Comparative Blood Transcriptome Analysis of Semi-Natural and Controlled Environment Populations of Yangtze Finless Porpoise.

The Yangtze finless porpoises (Neophocaena asiaeorientalis asiaeorientalis) living in different environments display significant differences in behavior and physiology. To compare and analyze gene expression differences between an ex situ population and a controlled environment population of the Yangtze finless porpoise, we sequenced the transcriptome of blood tissues living in a semi-natural reserve and an artificial facility, respectively. We identified 6860 differentially expressed genes (DEGs), of which 6603 were up-regulated and 257 were down-regulated in the controlled environment vs ex situ comparison. GO and KEGG enrichment analysis showed that the up-regulated genes in the controlled environment population were significantly associated with glucose metabolism, amino acid metabolism, and the nervous system, while those up-regulated in the ex situ population were significantly associated with energy supply and biosynthesis. Further analysis showed that metabolic and hearing-related genes were significantly affected by changes in the environment, and key metabolic genes such as HK, PFK, IDH, and GLS and key hearing-related genes such as OTOA, OTOF, SLC38A1, and GABBR2 were identified. These results suggest that the controlled environment population may have enhanced glucose metabolic ability via activation of glycolysis/gluconeogenesis, the TCA cycle, and inositol phosphate metabolism, while the ex situ population may meet higher energy requirements via enhancement of the amino acid metabolism of the liver and muscle and oxidative phosphorylation. Additionally, the acoustic behavior and auditory-related genes of Yangtze finless porpoise may show responsive changes and differential expression under different environment conditions, and thus the auditory sensitivity may also show corresponding adaptive characteristics. This study provides a new perspective for further exploration of the responsive changes of the two populations to various environments and provides a theoretical reference for further improvements in conservation practices for the Yangtze finless porpoise.

Water-Level Fluctuation Control of the Trophic Structure of a Yangtze River Oxbow.

Seasonal water-level fluctuations can profoundly impact nutrient dynamics in aquatic ecosystems, influencing trophic structures and overall ecosystem functions. The Tian-e-Zhou Oxbow of the Yangtze River is China's first ex situ reserve and the world's first successful case of ex situ conservation for cetaceans. In order to better protect the Yangtze finless porpoise, the effects of water-level fluctuations on the trophic structure in this oxbow cannot be ignored. Therefore, we employed stable isotope analysis to investigate the changes in the trophic position, trophic niche, and contribution of basal food sources to fish during the wet and dry seasons of 2021-2022. The research results indicate that based on stable isotope analysis of the trophic levels of different dietary fish species, fish trophic levels during the wet season were generally higher than those during the dry season, but the difference was not significant (p > 0.05). Fish communities in the Tian-e-Zhou Oxbow exhibited broader trophic niche space and lower trophic redundancy during the wet season (p < 0.05), indicating a more complex and stable food web structure. In both the wet and dry seasons, fish in the oxbow primarily relied on endogenous carbon sources, but there were significant differences in the way they were utilized between the two seasons (p < 0.05). In light of the changes in the trophic structure of the fish during the wet and dry seasons, and to ensure the stable development of the Yangtze finless porpoise population, we recommend strengthening the connectivity between the Tian-e-Zhou Oxbow and the Yangtze River.

Viral Diversity and Epidemiology in Critically Endangered Yangtze Finless Porpoises (Neophocaena asiaeorientalis asiaeorientalis).

