Comparison of Myosepta Development and Transcriptome Profiling between Blunt Snout Bream with and Tilapia without Intermuscular Bones

Intermuscular bones (IBs) are small spicule-like bones located in the myosepta of basal teleosts, which negatively affect the edibleness and economic value of fish. Blunt snout bream (Megalobrama amblycephala, with epineural and epipleural IBs) and tilapia (Oreochromis niloticus, without epineural and epipleural IBs) are two major aquaculture species and ideal models for studying the formation mechanisms of fish IBs. Here, we compared myosepta development between M. amblycephala and O. niloticus, based on histological analysis, transcriptome profiling, and expression analysis of bone-related genes. The histological results showed that dye condensation began to appear in the myosepta 20 days post hatching (dph) in M. amblycephala, and IBs could be clearly observed 50 dph in the myosepta, based on different staining methods. However, in O. niloticus, dye condensation was not observed in the myosepta from 10 to 60 dph. Differentially expressed genes (DEGs) at different developmental stages were screened by comparing the transcriptomes of M. amblycephala and O. niloticus, and KEGG analysis demonstrated that these DEGs were enriched in many bone-related pathways, such as focal adhesion, calcium, and Wnt signaling pathways. Quantitative PCR was performed to further compare the expression levels of some bone-related genes (scxa, scxb, runx2a, runx2b, bgp, sp7, col1a2, entpd5a, entpd5b, phex, alpl, and fgf23). All the tested genes (except for alpl) exhibited higher expression levels in M. amblycephala than in O. niloticus. A comparison of the dorsal and abdominal muscle tissues between the two species also revealed significant expression differences for most of the tested genes. The results suggest that scxa, scxb, runx2a, runx2b, entpd5a, col1a2, and bgp may play important roles in IB development. Our findings provide some insights into the molecular mechanisms of IB formation, as well as clues for further functional analysis of the identified genes to better understand the development of IBs.

The Sustainable Treatment Effect of Constructed Wetland for the Aquaculture Effluents from Blunt Snout Bream (Megalobrama amblycephala) Farm

In aquaculture, constructed wetland (CW) has recently attracted attention for use in effluent purification due to its low running costs, high efficiency and convenient operation,. However, less data are available regarding the long-term efficiency of farm-scale CW for cleaning effluents from inland freshwater fish farms. This study investigated the effectiveness of CW for the removal of nutrients, organic matter, phytoplankton, heavy metals and microbial contaminants in effluents from a blunt snout bream (Megalobrama amblycephala) farm during 2013–2018. In the study, we built a farm-scale vertical subsurface flow CW which connected with a fish pond, and its performance was evaluated during the later stage of fish farming. The results show that CW improved the water quality of the fish culture substantially. This system was effective in the removal of nutrients, with a removal rate of 21.43–47.19% for total phosphorus (TP), 17.66–53.54% for total nitrogen (TN), 32.85–53.36% for NH4+-N, 33.01–53.28% NH3-N, 30.32–56.01% for NO3−-N and 42.75–63.85% for NO2−-N. Meanwhile, the chlorophyll a (Chla) concentration was significantly reduced when the farming water flowed through the CW, with a 49.69–62.01% reduction during 2013–2018. However, the CW system only had a modest effect on the chemical oxygen demand (COD) in the aquaculture effluents. Furthermore, concentrations of copper (Cu) and lead (Pb) were reduced by 39.85% and 55.91%, respectively. A microbial contaminants test showed that the counts of total coliform (TC) and fecal coliform (FC) were reduced by 55.93% and 48.35%, respectively. In addition, the fish in the CW-connected pond showed better growth performance than those in the control pond. These results indicate that CW can effectively reduce the loads of nutrients, phytoplankton, metals, and microbial contaminants in effluents, and improve the water quality of fish ponds. Therefore, the application of CW in intensive fish culture systems may provide an advantageous alternative for achieving environmental sustainability.

Transcriptome Analysis Reveals Sexual Disparities between Olfactory and Immune Gene Expression in the Olfactory Epithelium of Megalobrama amblycephala

The olfactory organ is an important chemoreceptor in vertebrates. However, the sexual disparities in gene expression patterns in the olfactory organ in fish remain unstudied. Here, we conducted a transcriptome analysis of the olfactory epithelium (OE) of male and female blunt snout bream (Megalobrama amblycephala) to identify the differences. The histological analysis showed that there were 22 leaf-like olfactory lamellaes on one side of the OE of the adult blunt snout bream. The sensory area of OE is enriched with ciliated receptor cells and microvilli receptor cells. The transcriptome analysis showed that only 10 out of 336 olfactory receptor genes (224 ORs, 5 V1Rs, 55 V2Rs, and 52 TAARs) exhibited significant expression differences between males and females, and most of the differentially expressed genes were related to the immune system. We also validated these results using qPCR: 10 OR genes and 6 immunity-related genes significantly differed between males and females. The FISH analysis results indicated that the ORs were mainly expressed at the edge of the olfactory lamellae. Collectively, our study reveals that gender is not an important factor influencing the expression of olfactory receptors, but the expression of immune genes varies greatly between the genders in blunt snout bream.

Identification and Characterization of Novel CircRNAs Involved in Muscle Growth of Blunt Snout Bream (Megalobrama amblycephala)

Circular RNAs (circRNAs), a novel class of endogenous RNAs, have been recognized to play important roles in the growth of animals. However, the regulatory mechanism of circRNAs on fish muscle growth is still unclear. In this study, we performed whole transcriptome analysis of skeletal muscles from two populations with different growth rates (fast-growing and slow-growing) of blunt snout bream (Megalobrama amblycephala), an important fish species for aquaculture. The selected circRNAs were validated by qPCR and Sanger sequencing. Pairs of circRNA–miRNA–mRNA networks were constructed with the predicted differentially expressed (DE) pairs, which revealed regulatory roles in muscle myogenesis and hypertrophy. As a result, a total of 445 circRNAs were identified, including 42 DE circRNAs between fast-growing (FG) and slow-growing (SG) groups. Many of these DE circRNAs were related with aminoglycan biosynthetic and metabolic processes, cytokinetic processes, and the adherens junction pathway. The functional prediction results showed that novel_circ_0001608 and novel_circ_0002886, competing to bind with dre-miR-153b-5p and dre-miR-124-6-5p, might act as competing endogenous RNAs (ceRNAs) to control MamblycephalaGene14755 (pik3r1) and MamblycephalaGene10444 (apip) level, respectively, thus playing an important regulatory role in muscle growth. Overall, these results will not only help us to further understand the novel RNA transcripts in M. amblycephala, but also provide new clues to investigate the potential mechanism of circRNAs regulating fish growth and muscle development.