Biofilm formation, antimicrobial assay, and toxin-genotypes of Clostridium perfringens type C isolates cultured from a neonatal Yangtze finless porpoise.

This is a culture-dependent study with the objective of pure culturing and characterizing pathogenic bacteria from the blowhole, lung, stomach and fecal samples of a neonatal crucially endangered Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) that died 27 days after birth. Bacteria were inoculated using a swab onto blood and MacConkey agar plates and representative isolates were identified through 16S rRNA gene sequence analysis. A total of three Clostridium perfringens type C strains from the fecal samples were isolated. Toxin genes, including cpa, cpb and cpb2, were detected by PCR amplification, whereas the etx, iap and cpe genes were not detected. Biofilm formation of the three strains was then examined. Only one strain was capable of biofilm formation. In addition, isolates showed strong resistance against the antibiotics amikacin (3/3), erythromycin (1/3), gentamicin (3/3), streptomycin (3/3), and trimethoprim (3/3), while sensitivity to ampicillin (3/3), bacitracin (3/3), erythromycin (2/3), penicillin G (3/3), and tetracycline (3/3). The results suggested C. perfringens type C could have contributed to the death of this neonatal porpoise.

Evaluating monitoring options for conservation: comparing traditional and environmental DNA tools for a critically endangered mammal.

While conservation management has made tremendous strides to date, deciding where, when and how to invest limited monitoring budgets is a central concern for impactful decision-making. New analytical tools, such as environmental DNA (eDNA), are now facilitating broader biodiversity monitoring at unprecedented scales, in part, due to time, and presumably cost, of methodological efficiency. Genetic approaches vary from conventional PCR (cPCR; species presence), to metabarcoding (community structure), and qPCR (relative DNA abundance, detection sensitivity). Knowing when to employ these techniques over traditional protocols could enable practitioners to make more informed choices concerning data collection. Using 12 species-specific primers designed for cPCR, eDNA analysis of the Yangtze finless porpoise (YFP; Neophocaena asiaeorientalis asiaeorientalis), a critically endangered aquatic mammal within the Yangtze River, we validated and optimized these primers for use in qPCR. We tested repeatability and sensitivity to detect YFP eDNA and subsequently compared the cost of traditional (visual and capture) sampling to eDNA tools. Our results suggest cPCR as the least expensive sampling option but the lack of PCR sensitivity suggests it may not be the most robust method for this taxon, predominately useful as a supplementary tool or with large expected populations. Alternatively, qPCR remained less expensive than traditional surveys, representing a highly repeatable and sensitive method for this behaviorally elusive species. Cost comparisons of surveying practices have scarcely been discussed; however, given budgetary constraints particularly for developing countries with limited local oversight but high endemism, we encourage managers to carefully consider the trade-offs among accuracy, cost, coverage, and speed for biodiversity monitoring.

Using environmental DNA to assess population-wide spatiotemporal reserve use.

Scientists increasingly rely on protected areas to assist in biodiversity conservation, yet the efficacy of these areas is rarely systematically assessed, often because of underfunding. Still, adaptive management strategies to maximize conservation success often rely on understanding the temporal and spatial dynamism of populations therein. Examination of environmental DNA (eDNA) is a time and cost-effective way to monitor species' distribution, and quantitative polymerase chain reaction (qPCR) provides information on organismal abundance. To date, however, such techniques remain underused for population assessments in protected areas. We determined eDNA concentration of the critically endangered Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) to describe its occurrence, range, and use of the Tian e-Zhou National Nature Reserve in Hubei, China, across seasons and hydrological depths. Despite the observation that total eDNA concentrations were highest in surface waters in summer, finless porpoise eDNA concentrations were significantly higher in deeper waters than in surface waters in summer. During the breeding season (spring), eDNA signals were site specific and restricted to the core area of the reserve. However, postbreeding eDNA concentrations were widespread across the reserve, encompassing sites previously thought to be unfrequented by the species. Our results suggest spatiotemporal idiosyncrasies in site, depth, and seasonal use of the reserve and a propensity for postbreeding population dispersal. With eDNA and qPCR we were able to assess an entire population's use of a protected area. Illuminating nuances in habitat use via eDNA could be valuable to set pragmatic conservation goals for this, and other, species.