The Yangtze finless porpoise (YFP) (Neophocaena asiaeorientalis asiaeorientalis) is a critically endangered freshwater cetacean, with about 1,249 individuals thought to be left in the wild. However, viral entities and viral diseases of YFPs remain obscure. In this study, anal swabs for virome analysis were collected during the physical examination of YFPs in the Tian-E-Zhou Oxbow (TEO) ex situ reserve. A total of 19 eukaryotic viral species belonging to 9 families, including Papillomaviridae, Herpesviridae, Picornaviridae, Picobirnaviridae, Caliciviridae, Retroviridae, Parvoviridae, Virgaviridae, and Narnaviridae, and other unclassified viruses were identified based on metasequencing. Among these detected viruses, a novel herpesvirus (NaHV), two different kobuviruses (NaKV1-2), and six different papillomaviruses (NaPV1 to -6) were considered potential risks to YFPs and confirmed by PCR or reverse transcription-PCR (RT-PCR). Most YFPs sampled were found to harbor one or more kinds of detected viral genomes (52/58 [89.7%]). Surveillance results demonstrated that kobuvirus and herpesvirus displayed obvious age distribution and PVs showed significant gender difference in YFPs. According to species demarcation criteria in individual genera in Papillomaviridae, two novel species (referred to as Omikronpapillomavirus 2 and 3) and four novel isolates of PV were identified in YFPs. Further evolutionary analysis suggested that NaPVs would occupy the mucosal niche and that virus-host codivergence mixed with duplications and host-switching events drives the evolution of cetacean PVs. Divergence times of PVs in YFP and other cetacean reflect the incipient speciation of YFPs. In summary, our findings revealed the potential viral entities, their prevalence, and their evolutionary history in YFPs, which raises an important issue regarding effects of viral infection on the fitness of YFPs. IMPORTANCE The Yangtze finless porpoise (YFP) is the only cetacean species in freshwater following the functional extinction of the baiji (Lipotes vexillifer). Health management, disease treatment, and other special measures are important for maintaining the existing YFP populations, especially in in situ and ex situ reserves. The discovery of potential viral entities and their prevalence in YFPs raises an important issue regarding the effects of viral infection on the fitness of YFPs and may contribute to the conservation of YFPs. The evolutionary history of papillomaviruses in YFP and other cetaceans reflects the phylogeny of their hosts and supports the status of incipient species, opening a window to investigate the evolutionary adaptation of cetaceans to freshwater as well as their phylogeny to remedy the deficiency of fossil evidence.

Accumulation and risk prioritization of psychoactive substances in the critically endangered Yangtze finless porpoise.

Psychoactive substances have been identified as a kind of emerging contaminants in aquatic environment and pose potential adverse effects on aquatic animals. Yangtze finless porpoise, a critically endangered species in China, is also facing the threat of psychoactive substances. In this study, the accumulation characteristics and risk prioritization of psychoactive substances were investigated in Yangtze finless porpoise collected from Poyang Lake (PYL) and Tian-E-Zhou Oxbow (TZO) in Yangtze River basin. The levels of psychoactive substances were detected in the range of below method detection limits (MDLs) to 98.4 ng/mL in the serum of Yangtze finless porpoise. Codeine (COD) and methamphetamine were identified as the major substances due to the highest residual levels with a median concentration of 0.72 ng/mL and 0.33 ng/mL, respectively. The total concentrations of psychoactive substances in the porpoise collected from TZO was significantly higher than those from PYL. Risk analysis based on effect ratio derived from the ratio of steady-state psychoactive substance serum concentration in the porpoise and human therapeutic plasma concentration revealed that COD was the substance with the highest risk among the psychoactive substances detected, followed by lysergic acid diethylamide (LSD), morphine, alprazolam (ALPZ) and lormetazepam. Location-specific risk prioritization of psychoactive substances found that the top 3 substances are LSD, lorazepam (LORZ) and ALPZ in PYL, and COD, LSD and LORZ in TZO. The results disclose the accumulation of psychoactive substances in Yangtze finless porpoise and suggest that the potential adverse effects should be concerned.

Underwater Noise Level Recordings from a Water Intake Pontoon and Possible Impacts on Yangtze Finless Porpoises in a Natural Reserve.

