Effects of sampling strategies and DNA extraction methods on eDNA metabarcoding: A case study of estuarine fish diversity monitoring.

Environmental DNA (eDNA) integrated with metabarcoding is a promising and powerful tool for species composition and biodiversity assessment in aquatic ecosystems and is increasingly applied to evaluate fish diversity. To date, however, no standardized eDNA-based protocol has been established to monitor fish diversity. In this study, we investigated and compared two filtration methods and three DNA extraction methods using three filtration water volumes to determine a suitable approach for eDNA-based fish diversity monitoring in the Pearl River Estuary (PRE), a highly anthropogenically disturbed estuarine ecosystem. Compared to filtration-based precipitation, direct filtration was a more suitable method for eDNA metabarcoding in the PRE. The combined use of DNeasy Blood and Tissue Kit (BT) and traditional phenol/chloroform (PC) extraction produced higher DNA yields, amplicon sequence variants (ASVs), and Shannon diversity indices, and generated more homogeneous and consistent community composition among replicates. Compared to the other combined protocols, the PC and BT methods obtained better species detection, higher fish diversity, and greater consistency for the filtration water volumes of 1 000 and 2 000 mL, respectively. All eDNA metabarcoding protocols were more sensitive than bottom trawling in the PRE fish surveys and combining two techniques yielded greater taxonomic diversity. Furthermore, combining traditional methods with eDNA analysis enhanced accuracy. These results indicate that methodological decisions related to eDNA metabarcoding should be made with caution for fish community monitoring in estuarine ecosystems.

Comparative Analysis of Complete Mitochondrial Genomes of Three Gerres Fishes (Perciformes: Gerreidae) and Primary Exploration of Their Evolution History.

Mitochondrial genome is a powerful molecule marker to explore phylogenetic relationships and reveal molecular evolution in ichthyological studies. Gerres species play significant roles in marine fishery, but its evolution has received little attention. To date, only two Gerres mitochondrial genomes were reported. In the present study, three mitogenomes of Gerres (Gerres filamentosus, Gerres erythrourus, and Gerres decacanthus) were systemically investigated. The lengths of the mitogenome sequences were 16,673, 16,728, and 16,871 bp for G. filamentosus, G. erythrourus, and G. decacanthus, respectively. Most protein-coding genes (PCGs) were initiated with the typical ATG codon and terminated with the TAA codon, and the incomplete termination codon T/TA could be detected in the three species. The majority of AT-skew and GC-skew values of the 13 PCGs among the three species were negative, and the amplitude of the GC-skew was larger than the AT-skew. The genetic distance and Ka/Ks ratio analyses indicated 13 PCGs were suffering purifying selection and the selection pressures were different from certain deep-sea fishes, were which most likely due to the difference in their living environment. The phylogenetic tree was constructed by molecular method (Bayesian Inference (BI) and maximum Likelihood (ML)), providing further supplement to the scientific classification of fish. Three Gerres species were differentiated in late Cretaceous and early Paleogene, and their evolution might link with the geological events that could change their survival environment.

eDNA metabarcoding as a promising conservation tool for monitoring fish diversity in a coastal wetland of the Pearl River Estuary compared to bottom trawling.

The difficulty of censusing fish diversity hampers effective management and conservation in estuarine and coastal ecosystems, especially wetland ecosystems. Improved noninvasive fish diversity monitoring programs are becoming increasingly crucial for coastal ecosystems. In this study, we investigated fish diversity and its seasonal variation in the Nansha wetland ecosystem using environmental DNA (eDNA) metabarcoding and bottom trawling, and the two approaches were compared. With the combination of the two methods, the identified fish taxa included 78 species within 60 genera and 33 families, and five nontarget taxa were only identified by eDNA metabarcoding. Compared to the two surveys, eDNA metabarcoding identified a significantly greater number of fish species per site and per season than bottom trawling (p < 0.05), with eDNA metabarcoding identifying 32.05% more fish species than bottom trawling. The overwhelming majority of the fish orders captured in the Nansha coastal wetland by bottom trawling were recovered from eDNA analysis, although certain taxa were not sampled due to limitations. Furthermore, the Whittaker index and relative abundance analysis of the two methods showed distinct differences between the sampling seasons, suggesting seasonal variations and reflecting the current or recent existence of fish species in the coastal ecosystem. Thus, our work provides more detailed seasonal data on biodiversity in the Nansha wetland of the Pearl River Estuary, which is essential for the long-term management and conservation of coastal biodiversity. Our study also adds to the evidence that the eDNA metabarcoding approach can be used in coastal environments to monitor a broad range of taxa and reflect seasonal fluctuations in fish diversity. As an emerging and transformative method, eDNA metabarcoding shows great potential for fish diversity monitoring in coastal wetland ecosystems.

Regulation of IL-17 by lncRNA of IRF-2 in the pearl oyster.

Long noncoding RNAs (lncRNAs), once thought to be nonfunctional, have recently been shown to participate in the multilevel regulation of transcriptional, posttranscriptional and epigenetic modifications and to play important roles in various biological processes, including immune responses. However, the expression and roles of lncRNAs in invertebrates, especially nonmodel organisms, remain poorly understood. In this study, by comparing a transcriptome to the PfIRF-2 genomic structure, we identified lncIRF-2 in the PfIRF-2 genomic intron. The results of the RNA interference (RNAi) and the nucleus grafting experiments indicated that PfIRF-2 might have a negative regulatory effect on lncIRF-2, and PfIRF-2 and lncIRF-2 may have a positive regulatory effect on PfIL-17. Additionally, lncIRF-2, PfIRF-2 and PfIL-17 were involved in responses to the nucleus graft. These results will enhance the knowledge of lncIRF-2, IRF-2, and IL-17 functions in both pearl oysters and other invertebrates.