Individual and group behavioral responses to nutritional state and context in a social fish.

Understanding how animal collectives and societies form and function is a fundamental goal in animal biology. To date, research examining fish shoaling behavior has focused mostly on the general principles and ecological relevance of the phenomeon, while the ways in which physiological state (e.g., nutrition) affects collective behavior remain overlooked. Here, we investigated the shoaling behavior of common minnows (Phoxinus phoxinus) with three different nutritional states (control treatment: fasting for 24 h, fasting treatment: fasting for 7 days, and digestion treatment: 1 h after satiation feeding) across two ecological contexts (i.e., without and with food). No effects of either nutritional state or context were found on swimming speed, but the acceleration was greater in the digestion group than in the control group, with that in the fasting group being intermediate. Similar to change tendency in group length and group width of shoals, both interindividual distance and nearest neighbor distance were also greater in the fasting group than in the digestion group, suggesting that fasting and digestion may exert opposite driving forces on group cohesion. However, neither nutritional state nor context influenced the group area, group speed, group percent time moving, or group polarization. Both the foraging efficiency and the percentage of food items consumed by the fish shoals were greater in the fasting and control groups than in the digestion group. Our study suggested that one week of hunger and the energetically demanding stage of food digestion tend to have opposite influences on group shape, while the social foraging context does not influence the individual and group behavior of fish.

Exposure to acute waterborne cadmium caused severe damage on lipid metabolism of freshwater fish, revealed by nuclear lipid droplet deposition in hepatocytes of rare minnow.

Cadmium (Cd) is a widely distributed aquatic toxic heavy metal with the potential to disrupt fish metabolism; however, more research is needed to clarify the underlying mechanisms. In the present study, rare minnows (Gobiocypris rarus) were used to detect the effects of cadmium on freshwater fish lipid metabolism and its underlying mechanism by histopathological observation, measurement of serum and liver biochemical indexes, and analysis of gene expression in terms of lipid oxidation, synthesis and transport. Here, severe damage, such as cytoplasmic lipid droplet (LD) accumulation, ectopic deposition of LDs, and the appearance of nuclear LDs (nLDs), was detected after exposure to 2.0 mg/L or higher concentrations (2.5 and 2.8 mg/L CdCl2) for 96 h. Other damage included abnormal increases in rough endoplasmic reticulum (RER) lamellae in a fingerprint or concentric circle pattern and necrosis of hepatocytes, and which was observed in the livers of fish exposed to 2.0 mg/L CdCl2.. Both hepatic and serum lipids, such as triglycerides and total cholesterol, were significantly increased after exposure to 2.0 mg/L CdCl2, as was serum lipase (LPS). Hepatic lipase and lipoprotein lipase remained unchanged, in accordance with the unchanged hepatic mRNA transcripts of PPARɑ. Furthermore, the mRNA transcripts of both SCD and SQLE were significantly decreased. Moreover, hepatic and serum low-density and high-density lipoprotein cholesterol showed significant changes, which were accompanied by a significant increase and decrease in hepatic APOAI and APOB100 mRNA levels, respectively. All the results indicate the presence of severe damage to hepatic lipid metabolism and that disrupted lipid transport may play a key role in the accumulation of hepatic LDs. In addition, the hepatic nLDs of nonmammalian vertebrates and their location across the nuclear envelope are intriguing, suggesting that large-size nLDs are a common marker for severe liver damage.

Identification of sex chromosome and sex-determining gene of southern catfish (Silurus meridionalis) based on XX, XY and YY genome sequencing.

Teleosts are important models to study sex chromosomes and sex-determining (SD) genes because they present a variety of sex determination systems. Here, we used Nanopore and Hi-C technologies to generate a high-contiguity chromosome-level genome assembly of a YY southern catfish (Silurus meridionalis). The assembly is 750.0 Mb long, with contig N50 of 15.96 Mb and scaffold N50 of 27.22 Mb. We also sequenced and assembled an XY male genome with a size of 727.2 Mb and contig N50 of 13.69 Mb. We identified a candidate SD gene through comparisons to our previous assembly of an XX individual. By resequencing male and female pools, we characterized a 2.38 Mb sex-determining region (SDR) on Chr24. Analysis of read coverage and comparison of the X and Y chromosome sequences showed a Y specific insertion (approx. 500 kb) in the SDR which contained a male-specific duplicate of amhr2 (named amhr2y). amhr2y and amhr2 shared high-nucleotide identity (81.0%) in the coding region but extremely low identity in the promotor and intron regions. The exclusive expression in the male gonadal primordium and loss-of-function inducing male to female sex reversal confirmed the role of amhr2y in male sex determination. Our study provides a new example of amhr2 as the SD gene in fish and sheds light on the convergent evolution of the duplication of AMH/AMHR2 pathway members underlying the evolution of sex determination in different fish lineages.

Roles of anti-Müllerian hormone and its duplicates in sex determination and germ cell proliferation of Nile tilapia.