Underwater noise pollution caused by human activities may affect freshwater cetaceans to different degrees. To analyze the impacts of water intake pontoons on Yangtze finless porpoises (Neophocaena asiaeorientalis), this study collected underwater noise data from such a pontoon in a nature reserve, plotted the power spectral density of acoustic signals, and calculated the root mean square sound pressure levels and the magnitude of sound source levels. The 1/3-octave sound pressure level root mean square values at the transient holding pens were <18.0 kHz, 39.5−60.0 kHz, which were slightly higher than the Yangtze finless porpoise hearing threshold curve values and therefore could be perceived. However, the results indicated that the porpoises would not develop a temporary hearing threshold shift. Meanwhile, pontoon noise did not interfere with the porpoises' high-frequency acoustic signal nor did it affect their echolocation; it significantly interfered with their low-frequency acoustic signal, however, and the mother−child communication of the finless porpoises was affected, but this effect was quickly compensated due to the limited space range of the holding pens. Through this study of Yangtze finless porpoises, this paper provides a reference for assessing whether human facilities have impacts on freshwater cetaceans.

A metagenomic insight into the Yangtze finless porpoise virome.

The Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis) inhabiting the Yantze River, China is critically endangered because of the influences of infectious disease, human activity, and water contamination. Viral diseases are one of the crucial factors that threatening the health of Yangtze finless porpoise. However, there are few studies which elaborate the viral diversity of Yangtze finless. Therefore, this study was performed to investigate the viral diversity of Yangtze finless by metagenomics. Results indicated that a total of 12,686,252 high-quality valid sequences were acquired and 2,172 virus reads were recognized. Additionally, we also obtained a total of 10,600 contigs. Phages was the most abundant virus in the samples and the ratio of DNA and RNA viruses were 69.75 and 30.25%, respectively. Arenaviridae, Ackermannviridae and Siphoviridae were the three most predominant families in all the samples. Moreover, the majority of viral genus were Mammarenavirus, Limestonevirus and Lambdavirus. The results of gene prediction indicated that these viruses play vital roles in biological process, cellular component, molecular function, and disease. To the best of our knowledge, this is the first report on the viral diversity of Yangtze finless porpoise, which filled the gaps in its viral information. Meanwhile, this study can also provide a theoretical basis for the establishment of the prevention and protection system for virus disease of Yangtze finless porpoise.

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.

The bacteria of Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) are site-specific and distinct from freshwater environment.

Bacteria play an essential role in the health of marine mammals, and the bacteria of marine mammals are widely concerned, but less is known about freshwater mammals. In this study, we investigated the bacteria of various body sites of Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) and analyzed their association with freshwater environmental bacteria. The bacterial community and function of Yangtze finless porpoise showed apparent site-specificity. Various body sites have distinct differences in bacteria and have their dominant bacteria. Romboutsia, Plesiomonas, Actinobacillus, Candidatus Arthromitus dominated in the intestine (fecal and rectal samples). Fusobacterium, Streptococcus, and Acinetobacter dominated in the oral. The dominant genera in the blowhole include Suttonella, Psychrobacter, and two uncultured genera. Psychrobacter, Flavobacterium, and Acinetobacter were dominant in the skin. The alpha diversity of intestinal (fecal and rectal) bacteria was the lowest, while that of skin was the highest. The oral and skin bacteria of Yangtze finless porpoise significantly differed between the natural and semi-natural conditions, but no sex difference was observed. A clear boundary was found between the animal and the freshwater environmental bacteria. Even the skin bacteria, which are more affected by the environment, are significantly different from the environmental bacteria and harbor indigenous bacteria. Our results provide a comprehensive preliminary exploration of the bacteria of Yangtze finless porpoise and its association with bacteria in the freshwater environment.

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.

Circulating concentrations of thyroid hormones and cortisol in wild and semi-natural Yangtze finless porpoise (Neophocaena asiaeorientalis).