Assembly and characterization of the MHC class I region of the Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis).

The Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis; YFP) is the sole freshwater subspecies of N. asiaeorientalis and is now critically endangered. Major histocompatibility complex (MHC) is a family of highly polymorphic genes that play an important immunological role in antigen presentation in the vertebrates. Currently, however, little is known about MHC region in the genome of the YFP, which hampers conservation genetics and evolutionary ecology study using MHC genes. In this work, a nucleotide sequence of 774,811 bp covering the YFP MHC class I region was obtained by screening a YFP bacterial artificial chromosome (BAC) library, followed by sequencing and assembly of positive BAC clones. A total of 45 genes were successfully annotated, of which four were MHC class I genes. There are high similarities among the four YFP MHC class I genes (>94%). Divergence in the coding region of the four YFP MHC class I genes is mainly localized to exons 2 and 3, which encode the antigen-binding sites of MHC class I genes. Additionally, comparison of the MHC structure in YFP to those of cattle, sheep, and pig showed that MHC class I genes are located in genome regions with regard to the conserved genes, and the YFP contains the fewest MHC class I genes among these species. This is the first report characterizing a cetacean MHC class I region and describing its organization, which would be valuable for further investigation of adaptation in natural populations of the YFP and other cetaceans.

Parentage-Based Group Composition and Dispersal Pattern Studies of the Yangtze Finless Porpoise Population in Poyang Lake.

Social behaviors are poorly known for the critically endangered Yangtze finless porpoise (YFP, Neophocaena asiaeorientalis asiaeorientalis). Here, group composition and dispersal patterns of the YFP population living in the Poyang Lake were studied by parentage-based pedigree analyses using 21 microsatellite loci and a 597 bp segment of the mitochondrial DNA control region. In this study, 21 potential mother-offspring pairs and six potential father-offspring pairs (including two potential parents-offspring pairs) were determined, among which 12 natural mother-offspring groups and a maternal group of three generations were found. No genetically-determined fathers were found associated with their offspring. This study also found that maternally related porpoises at the reproductive state tend to group together. This suggest maternal relationship and reproductive state may be factors for grouping in the YFP population. In natural mother-offspring groups, male offspring were all younger than two years old, which suggest male offspring may leave their mothers at approximately two years of age, or at least they were not in tight association with their mothers as they may have been under two years old. However, female offspring can stay longer with their mothers and can reproduce in the natal group.

De novo assembly and characterization of narrow-ridged finless porpoise renal transcriptome and identification of candidate genes involved in osmoregulation.

During the evolutionary transition from land to water, cetaceans have undergone numerous critical challenges, with osmoregulation being the major one. Two subspecies of the narrow-ridged finless porpoise (Neophocaena asiaeorientalis), the freshwater Yangtze finless porpoise (N. a. asiaeorientalis, NAA) and the marine East Asian finless porpoise (N. a. sunameri, NAS), provide excellent subjects to understand the genetic basis of osmoregulatory divergence between freshwater and marine mammals. The kidney plays an important and well-established role in osmoregulation in marine mammals and thus, herein, we utilized RNA-seq to characterize the renal transcriptome and preliminarily analyze the divergence between the NAA and the NAS. Approximately 48.98 million clean reads from NAS and 49.40 million clean reads from NAA were obtained by RNA-Seq. And 73,449 (NAS) and 68,073 (NAA) unigenes were assembled. Among these annotations, 22,231 (NAS) and 21,849 (NAA) unigenes were annotated against the NCBI nr protein database. The ion channel complex GO term and four pathways were detected as relevant to osmoregulation by GO and KEGG pathway classification of these annotated unigenes. Although the endangered status of the study species prevented analysis of biological replicates, we identified nine differentially expressed genes (DEGs) that may be vital in the osmoregulation of the narrow-ridged finless porpoise and worthwhile for future studies. Of these DEGs, the differential expression and distribution of the aquaporin-2 (AQP2) in the collecting duct were verified using immunohistochemical experiments. Together, this work is the first report of renal transcriptome sequencing in cetaceans, and it will provide a valuable resource for future molecular genetics studies on cetacean osmoregulation.

Genetic diversity and population structure of the critically endangered Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) as revealed by mitochondrial and microsatellite DNA.

