New insights into biologic interpretation of bioinformatic pipelines for fish eDNA metabarcoding: A case study in Pearl River estuary.

Environmental DNA (eDNA) metabarcoding is an emerging tool for monitoring biological communities in aquatic ecosystems. The selection of bioinformatic pipelines significantly impacts the results of biodiversity assessments. However, there is currently no consensus on the appropriate bioinformatic pipelines for fish community analysis in eDNA metabarcoding. In this study, we compared three bioinformatic pipelines (Uparse, DADA2, and UNOISE3) using real and mock (constructed with 15/30 known fish) communities to investigate the differences in biological interpretation during the data analysis process in eDNA metabarcoding. Performance evaluation and diversity analyses revealed that the choice of bioinformatic pipeline could impact the biological results of metabarcoding experiments. Among the three pipelines, the operational taxonomic units (OTU)-based pipeline (Uparse) showed the best performance (sensitivity: 0.6250 ± 0.0166; compositional similarity: 0.4000 ± 0.0571), the highest richness (25-102) and minimal inter-group differences in alpha diversity. It suggested the OTU-based pipeline possessed superior capability in fish diversity monitoring compared to ASV/ZOTU-based pipeline. Additionally, the Bray-Curtis distance matrix achieved the highest discriminative effect in the PCoA (43.3%-53.89%) and inter-group analysis (P < 0.01), indicating it was better at distinguishing compositional differences or specific genera of fish community at different sampling sites than other distance matrices. These findings provide new insights into fish community monitoring through eDNA metabarcoding in estuarine environments.

Deciphering assembly processes, network complexity and stability of potential pathogenic communities in two anthropogenic coastal regions of a highly urbanized estuary.

The existence of potential pathogens may lead to severe water pollution, disease transmission, and the risk of infectious diseases, posing threats to the stability of aquatic ecosystems and human health. In-depth research on the dynamic of potential pathogenic communities is of significant importance, it can provide crucial support for assessing the health status of aquatic ecosystems, maintaining ecological balance, promoting sustainable economic development, and safeguarding human health. Nevertheless, the current understanding of the distribution and geographic patterns of potential pathogens in coastal ecosystems remains rather limited. Here, we investigated the diversity, assembly, and co-occurrence network of potential pathogenic communities in two anthropogenic coastal regions, i.e., the eight mouths (EPR) and nearshore region (NSE), of the Pearl River Estuary (PRE) and a total of 11 potential pathogenic types were detected. The composition and diversity of potential pathogenic communities exhibited noteworthy distinctions between the EPR and NSE, with 6 shared potential pathogenic families. Additionally, in the NSE, a significant pattern of geographic decay was observed, whereas in the EPR, the pattern of geographic decay was not significant. Based on the Stegen null model, it was noted that undominant processes (53.36%/69.24%) and heterogeneous selection (27.35%/25.19%) dominated the assembly of potential pathogenic communities in EPR and NSE. Co-occurrence network analysis showed higher number of nodes, a lower average path length and graph diameter, as well as higher level of negative co-occurrences and modularity in EPR than those in NSE, indicating more complex and stable correlations between potential pathogens in EPR. These findings lay the groundwork for the effective management of potential pathogens, offering essential information for ecosystem conservation and public health considerations in the anthropogenic coastal regions.

Distinct stochastic processes drive bacterial community assembly and co-occurrence patterns with common antibiotic resistance genes in two highly urbanised coastal ecosystems of the Pearl River Estuary.

To comprehensively elucidate the ecology of the bacterial community and antibiotic resistance genes (ARGs) in urbanised coastal ecosystems, this study investigated the variations of bacterial community and five common types of ARGs, the impacting factors and assembly of bacterial community, as well as their co-occurrence relationships in two ecosystems of the Pearl River Estuary (PRE). The bacterial community composition and structure of the nearshore ecosystem (NSE) and the eight mouths of the PRE (EPR) markedly differed, with 38 phyla shared between these two ecosystems. The abundances of 10 ARGs and bacterial community diversity were significantly higher in the EPR than NSE. Moreover, 67.82% and 27.82% of the variation in the bacterial community was explained by spatial (44.42%/8.63%) and environmental (23.40%/19.19%) variables in the NSE and EPR, respectively. Significant distance-decay patterns were observed, and distinct stochastic processes (undominated processes or dispersal limitation) dominated bacterial community assembly in the NSE and EPR. Furthermore, co-occurrence patterns showed significant positive correlations between 48/182 ASVs belonging to 6/15 bacterial phyla and 8/11 ARGs in the NSE/EPR, with six common dominant hosts. These results clarify the drivers and mechanism shaping the bacterial community, providing further proof for potential ARG bacterial hosts in urbanised estuarine ecosystems.

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.