Our understanding about how environmental and biological variables may influence circulating thyroid and adrenal hormones in free-ranging cetaceans is limited. As such, we used liquid chromatography-mass spectrometry to determine concentrations of circulating cortisol and thyroid hormones (THs; tT3, tT4) in 132 Yangtze finless porpoises (YFPs) located in Poyang Lake, (PL, n = 92) and Tian-E-Zhou Oxbow reserve (TZO, n = 40). For overall hormone comparisons, animals were partitioned by age [juvenile and adult (male and non-pregnant, non-lactating female)], sex, season (winter or spring) and geographical location. Geographically, during winter, circulating THs were significantly higher in the PL versus TZO population. Seasonally, within PL, THs were significantly higher in the winter versus spring season. Animals were further binned into groups as follows: juvenile male (JM) and juvenile female (JF), adult male (AM), non-pregnant adult female, pregnant female and non-pregnant lactating female. Intra-group comparisons between locations showed a significant increase in JM THs at PL. Significant increases in THs during winter compared to spring were detected between JM and JF groups. Mean comparisons of cortisol within and between locations for each group identified a significant increase for TZO AM versus TZO pregnant female and JM and JF. Seasonally, in PL, only JF has significantly higher cortisol in winter versus spring. Finally, we established reference values of THs and cortisol for YFPs in different geographical locations. These references are important baselines from which the effects of environmental and biological variables on THs and cortisol may be evaluated.

Comparative analysis of microbial community structure between healthy and Aeromonas veronii-infected Yangtze finless porpoise.

BACKGROUND: The gut microbiota is a complex ecosystem, which is essential for the metabolism, health and immunity of host. Many diseases have been shown to be closely related to the alteration of intestinal flora. Aeromonas veronii as a conditioned pathogen can cause disease in Yangtze finless porpoise through intestinal infections. However, it is not clear whether the disease caused by Aeromonas veronii is related to changes of intestinal flora. In the current study, the diversity and composition of gut microbiota in the healthy and Aeromonas veronii-infected Yangtze finless porpoise were evaluated by high-throughput sequencing to further investigate the potential association between intestinal flora alteration and pathogen invasion. RESULTS: A total of 127,3276 high-quality sequences were achieved and 2465 operational taxonomic units (OTUs) were in common among all samples. The results of alpha diversity showed that there was no obvious difference in richness and diversity between healthy and Aeromonas veronii-infected Yangtze finless porpoise. Firmicutes, Bacteroidetes and Proteobacteria were the most dominant phyla in all samples. In addition, the healthy Yangtze finless porpoise exhibited higher abundance of Firmicutes and Fusobacteria than Aeromonas veronii-infected Yangtze finless porpoise, while, the level of Proteobacteria was decreased. At the genus level, Paeniclostridium and Paraclostridium were the predominant bacteria genera in the CK (healthy Yangtze finless porpoise) group. In the DIS (Aeromonas veronii-infected Yangtze finless porpoise) group, Lactobacillus and unidentified_Enterobacteriaceae were the dominant bacteria genera and the proportion of Paeniclostridium, Paraclostridium, Terrisporobacter, Cetobacterium, Candidatus Arthromitus, Terrabacter and Dechloromonas were reduced. CONCLUSIONS: In conclusion, our results showed that Aeromonas veronii infection can alter the gut microbiota of the Yangtze finless porpoise by affecting the number of harmful bacteria and beneficial bacteria.

Evoked-potential audiogram variability in a group of wild Yangtze finless porpoises (Neophocaena asiaeorientalis asiaeorientalis).

Hearing is considered the primary sensory modality of cetaceans and enables their vital life functions. Information on the hearing sensitivity variability within a species obtained in a biologically relevant wild context is fundamental to evaluating potential noise impact and population-relevant management. Here, non-invasive auditory evoked-potential methods were adopted to describe the audiograms (11.2-152 kHz) of a group of four wild Yangtze finless porpoises (Neophocaena asiaeorientalis asiaeorientalis) during a capture-and-release health assessment project in Poyang Lake, China. All audiograms presented a U shape, generally similar to those of other delphinids and phocoenids. The lowest auditory threshold (51-55 dB re 1 µPa) was identified at a test frequency of 76 kHz, which was higher than that observed in aquarium porpoises (54 kHz). The good hearing range (within 20 dB of the best hearing sensitivity) was from approximately 20 to 145 kHz, and the low- and high-frequency hearing cut-offs (threshold > 120 dB re l µPa) were 5.6 and 170 kHz, respectively. Compared with aquarium porpoises, wild porpoises have significantly better hearing sensitivity at 32 and 76 kHz and worse sensitivity at 54, 108 and 140 kHz. The audiograms of this group can provide a basis for better understanding the potential impact of anthropogenic noise.

