Metagenomic analysis reveals hidden links between gut microbes and habitat adaptation among cave and surface dwelling Sinocyclocheilus species

Intestinal microbes are closely related to vital host functions such as digestion and nutrient absorption,which play important roles in enhancing host adaptability.As a natural“laboratory”,caves provide an outstanding model for understanding the significance of gut microbes and feeding habits in the habitat adaptability of hosts.However,research on the relationship between gut microbes,feeding habits,and the adaptability of troglobites remains insufficient.In this study,we compared the characteristics of the intestinal microbes of Sinocyclocheilus cavefish and surface fish and further established the relationship between intestinal and habitat microbes.Furthermore,we conducted environmental DNA(eDNA)(metabarcoding)analysis of environmental samples to clarify the composition of potential food resources in the habitats of the Sinocyclocheilus cavefish and surface fish.Results showed that the structure of the Sinocyclocheilus gut microbes was more related to ecological type(habitat type)than phylogenetic relationships.While horizontal transfer of habitat microbes was a source of gut microbes,hosts also showed strong selection for inherent microbes as dominant microorganisms.Differences in the composition and structure of gut microbes,especially dominant microbes,may enhance the adaptability of the two Sinocyclocheilus fish types from the perspectives of food intake,nutrient utilization,and harmful substance metabolism,suggesting that food resources,predation patterns,intestinal flora,digestive and absorptive capacity,and feeding habits and preferences are linked to habitat adaptability.These results should facilitate our understanding of the significance of fish gut microbes to habitat adaptation and provide a new perspective for studying the adaptive mechanisms of cavefish.

Three-dimensional morphology of the Sinocyclocheilus hyalinus(Cypriniformes: Cyprinidae) horn based on synchrotron X-ray microtomography

Sinocyclocheilus is a cave-dwelling cyprinid genus endemic to southwest China. Several species possess a conspicuous horn on their head, which has been suggested as a constructive troglomorphic trait but lacks substantial evidence. We used non- invasive, high spatial resolution synchrotron X-ray microtomography to investigate the three-dimensional （3D） morphology of the horn of Sinocyclocheilus hyalinus, one of eight such troglobiotic species. 3D renderings demonstrated the osteological components, which were comprised of a rear wall comprised of the supraoccipital bone, a remaining frontal wall with numerous fenestrae, and the bottom continuous with the parietal and epiotic. A horn cavity occurred within the horn. The fenestrae in the frontal wall were continuous in the horn cavity and showed elaborate channeling, and were, connected to the cranial cavity by soft tissue. We tentatively called this configuration the ＂otocornual connection＂ due to its anatomic and putative functional similarity to the otolateralic connection in clupeids and loricariids, which provide an indirect pathway to enhance perception of underwater sound signals. This study provides a functional morphology context for further histological and physiological investigations of such horn structures in Sinocyclocheilus cavefish, and we suggest that the horn might enhance acoustic perception to compensate for visual loss in subterranean life, which warrants future physiological examination as lab-reared S. hyalinus become available.

Three-dimensional morphology of the Sinocyclocheilus hyalinus(Cypriniformes:Cyprinidae)horn based on synchrotron X-ray microtomography

Sinocyclocheilus is a cave-dwelling cyprinid genus endemic to southwest China.Several species possess a conspicuous horn on their head,which has been suggested as a constructive troglomorphic trait but lacks substantial evidence.We used non-invasive,high spatial resolution synchrotron X-ray microtomography to investigate the three-dimensional(3D)morphology of the horn of Sinocyclocheilus hyalinus,one of eight such troglobiotic species.3D renderings demonstrated the osteological components,which were comprised of a rear wall comprised of the supraoccipital bone,a remaining frontal wall with numerous fenestrae,and the bottom continuous with the parietal and epiotic.A horn cavity occurred within the horn.The fenestrae in the frontal wall were continuous in the horn cavity and showed elaborate channeling,and were,connected to the cranial cavity by soft tissue.We tentatively called this configuration the“otocornual connection”due to its anatomic and putative functional similarity to the otolateralic connection in clupeids and loricariids,which provide an indirect pathway to enhance perception of underwater sound signals.This study provides a functional morphology context for further histological and physiological investigations of such horn structures in Sinocyclocheilus cavefish,and we suggest that the horn might enhance acoustic perception to compensate for visual loss in subterranean life,which warrants future physiological examination as lab-reared S.hyalinus become available.

Molecular phylogeny and historical biogeography of the cave fish genus Sinocyclocheilus(Cypriniformes:Cyprinidae)in southwest China

Modern accumulations of genetic data offer unprecedented opportunities for understanding the systematic classification and origins of specific groups of organisms.The genus Sinocyclocheilus is among the most cave abundant genera in Cyprinidae,with 76 recognized species,belonging to 4 species groups.Recent phylogenetic studies have shown that the classification of species groups within the genus Sinocyclocheilus remains controversial.In this study,we constructed a sequence supermatrix of 26 species from 4 species groups of the genus Sinocyclocheilus using the mitochondrial genome to reveal phylogenetic relationships,historical biogeography and patterns of species diversification in the genus Sinocyclocheilus.Phylogenetic analysis strongly supports the monophyletic groups of the 3 species groups(S.jii,S.cyphotergous,and S.tingi groups)except the S.angularis group.Phylogenetic analysis showed that S.anshuiensis and S.microphthalmus,which were recognized as numbers of S.angularis group,formed a strongly supported independent clade.Therefore,we propose a new species group,the S.microphthalmus group,which contains S.anshuiensis and S.microphthalmus.Biogeographic reconstruction suggests that the living Sinocyclocheilus may have originated in north-central Guangxi at the late Eocene and dispersed outward after a vicariance at 32.31 Million years ago(Ma).Early diversification is focused on the late Oligocene(ca.25 Ma),which is related to the second uplift of the Qinghai−Tibetan Plateau and the lateral extrusion of the Indochina at the Oligocene/Miocene boundary.Our results suggest that 2 uplifts of the Qinghai−Tibetan Plateau and climate change in the Miocene may have influenced the diversification of the Sinocyclocheilus lineage.

