The role of dietary chromium supplementation in relieving heat stress of juvenile blunt snout bream Megalobrama amblycephala.

The present study assessed the role of dietary chromium (Cr) supplementation in relieving heat stress (HS) of juvenile blunt snout bream Megalobrama amblycephala. The supplemented Cr contents by chromium picolinate (Cr-Pic) was 0 mg/kg (control group), 0.4 mg/kg, 1.6 mg/kg and 12.0 mg/kg, respectively. The fish continued to be fed four diets at suitable temperatures (26 °C) for 2 weeks, and then the temperature was then heated up to 33 °C through thermo-regulated system. The results showed that Cr supplementation had no significant effect on the immune indices and antioxidant indices before HS (P > 0.05). However, Cr supplementation played an important role in relieving HS. After HS, compared with the control group, 1.6 mg/kg and 12.0 mg/kg Cr supplementation groups significantly lowered the plasma glucose level and aspartate transaminase (AST) activity (P < 0.05), and 0.4 mg/kg and 1.6 mg/kg Cr supplementation groups significantly lowered alanine aminotransferase (ALT) activity (P < 0.05). 0.4 mg/kg and 1.6 mg/kg supplementation groups significantly improved hepatic total superoxide dismutase (T-SOD) activity (P < 0.05). Furthermore, 0.4mg/kg-12.0 mg/kg Cr supplementation groups significantly improved the activities of hepatic glutathione peroxidase (GPx) and catalase (CAT) and lowered hepatic malondialdehyde (MDA) level in liver (P < 0.05). The mRNA levels of hepatic copper zinc superoxide dismutase (Cu/Zn-SOD), CAT and GPx were significantly improved in 0.4mg/kg-12.0 mg/kg supplementation Cr groups (P < 0.05), however, there was no significant variation of hepatic manganese superoxide dismutase (Mn-SOD) mRNA levels under different levels of supplementation (P > 0.05). Significantly lower mRNA levels of hepatic pro-inflammatory cytokines observed in 0.4mg/kg-12.0 mg/kg Cr supplementation groups including tumour necrosis factor-α (TNF-α), interleukin 1ß (IL-1ß) and interleukin 8 (IL-8) (P < 0.05), and 0.4mg/kg-12.0 mg/kg Cr supplementation significantly improved the relative expressions of hepatic heat shock protein 70 (HSP70) and heat shock protein 90 (HSP90) (P < 0.05). The present study indicated that dietary Cr supplementation might have no significant effect on immune capacity and antioxidant capacity under normal physiological conditions, whereas it played an important role in relieving HS.

Endogenic upregulations of HIF/VEGF signaling pathway genes promote air breathing organ angiogenesis in bimodal respiration fish.

Air-breathing has evolved independently serval times with a variety of air-breathing organs (ABOs) in fish. The physiology of the air-breathing in bimodal respiration fish has been well understood, while studies on molecular mechanisms of the character are very limited. In the present study, we first determined the gill indexes of 110 fish species including 25 and 85 kinds of bimodal respiration fishes and non-air-breathing fishes, respectively. Then combined with histological observations of gills and ABOs/non-ABOs in three bimodal respiration fishes and two non-air breathing fishes, we found that the bimodal respiration fish was always of a degeneration gill and a well-vascularized ABO. Meanwhile, a comparative transcriptome analysis of posterior intestines, namely a well vascularized ABO in Misgurnus anguillicaudatus and a non-ABO in Leptobotia elongata, was performed to expound molecular variations of the air-breathing character. A total of 5,003 orthologous genes were identified. Among them, 1,189 orthologous genes were differentially expressed, which were enriched in 14 KEGG pathways. More specially, the expressions of hemoglobin genes and various HIF/VEGF signaling pathway genes were obviously upregulated in the ABO of M. anguillicaudatus. Moreover, we found that HIF-1α, VEGFAa, and MAP2K1 were co-expressed dramatically higher in ABOs of bimodal respiration fishes than those of non-ABOs of non-air-breathing fishes. These results indicated that the HIF/VEGF pathway played an important role in ABO angiogenesis/formation to promote fish to do aerial respiration. This study will contribute to our understanding of molecular mechanisms of air-breathing in fish.

Fibronectin 1B Gene Plays an Important Role in Loach Barbel Air-Breathing.