Duplicates of amh are crucial for fish sex determination and differentiation. In Nile tilapia, unlike in other teleosts, amh is located on X chromosome. The Y chromosome amh (amhΔ-y) is mutated with 5 bp insertion and 233 bp deletion in the coding sequence, and tandem duplicate of amh on Y chromosome (amhy) has been identified as the sex determiner. However, the expression of amh, amhΔ-y, and amhy, their roles in germ cell proliferation and the molecular mechanism of how amhy determines sex is still unclear. In this study, expression and functions of each duplicate were analyzed. Sex reversal occurred only when amhy was mutated as revealed by single, double, and triple mutation of the 3 duplicates in XY fish. Homozygous mutation of amhy in YY fish also resulted in sex reversal. Earlier and higher expression of amhy/Amhy was observed in XY gonads compared with amh/Amh during sex determination. Amhy could inhibit the transcription of cyp19a1a through Amhr2/Smads signaling. Loss of cyp19a1a rescued the sex reversal phenotype in XY fish with amhy mutation. Interestingly, mutation of both amh and amhy in XY fish or homozygous mutation of amhy in YY fish resulted in infertile females with significantly increased germ cell proliferation. Taken together, these results indicated that up-regulation of amhy during the critical period of sex determination makes it the sex-determining gene, and it functions through repressing cyp19a1a expression via Amhr2/Smads signaling pathway. Amh retained its function in controlling germ cell proliferation as reported in other teleosts, while amhΔ-y was nonfunctionalized.

Individual variation in metabolic rate, locomotion capacity and hypoxia tolerance and their relationships in juveniles of three freshwater fish species.

Individual variations in metabolic rate, locomotion capacity and hypoxia tolerance and their relationships were investigated in three cyprinid species [crucian carp (Carassius auratus), common carp (Cyprinus carpio) and qingbo (Spinibarbus sinensis), in 60 individuals of each species]. Either the active metabolic rate (AMR) and critical swimming speed (Ucrit) (30 individuals) or critical oxygen tension (Pcrit) and loss of equilibrium (LOE) (30 individuals) were measured in each species after measuring the resting metabolic rate (RMR). Both the AMR and Ucrit were found to be significantly and positively correlated with the RMR in all three cyprinid species, indicating that high-RMR individuals have high aerobic capacity and thus good swimming performance. Pcrit was positively correlated with the RMR in all three species, whereas the LOE was highly positively correlated, weakly positively correlated and not correlated with the RMR in qingbo, common carp and crucian carp, respectively, possibly due to specialized morphological and biochemical adaptations involved in hypoxia tolerance in crucian and common carp. Crucian carp showed relatively poor swimming performance, i.e., a low Ucrit (relatively high variation), strong hypoxia tolerance, and low LOE (relatively low variation); qingbo showed relatively good swimming performance (relatively low variation) and weak hypoxia tolerance (relatively high variation); and common carp showed moderate swimming performance and relatively strong hypoxia tolerance (moderate variation). These interspecific differences may be due to the different lifestyles of these cyprinid fishes based on their associated fast-slow-flow regime and are outcomes of long-term selection.

Emergence of breeding tubercles and puberty onset in male zebrafish: evidence for a dependence on body growth.

The presence of breeding tubercles (BTs) on the pectoral fins has been investigated as a typical male secondary sexual characteristic (SSC) that distinguish males from females in adult zebrafish. Nonetheless, the earliest occurrence of these tubercles and its association with puberty onset and body growth remain unclear. In this study, using morphological, histological and statistical analyses, the authors examined the first appearance of BTs and puberty onset in male zebrafish, with particular emphasis on the potential impact of body growth on them. The results of this study revealed that BTs distributed along the first five branched pectoral fin rays were the earliest manifestation of male SSCs, which is significantly strongly correlated with body weight (R2  = 0.9609, P < 0.001), and could be used as a "gold standard" for the earliest sex distinction (<0.1 g in weight). Using the first appearance of BTs (<0.20 mm2 ) as a metric, the authors established that male puberty commenced at a body weight of c. 0.056 ± 0.015 g or a standard length of 10.99 ± 1.051 mm (mean ± S.D.). In this study, the authors thus established a simple method that can be used to sex live zebrafish at the pubertal stage and provides the first evidence for the relationship of BTs and male puberty initiation with body growth. These findings will accordingly lay a foundation for exploring mechanisms of the SSCs and male puberty onset in zebrafish and other teleost fish.

Effects of subchronic exposure to waterborne cadmium on H-P-I axis hormones and related genes in rare minnows (Gobiocypris rarus).