Changes in the fecal microbiome of the Yangtze finless porpoise during a short-term therapeutic treatment.

The fecal microbiome is an integral part of aquatic mammals, like an inner organ. But we know very little about this inner organ of the threatened aquatic species, Yangtze finless porpoise (YFP). Four YFPs were placed into a purse seine for skin ulceration treatment, and this opportunity was taken to nurse the animals closer. In particular, we collected the feces of the YFPs before and after the paired healing and therapeutic treatment, along with samples of their fish diet and water habitat, to explore the changes in their fecal microbiome. Firmicutes (20.9-96.1%), Proteobacteria (3.8-78.7%), Actinobacteria (0.1-35.0%) and Tenericutes (0.8-17.1%) were the most dominant phyla present in the feces. The proportion of Proteobacteria and Actinobacteria increased after the treatment. Firmicutes showed a significant decrease, and most potential pathogens were absent, which reflected the administration of ciprofloxacin hydrochloride. Moreover, environmental shifts can also contribute to changes in the fecal microbiome. These results indicate that certain microbial interactions can be affected by environmental shifts, dietary changes and health-care treatments, which can also help maintain the internal environment of YFPs. These findings will inform the future enhanced protection and management of endangered YFPs and other vulnerable aquatic animals.

Non-invasive Fecal Steroid Measurements for Monitoring the Reproductive Status of a Critically Endangered Yangtze Finless Porpoises (Neophocaena asiaeorientalis asiaeorientalis).

Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) is a critically endangered freshwater cetacean dwelling in the Yangtze River and its adjoining lakes. Affected mainly by the various anthropogenic activities in this region, its population decreased dramatically in the past two decades. To protect this animal from extinction, captive breeding program is an important way to provide basic knowledge for wild population conservation. Non-invasive fecal steroid radioimmunoassay technique was validated in three captive Yangtze finless porpoises for the first time in this study. The seasonality of one captive male and the reproductive status of two females were investigated by longitudinal monitoring their fecal reproductive steroid hormone metabolites. Pregnancy could be diagnosed by an abrupt increase in fecal progesterone metabolites. In late pregnancy (4 months before birth), a significant decrease of fecal progesterone metabolites was observed, which might be referenced for the expectation of parturition date. Seven estrous cycles were recognized in one breeding season of a non-pregnant female judged by the variation of fecal progesterone metabolite levels. The fecal progesterone metabolite level was proved a reliable and precise indicator for estrus and pregnancy diagnosis.

Hematologic and Biochemical Reference Interval Development and the Effect of Age, Sex, Season, and Location on Hematologic Analyte Concentrations in Critically Endangered Yangtze Finless Porpoise (Neophocaena asiaeorientalis ssp. asiaeorientalis).

In this study, references intervals for 49 clinicopathological parameters were established for the critically endangered Yangtze finless porpoise (YFP) (Neophocaena phocaenoides asiaorientalis). Both from the wild (Poyang Lake) and seminatural (Tian-E-Zhou Oxbow) populations, individual blood samples from 188 animals were collected from 2009 to 2017 and from 2002 to 2015, respectively. For reference interval determination, we used a non-parametric bootstrap-based procedure to determine the 95th percentiles and the associated 90% confidence interval for each analyte. Our results indicated a need to partition the analyte concentrations by sex, age group, or pregnancy; however, we did not find a need to partition results by location. We then used a linear mixed model to determine if evidence existed for mean differences between location with sex and season as covariates and age group as the clustered random variable on mean hematological parameters in the YFP. We found that 88% of the analytes were significantly different between locations. Within the covariates, sex and season showed 31 and 69% significant difference in mean distributions, respectively. Additionally, age group provided a significant source of variation in 25% of the analytes. In summary, our finding suggests that analytes should be grouped according to sex, age, and reproductive status (non-pregnant and non-lactating, pregnant and lactating). Furthermore, we have provided the first set of reference intervals for 49 clinicopathological parameters that could provide guidelines for the initial evaluation of individuals during health assessments.