Loach (Misgurnus anguillicaudatus) is well known to perform air-breathing through the posterior intestine and skin. However, we find here for the first time a unique central vascular structure in the loach barbel, with a blood-gas diffusion distance as short as that of the posterior intestine. Under acute hypoxia, the distance of loach barbels became significantly shorter. Moreover, barbel removal significantly decreased air-breathing frequency of the loach. These findings imply that the barbel is another air-breathing organ of the loach. For further investigation of loach barbel air-breathing, a transcriptome analysis of barbels with air exposure treatment was performed. A total of 2546 differentially expressed genes (DEGs) between the T-XU (air exposure) and C-XU (control) group were identified, and 13 key DEGs related to barbel air-breathing were screened out. On this foundation, sequence, expression, and location analysis results indicated an important positive role of fibronectin 1b (fn1b) in loach barbel air-breathing. We further generated an fn1b-depletion loach (MT for short) using the CRISPR/Cas9 technique. It was indicated that depletion of fn1b could weaker barbel air-breathing ability. In conclusion, due to nonlethal and regenerative characteristics, the loach barbel, a newly discovered and fn1b-related fish air-breathing organ, can be a good model for fish air-breathing research.

The fast and the tendinous? Locomotor modifications of the caudal peduncle in Gila spp. from the American Southwest.

In the American Southwest, the fishes within the genus Gila evolved in an environment with seasonal rainstorms that caused stochastic flooding. Some species within this genus, such as bonytail (Gila elegans), possess locomotor morphologies that are similar to those seen in high-performance swimmers such as tuna and lamnid sharks. These shared features include a shallow caudal peduncle, lunate tail, and mechanisms to transmit force from the anterior musculature to the tail fin. We compared the skeletal anatomy of the caudal region of bonytail to roundtail chub (Gila robusta) and humpback chub (Gila cypha) to determine which vertebral elements have been modified to create a shallow peduncle. We also tested the tensile strength of the red (slow oxidative) axial muscle by performing a standard stress test. If the muscle can withstand a large load, this suggests it may play a tendon-like role in transmitting force from the anterior muscle to the hypural plate of the tail. Lastly, we measured the collagen content of the red axial muscle (visualized using serial sections and Masson's trichrome stain) to determine if increased tensile strength is associated with increased collagen content. We found bonytail caudal peduncles are characterized by acute vertebral spines and have red axial muscle that can resist tearing under tension. Roundtail chub peduncles are characterized by relatively more obtuse angles and the red muscle tears easily under tension. Humpback chub possess an intermediate morphology, with relatively obtuse vertebral spine angles and the red muscle can resist tearing under tension. Bonytail have increased collagen content in posterior red axial muscle compared to the anterior musculature also suggesting a tendon-like role of the posterior red muscle. In combination with previous studies of swimming performance, our findings suggest that the axial musculature of bonytail may play a role in transmitting force directly to the shallow peduncle in a manner similar to that of the great lateral tendon of scombrids.

No evidence of physiological declines with age in an extremely long-lived fish.

Although the pace of senescence varies considerably, the physiological systems that contribute to different patterns of senescence are not well understood, especially in long-lived vertebrates. Long-lived bony fish (i.e., Class Osteichthyes) are a particularly useful model for studies of senescence because they can readily be aged and exhibit some of the longest lifespans among vertebrates. In this study we examined the potential relationship between age and multiple physiological systems including: stress levels, immune function, and telomere length in individuals ranging in age from 2 to 99 years old in bigmouth buffalo (Ictiobus cyprinellus), the oldest known freshwater teleost fish. Contrary to expectation, we did not find any evidence for age-related declines in these physiological systems. Instead, older fish appeared to be less stressed and had greater immunity than younger fish, suggesting age-related improvements rather than declines in these systems. There was no significant effect of age on telomeres, but individuals that may be more stressed had shorter telomeres. Taken together, these findings suggest that bigmouth buffalo exhibit negligible senescence in multiple physiological systems despite living for nearly a century.

Effects of Pesticide Use on the Distributions of Grey Herons (Ardea cinerea) and Great Egrets (Ardea alba) in Rice Fields of the Republic of Korea.

