives. We also discuss reasons of the reduced regenerative capacity in higher vertebrate, the roles of inflammation during regeneration, and the difference between organogenesis and regeneration.