Trace elements accumulation in the Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) - A threat to the endangered freshwater cetacean.

As a freshwater cetacean with a population of only approximately 1000 individuals, the Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) is threatened by water pollution. However, studies of contaminants accumulated in the Yangtze finless porpoise remain limited. In this study, concentrations of 11 trace elements in different tissues sampled from 38 Yangtze finless porpoise individuals were determined. The elements V, Ni, Zn, and Pb were mostly accumulated in the epidermis, Cr, Mn, Cu, Se, and Hg were mostly accumulated in the liver, while As and Cd were mostly accumulated in the blubber and kidney, respectively. The results show that trace elements concentrations in the epidermis do not reliably indicate concentrations in internal tissues of the Yangtze finless porpoises. Positive correlations between different trace elements concentrations in tissues with the highest concentrations suggested the similar mechanism of metabolism or uptake pathway of those elements. Concentrations of As, Se, Cd, Hg, and Pb in the tissues with the highest concentrations were significantly positively correlated with the body length. Furthermore, significantly higher trace elements concentrations were measured in the reproductive organs of females (ovaries) than males (testis). However, no significant difference of trace elements concentrations between habitats was found. In consideration of higher Hg and Cd level in Yangtze finless porpoises compared to other small cetaceans, the potential risk of Hg (in particular) and Cd toxicity to Yangtze finless porpoises needs further attention.

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.

Immune Responses of the Critically Endangered Yangtze Finless Porpoises (Neophocaena asiaeorientalis ssp. asiaeorientalis) to Escalating Anthropogenic Stressors in the Wild and Seminatural Environments.

Increasing anthropogenic stressors are potential threats to biodiversity conservation and management of Yangtze finless porpoises (YFPs). The objective of this study was to indirectly compare the habitat quality of a natural reserve, Poyang Lake and a seminatural reserve, the Tian-E-Zhou Oxbow (TZO) in terms of anthropogenic stressors by investigating different stress and immunological parameters in the blood of YFPs. Samples from a total of 74 YFPs from the TZO (n = 43) and Poyang Lake (n = 31) were collected and analyzed. The animals were divided into ontogenetic groups: male calf, female calf, juvenile female, juvenile male, and adult male, and reproductive groups: pregnant female, lactating female, and pregnant plus lactating. The blood from all the animals was analyzed for general stress (HSP14, SOD1, TXN, and FTL), metabolic stress (ACAT2 and THRA), and immunity-related genes (IL12p40, IFNγ, TNFα; IL1α, IL1ra, COX2, CRPL, IL4, and IL8) using qPCR. YFPs living in Poyang Lake showed an increased relative expression pattern for IFNγ, IL1ra, IL4, ACAT2, and CRPL across all the ontogenetic groups with significantly higher expression in adult males. In contrast, YFPs living in the TZO showed a significantly higher expression in 13 of 15 genes analyzed in the male calf group. Across the reproductive states for porpoises living in Poyang Lake, eight of the 15 genes in the pregnant female and three of the 15 genes in the pregnant plus lactating group had a significantly higher expression level. However, in YFPs living in the TZO, eight of the 15 genes showed significantly higher expression in the pregnant and lactating groups. There was significantly a higher expression of most of the genes in porpoises living in the TZO compared to the age-matched groups from porpoises living in Poyang Lake. The exception was the pregnant female group. The higher relative expression of stress and immune genes in the TZO porpoise population compared to porpoises living in Poyang Lake suggests the effects of worsening habitat quality, possibly indicating water pollution and lack of feeding resources.