The H (hypothalamic)-P (pituitary)-I (interrenal) axis is critical in the stress response and other activities of fish. To further investigate cadmium (Cd) toxicity on the H-P-I axis and to identify its potential regulatory genes in fish, the adult female rare minnows (Gobiocypris rarus) were exposed to subchronic (5weeks) levels of waterborne Cd in the present study. This kind of treatment caused dose-dependent decline in fish growth, with significance in the high dose group (100µg/L). Correspondingly, low dose (5-50µg/L) waterborne Cd disrupted the endocrine system of H-P-I axis just at the secretion level, while high dose Cd disrupted both the secretion and synthesis of cortisol and its downstream signals in rare minnows, revealed by the significantly upregulation and positive correlation of corticosteroidogenic genes including MC2R, StAR, CYP11A1, and CYP11B1 in the kidney (including the interrenal tissue) (P<0.05), and the significant alteration of Glcci1, Hsp90AA and Hsp90AB in the hepatopancreas, gill and intestine as well (P<0.05). The expression of Glcci1 was significantly decreased in hepatopancreas, gill and intestine of tested fish following treatment, and its positive correlation with GR (Glucocorticoid receptor) suggested its potential regulation on the cortisol and/or H-P-I axis in fish. The expression of FKBP5 in the intestine was positively and significantly correlated with that of Hsp90AA (P<0.05), and the Hsp90AB transcript in the hepatopancreas was positively correlated with that of Hsp90AA (P<0.05), which indicated that Hsp90AA and Hsp90AB were more likely to serve as cofactors of GR and FKBP5 in response to Cd exposure.

Transcriptome profiling analysis of rare minnow (Gobiocypris rarus) gills after waterborne cadmium exposure.

Rare minnow (Gobiocypris rarus) is a widely used experimental fish in risk assessments of aquatic pollutants in China. Cadmium (Cd) is one of the most toxic heavy metals in the world; however, few studies have used fish gills, a multi-functional organ. In this study, we characterized the differential expression of adult female rare minnow gills after sub-chronic waterborne Cd (75µg/L CdCl2) exposure for 35d. A total of 452 genes (209 up-regulated and 243 down-regulated) were identified by gene expression profiling using RNA-Seq before and after treatment. Of these differentially expressed genes, 75, 21, and 54 differentially expressed genes are related to ion transport, oxidation-reduction processes, and the immune response, respectively. The results of GO and KEGG enrichment analyses, together with the altered transcript levels of major histocompatibility complex (MHC) class I and class II molecules and the significant increases in the levels of serum tumor necrosis factor α (TNF-α), interleukin 1ß (IL1ß) and nuclear factor-κB (NF-κB), indicated a disruption of the immune system, particularly the induction of inflammation and autoimmunity. The significant down-regulation of coagulation factor XIII A1 polypeptide (F13A1), tripartite motif-containing protein 21 (TRIM21), and Golgi-associated plant pathogenesis-related protein (GAPr) during both acute (≤96h) and sub-chronic (35d) waterborne Cd exposure, as well as their dosage dependence, suggested that these three genes could be used as sensitive biomarkers for aquatic Cd risk assessment.

[Toxic effects of CdSe/ZnS QDs to zebrafish embryos].

The toxic effects of CdSe/ZnS QDs on zebrafish (Danio rerio) embryos at different developmental stages were investigated in this study. The voluntary movement frequency, body length, hatching rate, mortality and malformation rate, SOD activities, MDA contents, mRNA expression of metallothionein (MT) and heat stress protein 70 (Hsp70) were used as indicators. The results showed that the EC50 was 316.994 nmol x L(-1) for zebrafish embryos (72 hpf) when exposed to CdSe/ZnS QDs. After the CdSe/ZnS QDs exposure, the embryos showed a significant increase in mortality and malformation rate, a decrease in hatching rate and body length, an advance in hatching time, and a changing in the spontaneous movement frequency, and many other toxic effects, such as the condensation of embryonic eggs, the formation of pericardial cysts and curvature of the spine. Moreover, it was found that the MDA contents in the embryos in CdSe/ZnS QDs groups were significantly increased, and the SOD activities were changed. In addition, the mRNA expression level of MT and Hsp70 were up-regulated. All the information suggests that exposure of CdSe/ZnS QDs can cause toxic effects on zebrafish embryos, and the effects may be related to the releasing of Cd2+, particle size and oxidative stress.

Silk structure and degradation.

To investigate the structure of silk and its degradation properties, we have monitored the structure of silk using scanning electron microscopy and frozen sections. Raw silk and degummed raw silk were immersed in four types of degradation solutions for 156 d to observe their degradation properties. The subcutaneous implants in rats were removed after 7, 14, 56, 84, 129, and 145 d for frozen sectioning and subsequent staining with hematoxylin and eosin (H.E.), DAPI, Beta-actin and Collagen I immunofluorescence staining. The in vitro weight loss ratio of raw silk and degummed raw silk in water, PBS, DMEM and DMEM containing 10% FBS (F-DMEM) were, respectively, 14%/11%, 12.5%/12.9%, 11.1%/14.3%, 8.8%/11.6%. Silk began to degrade after 7 d subcutaneous implantation and after 145 d non-degraded silk was still observed. These findings suggest the immunogenicity of fibroin and sericin had no essential difference. In the process of in vitro degradation of silk, the role of the enzyme is not significant. The in vivo degradation of silk is related to phagocytotic activity and fibroblasts may be involved in this process to secrete collagen. This study also shows the developing process of cocoons and raw silk.