滇池金线鲃、西畴金线鲃及其杂交F1代的形态分析

根据传统形态度量学和框架结构形态学法测量了15月龄的杂交F1代(滇池金线鲃Sinocyclocheilus grahami♀×西畴金线鲃S.xichouensis♂)以及滇池金线鲃(S.grahami)和西畴金线鲃(S.xichouensis)的10项传统可量性状数据和24项框架结构数据以及鳍条、侧线鳞和脊椎骨等可数性状数据。并用方差分析和多元统计分析法(聚类分析,判别分析和主成分分析)对三者的传统可量性状和框架结构数据进行分析。结果显示:三者的可数性状差别不大,杂交F1代体表花纹与双亲相比发生一定变化。聚类分析结果表明,杂交F1代的形态与母本滇池金线鲃形态较为相似。采用逐步判别分析方法建立了3种鱼的判别函数,判别准确率较高(90%,90%,100%)。主成分分析结果及主成分散点图显示:三者均有重叠区域不容易从形态上清楚分开,但杂交F1代明显更接近母本滇池金线鲃。结合方差分析和主成份分析发现杂交F1代与双亲不同的性状主要集中在头部和尾部。

冷休克法诱导乌蒙山金线鲃三倍体及其生长分析

为了研究冷休克法诱导乌蒙山金线鲃(Sinocyclocheilus wumengshanensis)三倍体的方法,试验分析了不同参数的诱导效果及三倍体生长特征。结果显示:冷休克2~3℃、受精后5 min开始诱导、诱导25 min可获得受精率64.55%、孵化率60.61%、畸形率26.54%,三倍体率100%。诱导组平均孵化时间为4 d 14 h~6 d 19 h,低于对照组7 d 2 h。诱导组初孵仔鱼畸形率为17.19%~71.25%。受精后3 min与5 min开始诱导、持续诱导30 min,仔鱼死亡率最大分别为24.70%与39.38%。3~60日龄三倍体与二倍体的体重、体长均无显著性差异,180日龄三倍体的体长与体重分别为(74.00±1.58)mm、(4.46±0.30)g,分别是二倍体的1.09倍与1.27倍,体重显著大于二倍体。

滇池金线鲃、西畴金线鲃及其杂交F1代抵抗小瓜虫与车轮虫能力的探究

用小瓜虫（Ichthyophthirius multifiliis）和车轮虫（Trichodina spp.）对滇池金线鲃（Sinocyclocheilus grahami）和西畴金线鲃（S.xichouensis）及其杂交F1代分别进行了两次人工感染实验。18月龄的杂交F1代、滇池金线鲃（Sinocyclocheilus grahami）和西畴金线鲃（S.xichouensis）感染小瓜虫后平均死亡率分别为（50.00±11）%、（56.70±23）%、（80.0±18）%。经方差检验杂交F1代死亡率显著低于父本西畴金线鲃。6月龄杂交F1代、滇池金线鲃、西畴金线鲃感染车轮虫后的平均死亡率分别为（40.56±13）%、（56.11±5）%、（42.78±13）%。经方差检验杂交F1代死亡率显著低于母本滇池金线鲃。在感染小瓜虫和车轮虫后,三种鱼的死亡量均呈现激增后又显著回落的趋势,死亡高峰在第4天至第7天。本次感染实验的小瓜虫和车轮虫主要分布于病鱼体表：躯干分布最多57%-79%,其次为鳍12%-28%,头部分布较少4%-15%。与本研究中车轮虫几乎于鳃丝无分布（0%-3%）不同,有大约10%-20%的小瓜虫分布于鳃丝上。

Brain of the blind:transcriptomics of the golden-line cavefish brain

The genus Sinocyclocheilus (golden-line barbel) includes 25species of cave-dwelling blind fish (cavefish)and more than 30 surface-dwelling species with normal vision.Cave environments are dark and generally nutrient-poor with few predators.Cavefish of several genera evolved convergent morphological adaptations in visual,pigmentation,brain,olfactory,and digestive systems. We compared brain morphology and gene expression patterns in a cavefish Sinocyclocheilus anophthalmus with those of a closely related surface-dwelling species S.angustiporus.Results showed that cavefish have a longer olfactory tract and a much smaller optic tectum than surface fish.Transcriptomics by RNA-seq revealed that many genes upregulated in cavefish are related to lysosomes and the degradation and metabolism of proteins,amino acids,and lipids.Genes down- regulated in cavefish tended to involve "activation of gene expression in cholesterol biosynthesis" and cholesterol degradation in the brain.Genes encoding Srebfs (sterol regulatory element- binding transcription factors)and Srebf targets,including enzymes in cholesterol synthesis,were downregulated in cavefish brains compared with surface fish brains.The gene encoding Cyp46a1, which eliminates cholesterol from the brain,was also downregulated in cavefish brains,while the total level of cholesterol in the brain remained unchanged.Cavefish brains misexpressed several genes encoding proteins in the hypothalamus-pituitary axis,including Trh,Sst,Crh,Pomc,and Mc4r.These results suggest that the rate of lipid biosynthesis and breakdown may both be depressed in golden-line cavefish brains but that the lysosome recycling rate may be increased in cavefish;properties that might be related to differences in nutrient availability in caves.