Pesticide use is known to have a negative impact on the habitat use of birds. The decomposition of residual pesticides causes a drastic decrease in the biomass of wildlife food sources, indirectly affecting the ecosystem. In this study, we investigated the effects of pesticide use on the distributions of grey herons (Ardea cinerea) and great egrets (Ardea alba) in rice fields in the Republic of Korea. From early May to mid-June in 2015 and 2016, we recorded the abundance of these birds and their prey (loaches, other fish, tadpoles, and benthic invertebrates) and investigated their dependence on rice agricultural practices (eco-friendly vs conventional) and field types (harrowed, plowed, or transplanted). We found that both grey herons and great egrets preferentially used transplanted fields. Grey herons were observed more in conventional rice fields, while great egrets were observed more in eco-friendly rice fields. This may be driven by the distribution of their preferred prey types; we observed a higher density of tadpoles (the prey type favored by grey herons) in conventional fields and a higher density of loaches (the preferred prey of great egrets) in eco-friendly fields. Pesticides drive these patterns both directly and indirectly; pesticide use in conventional rice fields directly suppresses the abundance of loaches, which frees the tadpole population from predation pressures and indirectly boosts their abundance in conventional rice fields. Our findings suggest that the distributions of grey herons and great egrets vary depending on food availability and are directly and indirectly influenced by pesticide use.

Yunnanilus chuanheensis, a new loach species (Cypriniformes: Nemacheilidae) from the upper Lixianjiang River in Yunnan, China.

A new loach species (Cypriniformes: Nemacheilidae: Yunnanilini), Yunnanilus chuanheensis sp. nov., was caught from Chuanhe in the upper reaches of the Lixianjiang River, a tributary of the Red River in Nanjian County, Yunnan Province, China. This species is a member of the traditional Y. pleurotaenia species group based on the presence of a lateral line and cephalic lateral-line canals. It can be distinguished from other species in the Y. pleurotaenia species group by the following characters: lips without papillae, anterior and posterior nostrils separated, whole body scaled, eye diameter smaller than interorbital width, outer gill raker absent on first gill arch, eye diameter greater than 18% of head length, 10-11 inner gill rakers on first gill arch, and lateral line not extending to vertical through dorsal fin insertion. To the best of our knowledge, this is the first Yunnanilus species recorded from the Red River drainage.

Development of a Mucus Gland Bioreactor in Loach Paramisgurnus dabryanus.

Most currently available bioreactors have some defects in the expression, activity, or purification of target protein and peptide molecules, whereas the mucus gland of fish can overcome these defects to become a novel bioreactor for the biopharmaceutical industry. In this study, we have evaluated the practicability of developing a mucus gland bioreactor in loach (Paramisgurnus dabryanus). A transgenic construct pT2-krt8-IFN1 was obtained by subcloning the promoter of zebrafish keratin 8 gene and the type I interferon (IFN1) cDNA of grass carp into the SB transposon. The IFN1 expressed in CIK cells exhibited an antiviral activity against the replication of GCRV873 and activated two genes downstream of JAK-STAT signaling pathway. A transgenic loach line was then generated by microinjection of the pT2-krt8-IFN1 plasmids and in vitro synthesized capped SB11 mRNA. Southern blots indicated that a single copy of IFN1 gene was stably integrated into the genome of transgenic loach. The expression of grass carp IFN1 in transgenic loaches was detected with RT-PCR and Western blots. About 0.0825 µg of grass carp IFN1 was detected in 20 µL mucus from transgenic loaches. At a viral titer of 1 × 103 PFU/mL, plaque numbers on plates containing mucus from transgenic loaches reduced by 18% in comparison with those of the control, indicating that mucus of IFN1-transgenic loaches exhibited an antiviral activity. Thus, we have successfully created a mucus gland bioreactor that has great potential for the production of various proteins and peptides.

Effect of hypoxia and air-breathing restricted on respiratory physiology of air-breathing loach (Paramisgurnus dabryanus).