Ecological surveys have indicated that the population of the critically endangered Yangtze finless porpoise (YFP, Neophocaena asiaeorientalis asiaeorientalis) is becoming increasingly small and fragmented, and will be at high risk of extinction in the near future. Genetic conservation of this population will be an important component of the long-term conservation effort. We used a 597 base pair mitochondrial DNA (mtDNA) control region and 11 microsatellite loci to analyze the genetic diversity and population structure of the YFP. The analysis of both mtDNA and microsatellite loci suggested that the genetic diversity of the YFP will possibly decrease in the future if the population keeps declining at a rapid rate, even though these two types of markers revealed different levels of genetic diversity. In addition, mtDNA revealed strong genetic differentiation between one local population, Xingchang-Shishou (XCSS), and the other five downstream local populations; furthermore, microsatellite DNA unveiled fine but significant genetic differentiation between three of the local populations (not only XCSS but also Poyang Lake (PY) and Tongling (TL)) and the other local populations. With an increasing number of distribution gaps appearing in the Yangtze main steam, the genetic differentiation of local populations will likely intensify in the future. The YFP is becoming a genetically fragmented population. Therefore, we recommend attention should be paid to the genetic conservation of the YFP.

Passive eDNA sampling facilitates biodiversity monitoring and rare species detection.

Environmental DNA (eDNA) technology has revolutionized biomonitoring, but challenges remain regarding water sample processing. The passive eDNA sampler (PEDS) represents a viable alternative to active, water filtration-based eDNA enrichment methods, but the effectiveness of PEDS for surveying biodiverse and complex natural water bodies is unknown. Here, we collected eDNA using filtration and glass fiber filter-based PEDS (submerged in water for 1 d) from 27 sites along the final reach of the Yangtze River and the coast of the Yellow Sea, followed by eDNA metabarcoding analysis of fish biodiversity and quantitative PCR (qPCR) for a critically endangered aquatic mammal, the Yangtze finless porpoise. We ultimately detected 98 fish species via eDNA metabarcoding. Both eDNA sampling methods captured comparable local species richness and revealed largely similar spatial variation in fish assemblages and community partitions between the river and sea sites. Notably, the Yangtze finless porpoise was detected only in the metabarcoding of eDNA collected by PEDS at five sites. Also, species-specific qPCR revealed that the PEDS captured porpoise eDNA at more sites (7 vs. 2), in greater quantities, and with a higher detection probability (0.803 vs. 0.407) than did filtration. Our results demonstrate the capacity of PEDS for surveying fish biodiversity, and support that continuous eDNA collection by PEDS can be more effective than instantaneous water sampling at capturing low abundance and ephemeral species in natural waters. Thus, the PEDS approach can facilitate more efficient and convenient eDNA-based biodiversity surveillance and rare species detection.

Nested clade phylogeographical analysis of the finless porpoise (Neophocaena phocaenoides) inhabiting Chinese and Japanese coasts.

The finless porpoise (Neophocaena phocaenoides) is a small cetacean whose survival is largely affected by human activity. The characteristics and structures of 3 populations in China and 4 to 5 populations in Japan have been well documented, although their history and origins remain poorly understood. In this study, nested clade phylogeographical analysis was applied to mtDNA sequences from finless porpoises to delineate the historical factors shaping the divergence pattern of this species. The sampling range covered most habitats of the finless porpoise, including the Chinese and Japanese coasts and the Yangtze River. A nested cladogram was constructed, and all 3 (2-step) clades were linked together without interior or tip status. Our data indicated that haplotype C was the most common among most individuals and populations, which could be the most ancient haplotype among all of the sampled types. As populations expanded to reside in different areas, ancient types with haplotype C mutated to other haplotypes, which were kept endemic by geological barriers that changed during glacial cycles that retarded gene flow. Populations in Japanese Pacific coasts and inland sea and post-arc marginal seacoasts and the Yangtze River were formed by the residual individuals left when allopatric fragmentation occurred in the ancestral population. The existence of highly endemic haplotypes and high genetic diversity from the Ariake Sound, Tachibana Bay, and the Yellow Sea indicated a relatively isolated state in these areas during the evolutionary history of the finless porpoise. The phylogenetic pattern revealed in the present study provided a better understanding of the biogeographical events that affected the finless porpoise within this region.