Physiological consequences of biologic state and habitat dynamics on the critically endangered Yangtze finless porpoises (Neophocaena asiaeorientalis ssp. asiaeorientalis) dwelling in the wild and semi-natural environment.

The objectives of this study were to investigate the effects of habitat and biological state on the physiology of critically endangered wild and semi-natural Yangtze Finless Porpoises (YFPs; Neophocaena asiaeorientalis ssp. asiaeorientalis) by measuring and comparing serum biochemical parameters. A total of 168 YFPs were sampled, 68 living in the semi-natural (Tian-E-Zhou Oxbow) and 98 living in the wild (Poyang Lake, PL) environment. The YFPs in the Tian-E-Zhou Oxbow were sampled from 2002 to 2015 and in the PL from 2009 to 2017. Each population was divided into Juvenile Male, Juvenile Female, Adult Male, Pregnant and Lactating Female life history categories. Overall, with location, 19/33 of the analytes and with season 18/33 of the analytes were significantly different. Similarly, within each location, 15/33 of the analytes changed with time in PL while only 8/33 changed with time in Tian-E-Zhou Oxbow, respectively. Finally, 15/33 of the analytes demonstrated significant differences between the different age and sex groups of animals. In our study, a significant variation, as well as an increasing and decreasing pattern of several parameters in both populations, suggest a worsening ecological environment of both habitats. This study will help in health assessment, improving conservation and management practices, a crucial requisite for biodiversity conservation.

The Possible Effects of High Vessel Traffic on the Physiological Parameters of the Critically Endangered Yangtze Finless Porpoise (Neophocaena asiaeorientalis ssp. asiaeorientalis).

Background: Poyang is the largest freshwater lake in China, where the acoustic environment and space for the critically endangered Yangtze finless porpoises (YFPs) has been altered by heavy vessel traffic and dredging activities. The density of vessel and the rate of dredging increases annually, especially in the area with the highest density of YFPs. The heavy vessel traffic can cause an increase in the physical activities and direct physical injuries to the YFPs. Furthermore, noise is a potent stressor to all cetaceans irrespective of age and can compromise all their physiological functions. The objective of this study was to examine the possible effects of heavy vessel traffic and dredging on the biochemistry, hematology, adrenal, thyroid, and reproductive hormones of two different YFP populations. One population was living in Poyang Lake and the second living in the Tian-E-Zhou Oxbow which is a semi-natural resserve. Results: The results showed statistically significantly higher levels of serum cortisol, fT3, fT4, and lowered testosterone in both adult and juvenile YFPs living in Poyang Lake vs. adult YFPs living in the Tian-E-Zhou Oxbow. The serum biochemical parameters (Aspartate Amino Transferase, Alkaline Phosphatase, High Density Lipoprotein cholesterol ratio, Globulin, Uric acid, Glucose, K+, and Amylase) and the hematology parameters (Red Blood Cells, Hematocrit, Mean Corpuscular Volume, White Blood Cells, and Eosinophils) were statistically significantly higher in the adult Poyang Lake YFPs vs. adult Tian-E-Zhou Oxbow YFPs. On the other hand, adult males of the Tian-E-Zhou Oxbow also showed significantly higher levels of the serum biochemical parameters (Total Cholesterol, Light Density Lipoprotein cholesterol, Direct Bilirubin, Albumin, Lactate Dehydrogenase, CO2, and Na+) and the blood parameters (Mean Corpuscular Hemoglobin and Mean Corpuscular Hemoglobin Concentration). In Poyang Lake YFPs, various parameters showed significantly positive (fT4, amylase, neutrophil, Ca+2) or negative (total protein, lymphocyte) correlations with cortisol levels. Conclusions: The hyperactivity of adrenal glands in response to heavy vessel traffic and dredging resulted in significantly elevated cortisol levels in Poyang Lake YFPs. The higher cortisol level could possibly have affected various hormonal, hematological, and biochemical parameters, and ultimately the YFPs physiology.