This aim of this study was to determine the respiratory physiology response in the gill and gut of Paramisgurnus dabryanus under different breathing treatment patterns. The experimental design included the following three conditions: a control group without any stress treatments, an inhibited group with intestinal respiration inhibited, and an air-exposed group with gill respiration inhibited. The results indicated that the total static metabolic rate in the air-exposed group (188.92 ± 13.67 mg h-1 kg-1) was much higher than that of the other group after 7 days, decreased significantly after the first day of recovery (81.64 ± 7.85 mg h-1 kg-1). The air metabolic rate in the air-exposed group increased significantly after 7 days (P < 0.05). There was no significant difference among the groups. Histological observation on the gill and hindgut of P. dabryanus showed that the gill filament area of inhibited group became larger, while the gill structure of air exposed group showed some damage. The number of capillariesin the hindgut mucosal epithelial in air-exposed group showed a rapidly increase (P < 0.05). Likewise, the gas diffusion distance (1.24 ± 0.36 µm) became significantly shorter (P < 0.05). Lactate dehydrogenase activity of gill in the air-exposed group (846.68 ± 88.78 U mg-1 protein) significantly increased after 7 days whereas succinate dehydrogenase (1.02 ± 0.21 U mg-1 protein) and Na+/K+ ATPase (0.57 ± 0.20 U mg-1 protein) activity decreased significantly (P < 0.05). However, there was no significant change in the hindgut. After recovery, there was no significant difference in lactate dehydrogenase, succinate dehydrogenase, and Na+/K+ ATPase activity in the gill or hindgut in groups. P. dabryanus had a high viability in air-exposed condition. When recovery occurred under normoxic conditions, the physical levels of respiration returned back to the normal level quickly.

Taxonomic diversity pattern and composition of fish species in the upper reaches of Ganjiang River, Jiangxi, China.

Maintaining fish diversity is essential for environmental protection. To characterize the fish composition of the tributaries and mainstream in the upper reaches of the Ganjiang River, we identified seventy-five species of 15 families in 14 sampling sites. These data were analyzed using the inclusion index at the taxonomic level (TINCLi), the importance value index (IVI), the taxonomic diversity indices (Δ+ and Λ+), cluster analysis and non-metric multidimensional scaling (nMDS). The results showed that the most common and dominant order was the Cypriniformes, represented most frequently by Hemiculter leucisculus among the sample sites. Most fishes were omnivorous, mountain stream or settled fish that lay sinking or viscid eggs. Most sites showed a trend of more discrete distribution from high latitude to low latitude. According to the taxonomic diversity indices, the fish taxonomic composition in the upper reaches of Ganjiang River is uneven. The Bray-Curtis resemblance matrix and nMDS showed that the habitats of the Ganjiang River were divided into four areas. The results will provide information needed for freshwater fish resource protection in the upper reaches of the Ganjiang River.

Gene Flow and Individual Relatedness Suggest Population Spatial Connectivity of Sinogastromyzon sichangensis (Cypriniformes: Balitoridae) in the Chishui River, China.

Sinogastromyzon sichangensis is a hillstream loach endemic to the upper Yangtze River, China. It is unclear whether this fish lives in a very restricted area or may be dispersed over a long distance. In the present study spatial connectivity of populations of S. sichangensis was investigated based on 343 individuals collected from 12 sites of Chishui River and employing 22 microsatellite loci. The results of genetic diversity analysis showed that observed heterozygosity (HO) and polymorphism information content (PIC) ranged from 0.5653 to 0.6999 and 0.8513 to 0.8819, respectively. Population structure analysis suggested that S. sichangensis had an unclear genetic structure. AMOVA showed that 69.36% of genetic variation was attributed to differentiation within individuals and all the pairwise genetic differentiation indices (FST) were low (mean FST = 0.0344), indicating weak differentiation among these populations. Estimation of gene flow showed frequent flow among populations, and contemporary levels (mean contemporary migration rate, mc = 0.0131) were approximately equal to historical levels (mean historical migration rate, mh = 0.0147). Individual relatedness analysis revealed a high level of sibship within and among different populations. The frequent gene flow and widespread sibship were due to S. sichangensis laying drifting eggs which travel for a long distance until hatching, after which the juveniles or adults migrate upstream. The results of unclear geographic structure and frequent exchange also indicate that it is necessary to decrease the negative impacts of anthropogenic activities on the connectivity of rivers to protect the migration routes of S. sichangensis.

Comparison of artificial spawning effectiveness of hCG, CPH and GnRHa in combination with dopamine inhibitors in a wild strain of ide Leuciscus idus (L.) in hatchery conditions.