Multilocus DNA metabarcoding diet analyses of small cetaceans: a case study on highly vulnerable humpback dolphins and finless porpoises from the Pearl River Estuary, China.

Accurate diet identification of top predators is crucial to fully understand their ecological roles. Compared to terrestrial animals, gathering dietary information from cetaceans is notoriously difficult. Here, we applied a multilocus metabarcoding approach to investigate the diet of vulnerable Indo-Pacific humpback dolphins and Indo-Pacific finless porpoises from the Pearl River Estuary (PRE), China. Our analyses identified 21 prey fish species from the 42 humpback dolphin stomachs, as well as 10 species of fish and 1 species of cephalopod from the 13 finless porpoise stomachs. All of the taxa were assigned to the species level, highlighting that the multimarker approach could facilitate species identification. Most of the prey species were small- and medium-sized fishes that primarily fed on zooplankton. The calculated similarity index revealed a moderated dietary overlap between the 2 cetaceans, presumably due to the feeding of the 2 predators in association with fishing vessels in the PRE. A more diverse diet was observed in humpback dolphins in the closed fishing season compared to the fishing season, implying the influence on the dolphin diet due to the availability of commercial fishery resources. However, according to the results of species rarefaction curves, our findings on the feeding habits of the 2 cetaceans are still limited by insufficient sample size and therefore should be interpreted with caution. This study represents a first attempt to apply the multilocus DNA metabarcoding technique in the diet analysis of small cetaceans, although more efforts are needed to improve this type of analysis.

High level population differentiation of finless porpoises (Neophocaena phocaenoides) in Chinese waters revealed by sequence variability of four nuclear introns.

In the present study, sequence variations at four nuclear introns which were respectively from the parathyroid hormone-like (PTH) gene, isolate Pdalz1692 interferon (IFN) gene, peripherin-like (RDS) gene, and tyrosine kinase receptor-like (KIT) gene, were examined to analyze genetic diversity and population structure of the finless porpoise (Neophocaena phocaenoides) in Chinese waters. High among-population differentiation was revealed, with a significant genetic structure between populations (PTH: F(ST) = 0.29, P < 0.001; IFN1@: F(ST) = 0.23, P < 0.001; RDS: F(ST) = 0.12, P < 0.001; KIT: F(ST) = 0.16, P < 0.001) shown by the analysis of molecular variance. Although common haplotypes accounted for more than one half of all samples examined, many haplotypes were found to be population-specific. The Tajima's D, Fu's tests and mismatch distributions all suggested a recent colonization and population expansion of finless porpoises in Chinese waters. In view of special reference to the conservation priority of the Yangtze finless porpoises, special protection measures must be taken urgently for this population.

Gapless genome assembly of East Asian finless porpoise.

In recent years, conservation efforts have increased for rare and endangered aquatic wildlife, especially cetaceans. However, the East Asian finless porpoise (Neophocaena asiaeorientalis sunameri), which has a wide distribution in China, has received far less attention and protection. As an endangered small cetacean, the lack of a chromosomal-level reference for the East Asian finless porpoise limits our understanding of its population genetics and conservation biology. To address this issue, we combined PacBio HiFi long reads and Hi-C sequencing data to generate a gapless genome of the East Asian finless porpoise that is approximately 2.5 Gb in size over its 21 autosomes and two sex chromosomes (X and Y). A total of 22,814 protein-coding genes were predicted where ~97.31% were functionally annotated. This high-quality genome assembly of East Asian finless porpoise will not only provide new resources for the comparative genomics of cetaceans and conservation biology of threatened species, but also lay a foundation for more speciation, ecology, and evolutionary studies. Measurement(s) Neophocaena asiaeorientalis sunameri • Gapless genome assembly • sequence annotation Technology Type(s) MGISEQ. 2000 • PacBio HiFi Sequencing • Hi-C Sample Characteristic - Organism Neophocaena asiaeorientalis sunameri Sample Characteristic - Environment seawater Sample Characteristic - Location Yellow Sea near Lianyungang City, Jiangsu Province, China.

Population Structure of Finless Porpoise (Neophocaena phocaenoides) Discovered off Coastal Waters, Republic of Korea.