Changes in protist communities in drainages across the Pearl River Delta under anthropogenic influence.

Drainages in the Pearl River Delta urban agglomeration (PRDUA) host vital aquatic ecosystems and face enormous pressures from human activities in one of the largest urban agglomerations in the world. Despite being crucial components of aquatic ecosystems, the interactions and assembly processes of the protistan community are rarely explored in areas with serious anthropogenic disturbance. To elucidate the mechanisms of these processes, we used environmental DNA sequencing of 18S rDNA to investigate the influence of environmental factors and species interactions on the protistan community and its assembly in drainages of the PRDUA during summer. The protistan community showed a high level of diversity and a marked spatial pattern in this region. Community assembly was driven primarily by stochastic processes based on the Sloan neutral community model, explaining 74.28%, 75.82%, 73.67%, 74.40% and 51.24% of community variations in the BJ (Beijiang), XJ (Xijiang), PRD (Pearl River Delta), PRE (Pearl River Estuary) areas and in total, respectively. Meanwhile, environmental variables including temperature, pH, dissolved oxygen, transparency, nutrients and land use were strongly correlated with the composition and assembly of the protistan community, explaining 40.40% of variation in the protistan community. Furthermore, the bacterial community was simultaneously analysed by the 16S rDNA sequencing. Co-occurrence network analysis revealed that species interactions within bacteria (81.41% positive) or protists (82.80% positive), and those between bacteria and protists (50% positive and 50% negative) impacted the protistan community assembly. In summary, stochastic processes dominated, whereas species interactions and environmental factors also played important roles in shaping the protistan communities in drainages across the PRDUA. This study provides insights into the ecological patterns, assembly processes and species interactions underlying protistan dynamics in urban aquatic ecosystems experiencing serious anthropogenic disturbance.

Complete Mitogenomes of Three Carangidae (Perciformes) Fishes: Genome Description and Phylogenetic Considerations.

Carangidae are ecologically and economically important marine fish. The complete mitogenomes of three Carangidae species (Alectis indicus, Decapterus tabl, and Alepes djedaba) were sequenced, characterized, and compared with 29 other species of the family Carangidae in this study. The length of the three mitogenomes ranged from 16,530 to 16,610 bp, and the structures included 2 rRNA genes (12S rRNA and 16S rRNA), 1 control region (a non-coding region), 13 protein-coding genes, and 22 tRNA genes. Among the 22 tRNA genes, only tRNA-Ser (GCT) was not folded into a typical cloverleaf secondary structure and had no recognizable DHU stem. The full-length sequences and protein-coding genes (PCGs) of the mitogenomes of the three species all had obvious AT biases. The majority of the AT-skew and GC-skew values of the PCGs among the three species were negative, demonstrating bases T and C were more plentiful than A and G. Analyses of Ka/Ks and overall p-genetic distance demonstrated that ATP8 showed the highest evolutionary rate and COXI/COXII were the most conserved genes in the three species. The phylogenetic tree based on PCGs sequences of mitogenomes using maximum likelihood and Bayesian inference analyses showed that three clades were divided corresponding to the subfamilies Caranginae, Naucratinae, and Trachinotinae. The monophyly of each superfamily was generally well supported. The divergence time analyses showed that Carangidae evolved during three geological periods, the Cretaceous, Paleogene, and Neogene. A. indicus began to differentiate from other species about 27.20 million years ago (Mya) in the early Miocene, while D. tabl (21.25 Mya) and A. djedaba (14.67 Mya) differentiated in the middle Oligocene.

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.

Deep Insights into Gut Microbiota in Four Carnivorous Coral Reef Fishes from the South China Sea.

Investigations of gut microbial diversity among fish to provide baseline data for wild marine fish, especially the carnivorous coral reef fishes of the South China Sea, are lacking. The present study investigated the gut microbiota of four carnivorous coral reef fishes, including Oxycheilinus unifasciatus, Cephalopholis urodeta, Lutjanus kasmira, and Gnathodentex aurolineatus, from the South China Sea for the first time using high-throughput Illumina sequencing. Proteobacteria, Firmicutes, and Bacteroidetes constituted 98% of the gut microbiota of the four fishes, and 20 of the gut microbial genera recovered in this study represent new reports from marine fishes. Comparative analysis indicated that the four fishes shared a similar microbial community, suggesting that diet type (carnivorous) might play a more important role in shaping the gut microbiota of coral reef fishes than the species of fish. Furthermore, the genera Psychrobacter, Escherichia-Shigella, and Vibrio constituted the core microbial community of the four fishes, accounting for 61-91% of the total sequences in each fish. The lack of the genus Epulopiscium in the four fishes was in sharp contrast to what has been found in coral reef fishes from the Red Sea, in which Epulopiscium was shown to be the most dominant gut microbial genus in seven herbivorous coral reef fishes. In addition, while unique gut microbial genera accounted for a small proportion (8-13%) of the total sequences, many such genera were distributed in each coral reef fish species, including several genera (Endozoicomonas, Clostridium, and Staphylococcus) that are frequently found in marine fishes and 11 new reports of gut microbes in marine fishes. The present study expands our knowledge of the diversity and specificity of gut microbes associated with coral reef fishes.

