Ovulation requires proteinases to promote the rupture of ovarian follicles. However, the identity of these proteinases remains unclear. In our previous studies using RNA-seq analysis of differential expressed genes, we found significant down-regulation of five metalloproteinases: adam8b (a disintegrin and metalloproteinase domain 8b), adamts8a (a disintegrin and metalloproteinase with thrombospondin motif 8a), adamts9, mmp2 (matrix metalloproteinase 2), and mmp9 in the nuclear progestin receptor knockout (pgr^−/−) zebrafish that have failed to ovulate. We hypothesize that these metalloproteinases are responsible for ovulation and are regulated by progestin and Pgr. In this study, we first determined the expression of these five metalloproteinases and adamts1 in preovulatory follicles at different times within the spawning cycle in pgr^−/− and wildtype (wt) zebrafish and under varying hormonal treatments. We found that transcripts of adam8b, adamts1, adamts9, and mmp9 increased drastically in the preovulatory follicular cells of wt female zebrafish, while changes of adamts8a and mmp2 were not significant. This increase of adam8b, adamts9, and mmp9 was significantly reduced in pgr^−/−, whereas expression of adamts1 was not affected in pgr^−/− zebrafish. Among upregulated metalloproteinases, adamts9 mRNA was found to be expressed specifically in follicular cells. Strong immunostaining of Adamts9 protein was observed in the follicular cells of wt fish, and this expression was reduced drastically in pgr^−/−. Interestingly, about an hour prior to the increase of metalloproteinases in wt fish, both Pgr transcript and protein increased transiently in preovulatory follicular cells. The results from in vitro experiments showed that adamts9 expression markedly increased in a dose, time and Pgr-dependent manner when preovulatory follicles were exposed to a progestin, 17α,20β-dihydroxy-4-pregnen-3-one (DHP). Taken together, our results provide the first evidence that upregulation of adamts9 occurs specifically in preovulatory follicular cells of zebrafish prior to ovulation. Progestin and its receptor (Pgr) are essential for the upregulation of metalloproteinases.

Background: Given the lack of genetic background, the source tracking unknown individuals of fish species with both farmed and wild populations often cannot be robustly achieved. The gut microbiome, which is shaped by both deterministic and stochastic processes, can serve as a molecular marker of fish host source tracking, particularly as an alternative to the yet-to-be-established host genetic marker. A candidate for testing the feasibility is the large yellow croaker, Larimichthys crocea, which is carnivorous and ranks the top mariculture fish in China. Wild resource of this fish was depleted decades ago and might have potential problematic estimation because of escaping of farmed individuals. Results: The rectums of wild (n = 212) and farmed (n = 79) croakers from multiple batches were collected for the profiling of their gut bacterial communities. The farmed individuals had a higher alpha diversity and lower bacterial load than the wild individuals. The gut microbiota of the two sources exhibited divergence and high inter-batch variation, as featured by the dominance of Psychrobacter spp. in the wild group. Predicted functional capacity of the gut microbiome and representative isolates showed differences in terms of host source. This difference can be linked to the potential diet divergence between farmed and wild fishes. The non-stochastic distribution pattern of the core gut microbiota of the wild and farmed individuals supports the feasibility of microbiota-based host source tracking via the machine learning algorithm. A random forest classifier based on the divergence and non-stochastic assembly of the gut microbiome was robust in terms of host source tracking the individuals from all batches of croaker, including a newly introduced batch. Conclusions: Our study revealed the divergence of gut microbiota and related functional profiles between wild and farmed croakers. For the first time, with representative datasets and non-stochastic patterns, we have verified that gut microbiota can be robustly applied to the tracking of host source even in carnivorous fish.

Toll-like receptors (TLRs) are major molecular pattern recognition receptors, which are essential for triggering a series of innate immune responses against invading pathogens by recognizing their evolutionary conserved molecular patterns. The mudskipper, Boleophthalmus pectinirostris is exceptional among fishes due to its amphibious lifestyle and adaptation to living on mudflats. The whole-genome sequencing of B. pectinirostris has revealed that this species possesses an expansion of Tlr11 family [12 Tlr11 family genes (one tlr21, 4 tlr22, and 7 tlr23)] that we focused on in the present study. The full-length cDNA sequences of the 12 tlrs in B. pectinirostris were cloned and their deduced amino acid sequences possessed a typical TLR domain arrangement. Likelihood tests of selection revealed that these 12 Tlr11 family genes are under diversifying selection. A total of 13 sites were found to be positively selected by more than one evolution model, of which 11 were located in the ligand-binding ectodomain. The observed non-synonymous substitutions may have functional implications in antigen and pathogen recognition specificity. These 12 tlrs were highly expressed in immune-related tissues, i.e. spleen and kidney. Tlr21 and tlr22b transcripts were significantly up-regulated by LPS, whereas tlr22a, tlr22d, tlr23b, tlr23e, tlr23g were significantly up-regulated by poly(I:C) in the spleen or/and kidney, which implies that the expanded Tlr11 family genes may play roles in protecting the fish from the invasion of gram-negative bacteria and double-stranded RNA viruses. The results from the present study suggested that the expansion of Tlr11 family genes in B. pectinirostris may recognize ligands from various pathogens found in the intertidal zone.