The finless porpoise (Neophocaena phocaenoides Cuvier, 1829) is distributed in the coastal waters of Asia, throughout Indonesia to the east, and as far north as the Taiwan Strait. The finless porpoise has been declared critically endangered by the WWF (World Wide Fund for Nature), and in 2017 was rated vulnerable on the IUCN Red Threatened Species List. Since this species is distributed near the coast and has many interactions with humans, effective conservation of the species requires further studies into their genetic diversity and population. In this study, 45 samples were obtained from bycatch or stranded individuals in the East, South, and West Seas, where Korean porpoises were mainly distributed from 2017-2021. We compared 473 bp mtDNA sequences from the control region. Pairwise fixation indices (FST) revealed that the two populations differed significantly (FST = 0.4557, p = 0.000). In contrast to high levels of genetic differentiation, gene flow was identified as medium levels (Nm of 0.04-0.71). Our data suggest that finless porpoises may have undergone a historic differentiation event, and that finless porpoises in the three regions could be divided into two populations: West and East/South.

Relationship of stranded cetaceans in Thai territorial waters to global populations: Mitochondrial DNA diversity of Cuvier's beaked whale, Indo Pacific finless porpoise, pygmy sperm whale, and dwarf sperm whale.

Cetaceans inhabit oceans throughout the world. Four specific odontocetes, namely Cuvier's beaked whale (Ziphius cavirostris), Indo Pacific finless porpoise (Neophocaena phocaenoides), pygmy sperm whale (Kogia breviceps), and dwarf sperm whale (Kogia sima), have occasionally been found stranded along Thailand's coastal waters (the Andaman Sea and the Gulf of Thailand). Although shared haplotypes of each species for many locations have been found, and some species have revealed genetic structure through haplotype networks, cetaceans in Thai waters have never been investigated in terms of comparing haplotypes to those that have existed before. Herein, we have illustrated the matrilineally phylogeographic relationships among worldwide populations through Bayesian Phylogenetic tree computations using Markov Chain Monte Carlo (MCMC) and Median-Joining Networks (MJNs). Unique haplotypes of the control region mitochondrial DNA of Thai odontocetes were found for all species. Moreover, a high degree of worldwide haplotype diversity (hd) above 0.8 among the four species was detected, while the lowest degree of nucleotide diversity (π) was observed in the Indo Pacific finless porpoise (1.12% ± 0.184%). An expansion of the effective female population size worldwide of three odontocete species was detected using Bayesian Skyline Reconstruction, but this did not include the Indo Pacific finless porpoise. Because Thai seas are located within the Indo Polynesian province, where this biodiversity hotspot exists, we speculate that these odontocetes may also inhabit specific habitats within the Malay Peninsula and Thailand's territorial waters. Therefore, closer attention and monitoring of these cetacean populations will be necessary for future conservation efforts.

Detection of Cetacean Poxvirus in Peruvian Common Bottlenose Dolphins (Tursiops truncatus) Using a Pan-Poxvirus PCR.

Cetacean poxviruses (CePVs) cause 'tattoo' skin lesions in small and large cetaceans worldwide. Although the disease has been known for decades, genomic data for these poxviruses are very limited, with the exception of CePV-Tursiops aduncus, which was completely sequenced in 2020. Using a newly developed pan-pox real-time PCR system targeting a conserved nucleotide sequence located within the Monkeypox virus D6R gene, we rapidly detected the CePV genome in typical skin lesions collected from two Peruvian common bottlenose dolphins (Tursiops truncatus) by-caught off Peru in 1993. Phylogenetic analyses based on the sequencing of the DNA polymerase and DNA topoisomerase genes showed that the two viruses are very closely related to each other, although the dolphins they infected pertained to different ecotypes. The poxviruses described in this study belong to CePV-1, a heterogeneous clade that infects many species of dolphins (Delphinidae) and porpoises (Phocoenidae). Among this clade, the T. truncatus CePVs from Peru were more related to the viruses infecting Delphinidae than to those detected in Phocoenidae. This is the first time that CePVs were identified in free-ranging odontocetes from the Eastern Pacific, surprisingly in 30-year-old samples. These data further suggest a close and long-standing pathogen-host co-evolution, resulting in different lineages of CePVs.