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.

Mitochondrial genome of the Mackerel scad Decapterus macarellus (Perciformes: Carangidae).

The complete mitochondrial genome sequence was determined for the Mackerel scad Decapterus macarellus, one species of the economically important fish in Carangidae. The entire sequence of the genome was 16,544 bp in length, including the typical structure of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and 1 non-coding control region. Overall base compositions of the sequence were 27.3% of A, 30.4% of C, 25.3% of T and 17.0% of G, showing an obvious anti-G bias commonly observed in teleosts. The mitogenome of Decapterus macarellus had a quite high-sequence similarity (92.5%) with D. macrosoma, which was morphologically close to D. macarellus. The complete mitogenome sequence data of D. macarellus could provide useful information for taxonomic and phylogenetics studies.

Characterization of the mitochondrial genome of the Whitefin trevally Carangoides equula (Perciformes: Carangidae): a novel initiation codon for ATP6 gene.

The complete mitochondrial genome sequence of Carangoides equula (Perciformes: Carangidae) was determined. The size of the genome was 16,588 bp and overall base compositions of the sequence were 26.3% of A, 30.3% of C, 25.3% of T and 18.1% of G, showing an obvious anti G bias commonly observed in teleosts. The genome contained 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 2 non-coding regions (the control region and the light strand replication origin). Gene organization and gene order was similar to that observed in most other vertebrates. The initiation codon GTG was detected in the open reading frames of ATPase subunit 6, which was novel for ATP6 gene in Carangidae species. The mitogenome of C. equula shared 84.1% and 84.0% sequence similarity with two other Carangoides species Carangoides malabaricus and Carangoides armatus, respectively. The complete mitogenome sequence data of C. equula could provide useful information for taxonomic and phylogenetics studies.

Characterization of the mitochondrial genome of the Shortfin scad Decapterus macrosoma (Perciformes: Carangidae).

The complete mitochondrial genome sequence of Decapterus macrosoma has been obtained with overlapped polymerase chain reaction. The genome was 16,545 bp in length which contained 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and 2 non-coding regions (the control region and the light strand replication origin). Gene organization was similar to that observed in most other vertebrates. With the exception of the ND6 and eight tRNA genes, all other genes were encoded on the heavy strand. The genome was composed of 27.0% A, 30.4% C, 25.4% T and 17.2% G, showing an obvious anti-G bias commonly observed in teleosts. Two termination-associated sequence regions (TAS-1 and TAS-2), three central conserved sequence blocks (CSB-F, CSB-E and CSB-D), and three conserved sequence blocks (CSB-1, CSB-2 and CSB-3) were identified within the control region. The sequence data of D. macrosoma could provide useful information for the studies on molecular systematics and population genetics.

Organization and characterization of the mitochondrial genome of the Razorbelly scad Alepes kleinii (Perciformes: Carangidae).

The complete mitochondrial genome sequence of Alepes kleinii (Perciformes: Carangidae) was determined in this study. The entire sequence was 16,571 bp in length which contained 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 2 non-coding regions (the control region and the light strand replication origin). Gene organization and gene order were similar to that observed in most other vertebrates. Overall base compositions of the sequence were 28.0% of A, 28.5% of C, 27.0% of T, and 16.5% of G, showing an obvious anti-G bias commonly observed in teleosts. Apart from the ND6 and eight tRNA genes, all other genes were encoded on the heavy strand. Three types of initiation codons were detected in the open reading frames of the protein-coding genes, including codon ATA which was rarely detected in Carangidae species. The complete mitogenome sequence data of A. kleinii could provide useful information for taxonomic and phylogenetics studies.

Complete mitochondrial genome of the bigeye scad Selar crumenophthalmus (Perciformes: Carangidae).

The bigeye scad (Selar crumenophthalmus) is a widely distributed pelagic fish species with highly commercial values. Little information was available about its genetic characteristics. In the present study, we determined the complete mitochondrial genome of S. crumenophthalmus by combining sequences of overlapped fragments amplified by PCR using primers designed for Carangidae species. The entire sequence was 16,610 bp in length, which included a standard set of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 2 non-coding regions (the control region and the origin of light strand replication). The genome was composed of 27.2% A, 29.5% C, 26.5% T and 16.8% G, showing an obvious anti G bias commonly observed in teleosts. The complete mitogenome sequence of S. crumenophthalmus could contribute for the studies on phylogenetics, population structure and for developing fisheries management strategies.