Optimization of artificial reproduction, by increasing the survival rate of embryos and hatching rate, is of major importance for reducing genetic diversity, especially in fish captured from their natural habitat that subsequently spawn in hatcheries. The artificial reproduction of ide, Leuciscus idus (L.) was conducted in controlled conditions. The spawning agents included: different doses of human chorionic gonadotropin (hCG) that was compared with common carp pituitary homogenate (CPH), Ovopel - a commercial agent for induction of spawning that contains mammalian gonadotropin releasing hormone analogue (mGnRHa) with dopamine antagonists (DA): metoclopramide (MET) and Ovaprim - a commercial agent containing salmon gonadotropin releasing hormone analogue (sGnRHa) and a dopamine antagonists (DA): domperidone (DOM). There were no ovulations in females from control groups. There were no differences between the ovulation rates (90 %) or embryo survival (> 92 %) and hatching rates (> 91 %) when there was administration of hCG doses between 500 and 1000 IU/kg. When there was comparison of different spawning agents, the ovulation rate was 100 % for all treated groups. There were the shortest and longest latency times to the time of ovulation after administration of CPH (26 h) and hCG (79 h), respectively. The greatest embryo survival (> 93 %) and hatching (> 91 %) rates occurred as a result of hCG administration with these values being slightly greater than when there was treatment with Ovaprim. The association between latency time and hatching rate indicated that when there was a slower final oocyte maturation (FOM) there were greater hatching rates.

Clock Gene Expression in the Eye Exhibits Circadian Oscillation and Light Responsiveness but is Not Necessary for Nocturnal Locomotor Activity of Japanese Loach, Misgurnus anguillicaudatus.

There are few model fish that are both edible and suitable for use in the laboratory. The Japanese loach (Misgurnus anguillicaudatus) is a traditional food in Japan, but is highly neglected despite its great nutritional value. To understand its circadian system and photic input pathway for synchronization of physiological activities to environmental light-dark cycles, we measured locomotor activity under light-dark and constant dark (DD) conditions. Locomotor activity was found to be higher in the nighttime than daytime, and its rhythmicity was weakened under DD conditions. The nocturnal activity of the Japanese loach is mainly controlled by environmental light, rather than the circadian clock. We explored the circadian regulation and light-responsiveness of clock gene expression in the eyes of loaches. The daily expression profiles of its mRNA revealed that most of the examined Cry and Per genes were likely regulated by internal circadian and/or environmental light signals. Among the Opsin genes transcribed in the eye, we detected the retinal photopigment porphyropsin at the protein level, which was lower than in mice. This property of loach eyes prompted us to analyze the locomotor activities of eye-enucleated fish. As a result, they still showed nocturnal circadian activity. Thus, it is likely that extraocular photoreceptive tissue(s) also contribute to the photic input pathway, although loach eyes are a circadian photosensitive tissue. This suggests that the loach mainly uses not its vision but other stimuli, such as mechanical or chemical stimuli, detected by barbels, to coordinate its nocturnal behavior.

Sea water shaping the freshwater biota: Hidden diversity and biogeographic history in the Paracanthocobitis zonalternans species complex (Teleostei: Nemacheilidae) in western Southeast Asia.

Western Southeast Asia is hosting one of the world's most diverse faunas, and one of the reasons for this huge diversity is the complex geologic past of the area, increasing the frequency of isolation and expansion events over evolutionary time scale. As an example case, the present study reveals the phylogeny and biogeographic history of the Paracanthocobitis zonalternans species complex, small benthic freshwater fish (Teleostei: Nemacheilidae) that are commonly occurring across western Southeast Asia (from central Myanmar through western and southern Thailand to northern Malaysia). The group is particularly interesting since it occurs in three biogeographic subdivisions (Indian, Indochinese, Malay/Sundaic) and across all of the major biogeographic barriers in the region. Basing on mitochondrial and nuclear sequence data of 93 samples from about 50 localities we found six major clades, most with exclusive geographic distribution. Divergence time dated the origin of the P. zonalternans species complex to early Miocene (17.8 MYA) and a biogeographic analysis identified the Tenasserim region as the ancestral region. From this region the fish spread during periods of lowered global sea level, particularly during late Miocene (11-8 MYA) northwards into all Burmese river basins and southwards into south Thailand and northern Malaysia. Besides lowered global sea level periods, local stream capture events allowed the complex to expand, e.g. into the Mae Klong basin. Strong fragmentations during periods with elevated sea level during the Pliocene and Pleistocene repeatedly restricted populations to refuges and shaped the observed major lineages. Our results document a higher diversity within the P. zonalternans species complex than formerly believed and a strong impact of global sea level on its evolutionary history. Low sea levels promoted dispersal and elevated sea levels fragmentation events. A very similar impact of sea level changes can be expected in all stationary fauna (freshwater and terrestrial) in all non-mountainous coastal regions worldwide.