Integrated analysis of blood mRNAs and microRNAs reveals immune changes with age in the Yangtze finless porpoise (Neophocaena asiaeorientalis).

Populations of Yangtze finless porpoises (YFPs) have rapidly declined in recent decades, raising the specter of extinction. In order to protect YFPs, a greater understanding of their biology is needed, including studying how their immune functioning changes with age. Here, we systematically studied the hematologic and biochemical parameters, as well as mRNAs and miRNAs profiles of old, adult, and young YFPs. The lymphocyte (LYMPH), neutrophils (NEUT) and eosinophils (EOS) counts in old YFPs were lower than those in young or adult YFPs. When comparing old to adult YFPs, the latter showed higher expression of genes associated with the innate and adaptive immune systems, including complement components, major histocompatibility complex, interleukins, TNF receptors, and chemokines/cytokines. When comparing old to young YFPs, the most striking difference was in higher toll-like receptor signaling in the latter. When comparing adult to young YFPs, the former exhibited higher expression of genes related to adaptive immunity and the FoxO signaling pathway, but lower expression of genes associated with the PI3K-Akt signaling pathway. Negative miRNA-mRNA interactions were predicted in comparisons of the old and adult (326), old and young (316), adult and young (211) groups. Overall, these results delineate a progression from early innate immune function dominance to adaptive immune function enhancement (young to adult) and deterioration (adult to old), and the changes in miRNAs profile correlate with the effects of age on immune functions. This study is the first to observe the changes of immune function of Yangtze finless porpoise with age using transcriptome method, and the study's findings are of great significance for protecting this endangered species.

Sequence polymorphism and geographical variation at a positively selected MHC-DRB gene in the finless porpoise (Neophocaena phocaenoides): implication for recent differentiation of the Yangtze finless porpoise?

Sequence polymorphism at the MHC class II DRB locus was investigated in three finless porpoise (Neophocaena phocaenoides) populations in Chinese waters. Intragenic recombination and strong positive selection were the main forces in generating sequence diversity in the DRB gene. MHC sequence diversity changed significantly along the study period. Significant decrease in heterozygosity and lost alleles have been detected in the Yangtze River population and South China Sea population since 1990. Furthermore, there is a trend of increasing population differentiation over time. Especially, the genetic differentiation between the Yangtze River population and the Yellow Sea population was very low prior to 1990 (F (ST) = 0.036, P = 0.009), but became very significant after 1990 (F (ST) = 0.134, P < 0.001), suggesting a recent augmentation of genetic differentiation between both populations probably in a relatively short-term period. Porpoises from the Yangtze River displayed divergent frequencies of shared and private alleles from those displayed by two marine populations, which suggest that the former riverine population has been under a different selection regime (characteristic of a fresh water environment) than that of its marine counterparts.

Genetic and historic evidence for climate-driven population fragmentation in a top cetacean predator: the harbour porpoises in European water.

Recent climate change has triggered profound reorganization in northeast Atlantic ecosystems, with substantial impact on the distribution of marine assemblages from plankton to fishes. However, assessing the repercussions on apex marine predators remains a challenging issue, especially for pelagic species. In this study, we use Bayesian coalescent modelling of microsatellite variation to track the population demographic history of one of the smallest temperate cetaceans, the harbour porpoise (Phocoena phocoena) in European waters. Combining genetic inferences with palaeo-oceanographic and historical records provides strong evidence that populations of harbour porpoises have responded markedly to the recent climate-driven reorganization in the eastern North Atlantic food web. This response includes the isolation of porpoises in Iberian waters from those further north only approximately 300 years ago with a predominant northward migration, contemporaneous with the warming trend underway since the 'Little Ice Age' period and with the ongoing retreat of cold-water fishes from the Bay of Biscay. The extinction or exodus of harbour porpoises from the Mediterranean Sea (leaving an isolated relict population in the Black Sea) has lacked a coherent explanation. The present results suggest that the fragmentation of harbour distribution range in the Mediterranean Sea was triggered during the warm 'Mid-Holocene Optimum' period (approx. 5000 years ago), by the end of the post-glacial nutrient-rich 'Sapropel' conditions that prevailed before that time.