A preliminary study for identification of candidate AFLP markers under artificial selection for shell color in pearl oyster Pinctada fucata.

Pearl oyster Pinctada fucata is widely cultured to produce seawater pearl in South China, and the quality of pearl is significantly affected by its shell color. Thus the Pearl Oyster Selective Breeding Program (POSBP) was carried out for the shell color and growth traits. The black (B), gold (G), red (R) and white (W) shell strains with fast growth trait were achieved after five successive generation selection. In this study, AFLP technique was used to scan genome of four strains with different shell colors to identify the candidate markers under artificial selection. Eight AFLP primer combinations were screened and yielded 688 loci, 676 (98.26%) of which were polymorphic. In black, gold, red and white strains, the percentage of polymorphic loci was 90.41%, 87.79%, 93.60% and 93.31%, respectively, Nei's gene diversity was 0.3225, 0.2829, 0.3221 and 0.3292, Shannon's information index was 0.4801, 0.4271, 0.4825 and 0.4923, and the value of FST was 0.1805. These results suggested that the four different shell color strains had high genetic diversity and great genetic differentiation among strains, which had been subjected to the continuous selective pressures during the artificial selective breeding. Furthermore, six outlier loci were considered as the candidate markers under artificial selection for shell color. This study provides a molecular evidence for the inheritance of shell color of P. fucata.

Mitochondrial genome of the Western Pacific gizzard shad Nematalosa come (Clupeiformes: Clupeidae), with comparison to two Nematalosa species.

The complete mitochondrial genome sequence of Nematalosa come has been obtained with polymerase chain reaction, which contained 22 tRNA genes, 13 protein-coding genes, 2 rRNA genes and 2 non-coding regions (the control region and the light strand replication origin) with the total length of 16,644 bp. Gene organization was similar to that observed in most other vertebrates. Except for the ND6 genes and eight tRNA genes, all other mitochondrial genes were encoded on the heavy strand. The genome was composed of 26.1% A, 29.2% C, 24.8% T and 19.9% G, showing an obvious anti-G bias commonly observed in fishes. It shared 86.1% and 88.4% mitogenome sequence with Nematalosa nasus and Nematalosa japonica, respectively. The sequence date could provide useful information for the studies on molecular systematics and conservation genetics.

Complete mitochondrial genome of the Bloch's gizzard shad Nematalosa nasus (Clupeiformes: Clupeidae).

The complete mitochondrial genome sequence of Nematalosa nasus was determined in this paper. The entire sequence was 16,674 bp in length and the overall base compositions were 26.5% of A, 28.4% of C, 25.4% of T and 19.7% of G. The mitogenome contained 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and two non-coding regions (the control region and the light strand replication origin). Gene arrangement was similar to that observed in most other vertebrates. Except for the ND6 genes and eight tRNA genes, all other genes were encoded on the heavy strand. Within the control region, the extended termination associated sequence was identified, as well as four central conserved sequence blocks (CSB-F, CSB-E, CSB-D and CSB-C) and three conserved sequence blocks (CSB-1, CSB-2 and CSB-3).

Complete mitochondrial genome of Anodontostoma chacunda (Clupeiformes: Clupeidae): genome characterization and phylogenetic consideration.

The complete mitochondrial genome sequence of Anodontostoma chacunda was determined in this paper. The genome is 16,772 bp in length and contains 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 2 noncoding regions. With the exception of ND6 and eight tRNA genes, all other genes are encoded on the heavy strand. Gene organization is similar to that observed in most other vertebrates. Overall base compositions of mitogenome are 27.4% of A, 28.7% of C, 25.1% of T, and 18.8% of G. Phylogenetic analyses using the concatenated nucleotide sequence of 12 protein-coding genes on the heavy strand reveal that genus Anodontostoma is the sister group to the assemblage (Nematalosa, (Clupanodon, Konosirus)) within subfamily Dorosomatinae.

Mitochondrial genome of the Chinese gizzard shad Clupanodon thrissa (Clupeiformes: Clupeidae) and related phylogenetic analyses.

The complete mitochondrial genome sequence of the single species from the Genus Clupanodon, the Chinese gizzard shad, Clupanodon thrissa, was determined. The entire sequence is 16,692 bp in length, including the typical structure of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, 2 rRNA genes, and 2 noncoding regions (control region and L-strand replication origin). With the exception of ND6 and eight tRNA genes, all other genes are encoded on the heavy strand and the organization of genes is similar to that observed in most other vertebrates. Phylogenetic analyses using the protein coding gene sequences reveal that C. thrissa is most closely related to Konosirus punctatus within the family Clupeidae and subfamily Dorosomatinae is not a monophyletic group.