Transcriptome analysis identifies candidate genes associated with skin color variation in Triplophysa siluroides.

In vertebrates, skin pigmentation is the most diverse phenotypic trait, and it is produced by a complex biological process that is often genetically controlled. Recently, two different colors (the typical brown and orange varieties) of Triplophysa siluroides, a species restricted to Yellow River drainage in China, were discovered. In the present study, the skin, brain and liver transcriptomes of T. siluroides of both colors were sequenced to search for genes related to skin pigmentation. Transcriptome sequencing generated 1,484,197,774 clean reads, resulting in a total of 222.6 Gb of sequence. The reads were assembled into 470,788 unigenes with a mean length of 1550 bp and an N50 size of 2944 bp. Functional annotation of the unigene dataset showed that 214,507, 304,161, 112,886, 179,074, 180,064, 184,837 and 82,081 unigenes were significantly matched to entries in the Nr protein, Nt, KO, Swiss-Prot, Pfam, GO and KOG databases, respectively. A differential expression analysis revealed that 2774, 3552 and 1529 unigenes were upregulated and 2720, 2663 and 1103 unigenes were downregulated in the skin, brain and liver of orange-skinned T. siluroides, respectively. Several genes that play key roles in pigmentation, i.e., Agouti, Slc45a2, Cbs, Mift and Slc7a11, showed significantly differential expression between brown and orange fish. In addition, we detected 158,863 simple sequence repeats (SSRs) in the T. siluroides transcriptome, and a total of 201,338 single-nucleotide polymorphisms (SNPs) were discovered in the different transcriptomes. The present results will facilitate further study of the molecular mechanisms of skin pigmentation and marker-assisted breeding of fish with valuable skin colors.

Negative feedback concept in tagging: Ghost tags imperil the long-term monitoring of fishes.

Wildlife monitoring using passive telemetry has become a robust method for investigating animal migration. With increased use, this method progressively pollutes the environment with technological waste represented by so called ghost tags (PIT tags ending in the environment due to reproductive expulsions, shedding or animal mortality). However, their presence in the environment may lead to failed detections of living individuals. We used tagging data from studies of the asp Leuciscus aspius and the bleak Alburnus alburnus collected from 2014 to 2018 and located ghost tag positions on the monitored spawning site using portable backpack reader for their detection. We modelled virtual river-wide flat-bed antennas (widths 0.2, 0.4, 0.6 and 0.8 m) representing monitoring effort and estimated the probability of the presence of ghost tags within the antenna field. Of 3724 PIT tags used in the study, we detected on the spawning ground 173 ghost tags originating from long-term monitoring. The ghost tags accumulated in the environment in time, suggesting insufficient degradation rate or shift downstream from the research site. Number of ghost tags present on the spawning ground led to high probability of disabled readings of tagged fish passing through the antenna electro-magnetic field. We demonstrate how accumulated ghost tags may cause detection failures for focal species and incomplete data acquisition. We infer that intensive long-term monitoring using PIT tag technology may encumber future data acquisition or entail additional costs for clean-up.

Psilorhynchus nahlongthai, a new fish species (Teleostei: Psilorhynchidae) from the Brahmaputra drainage, northeast India.

Psilorhynchus nahlongthai, a new psilorhynchid fish, is described from the Diyung River, a tributary to the Kopili River (itself a southern tributary of the Brahmaputra drainage) in Assam, northeast India. It is placed in the Psilorhynchus balitora species group and can be easily distinguished from all other members of this group by a combination of the following characters: dense and prominent tuberculation on the head region; thick and long pre- and post-epiphyseal fontanelles on the neurocranium; 9 + 8 caudal-fin rays; and 34 (24 + 10) vertebrae. Genetic divergence between P. nahlongthai and members of the P. balitora species group from the Brahmaputra and neighbouring drainages, with K2P distances ranging 3.7%-14.7% and 7.4%-20.7% in the mitochondrial COI and cyt b gene datasets respectively, support its report as a new species.

Morphological features of mucous secretory organ and mucous secretion of loach Misgurnus anguillicaudatus skin for friction drag reduction.

We examined the functional morphology of loach Misgurnus anguillicaudatus skin by using synchrotron X-ray micro-computed tomography (SR-µCT) and high-contrast staining using osmium tetroxide or phosphotungstic acid (PTA), which enhances the image contrast of soft tissues. The captured high-spatial resolution images revealed that the surface ornamentations were stuck in the basement membrane of the loach scales. The ornamentations consisting of grooves (radii) and ridges (circuli) that can move freely and bend flexibly. The cross-sectional lateral microstructures of flat, concave and convex loach skins were observed from a live image of loach skin obtained through dark-field optical coherence tomography (OCT) imaging. The thickness of loach skin was changed with varying empty space between the mucous-cell layer and the scales by bending motion of loach. In addition, through direct measurement of drag reduction of loach skin, the mucous layer was found to have a strong influence on the reduction of skin friction. The present results enhance the understanding of the functional morphologies of mucous layer of loach to secrete mucus for skin friction reduction.

Characterisation of stem and proliferating cells on the retina and lens of loach Misgurnus anguillicaudatus.

The eye of the fish has a lifelong persistent neurogenesis unlike eye of mammals, so it's highly interesting to study retinal neurogenesis and its genetic control to give complete knowledge about the cause of this property in fish in comparison to mammals. We performed fluorescent in situ hybridisation for loach Misgurnus anguillicaudatus bmi1, msi1 and sox2 genes, which are used as an indicator of the sites of multipotent stem cells. Proliferating cell nuclear antigen (PCNA), bromodeoxyuridine (BRDU) and KI67 markers were used as indicators of proliferating cells and glial fibrillary acidic protein (GFAP) immunofluorescence was used for detection of the glial property of cells, as well as, immunohistochemistry detected the role of peroxisome proliferator-activated receptor (PPAR)α and γ in retinal neurogenesis. Our results determined that the lens and the retina of loach M. anguillicaudatus contain proliferative and pluripotent stem cells that have both glial and neuroepithelial properties, which add new cells continuously throughout life even without injury-induced proliferation. The PPARα has an essential function in providing energy supply for retinal neurogenesis more than PPARγ.

Expression of the kisspeptin/gonadotropin-releasing hormone (GnRH) system in the brain of female Chinese sucker (Myxocyprinus asiaticus) at the onset of puberty.

The kisspeptin-kisspeptin receptor (kissr)-gonadotropin-releasing hormone (GnRH) system plays a key role in regulating the onset of puberty in mammals. However, the role of this system in fish is still unclear. We examined the relative gene expression patterns for kiss1, kiss2, kissr2, sGnRH, and pjGnRH in all parts of the brains of Chinese sucker (Myxocyprinus asiaticus) females at the prepubertal and pubertal stages by using real-time PCR. We also analyzed the expression of kiss1 and GnRH1 via immunofluorescence. Two variants of kisspeptin; a variant of kissr (kissr2); and two variants of GnRH, pjGnRH (GnRH1), and sGnRH (GnRH3), were expressed in all parts of the brain. The mRNA expression of kiss1 was higher in the telencephalon, mesencephalon, and diencephalon at the pubertal stage than at the prepubertal stage, and the expression of kiss2 was higher in only the telencephalon. The expression of kissr2 was higher in all parts of the brain, except the medulla, at the pubertal stage than at the prepubertal stage. pjGnRH was highly expressed in all parts of the brain at the pubertal stage, whereas sGnRH expression showed no distinct changes, except in the epencephalon. Strong kiss1 and weak GnRH-1 immunoreactivity was observed in the pineal gland, lateral tuberal nucleus (NLT), and ventral part of the NLT in the diencephalon of the Chinese sucker females at the pubertal stage. Our results suggest that the kiss1-kissr2-pjGnRH system was expressed highly at the onset of pubertal female Chinese sucker.