Jinsong Zheng

Massive losses of taste receptor genes in toothed and baleen whales

Taste receptor genes are functionally important in animals, with a surprising exception in the bottlenose dolphin, which shows extensive losses of sweet, umami, and bitter taste receptor genes. To examine the generality of taste gene loss, we examined seven toothed whales and five baleen whales and sequenced the complete repertoire of three sweet/umami (T1Rs) and ten bitter (T2Rs) taste receptor genes. We found all amplified T1Rs and T2Rs to be pseudogenes in all 12 whales, with a shared premature stop codon in 10 of the 13 genes, which demonstrated massive losses of taste receptor genes in the common ancestor of whales. Furthermore, we analyzed three genome sequences from two toothed whales and one baleen whale and found that the sour taste marker gene Pkd2l1 is a pseudogene, whereas the candidate salty taste receptor genes are intact and putatively functional. Additionally, we examined three genes that are responsible for taste signal transduction and found the relaxation of functional constraints on taste signaling pathways along the ancestral branch leading to whales. Together, our results strongly suggest extensive losses of sweet, umami, bitter, and sour tastes in whales, and the relaxation of taste function most likely arose in the common ancestor of whales between 36 and 53 Ma. Therefore, whales represent the first animal group to lack four of five primary tastes, probably driven by the marine environment with high concentration of sodium, the feeding behavior of swallowing prey whole, and the dietary switch from plants to meat in the whale ancestor.

Population Genetic Structure of the Yangtze Finless Porpoise (Neophocaena phocaenoides asiaeorientalis): Implications for Management and Conservation

Understanding the population genetic structure is a prerequisite for conservation of a species. The degree of genetic variability characteristic of the mitochondrial DNA control region has been widely exploited in studies of population genetic structure and can be useful in identifying meaningful population subdivisions. To estimate the genetic profile of the Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis), an endangered freshwater population endemic to China, the complete mtDNA control region was examined in 39 individuals belonging to seven different stocks inhabiting the middle and lower reaches of the Yangtze River. Very low genetic diversity was found (nucleotide diversity 0.0011± 0.0002 and haplotypic diversity 0.65± 0.05). The mtDNA genetic pattern of the Yangtze population appears to indicate a founder event in its evolutionary history and to support the marine origin for this population. Analyses by Fst and Φst yielded statistically significant population genetic structure (Fst = 0.44, P < 0.05; Φst = 0.36, P < 0.05). These results may have significant implications for the management and conservation of the Yangtze finless porpoise in the future.

Analysis of the bacterial diversity in the fecal material of the endangered Yangtze finless porpoise, Neophocaena phocaenoides asiaeorientalis

The aim of this study was to determine the bacteria present in the fecal material of the endangered Yangtze finless porpoise, Neophocaena phocaenoides asiaeorientalis. Fecal samples were collected from 12 Yangtze finless porpoises living in the wild at Poyang Lake, located in Jiangxi Province, China. To determine the bacterial diversity, a 16S rRNA gene clone library using the bacterial PCR primers fD1 and rP2, was prepared. A total of 138 near-full-length sequences were analyzed and 39 operational taxonomic units (OTUs) were identified. Sequences showing ≥97% similarity were grouped together as an OTU. Six different phyla were identified in which 38 OTUs were classified. Most of the OTUs contained sequences belonged to the phylum Firmicutes (51.3%), followed by Tenericutes (17.9%), Proteobacteria (15.4%), Actinobacteria (7.7%), Deinococcus-Thermus (2.6%) and Cyanobacteria (2.6%). A phylum could not be assigned for one clone within one OTU (2.6%). It appears that the Yangtze finless porpoise has a more diverse range of bacteria compared to other aquatic mammals, such as seals.

Parentage determination of an isolated Yangtze finless porpoise population Neophocaena phocaenoides asiaeorientalis in the Yangtze Tian-e-Zhou Baiji National Natural Reserve based on molecular data

Reproductive behaviors are poorly known for the Yangtze finless porpoise Neophocaena phocaenoides asiaeorientalis. In this study, the parentage of an isolated Yangtze finless porpoise population inhabiting the Yangtze Tian-e-Zhou Baiji National Natural Reserve is determined by analysis of microsatellite loci and mitochondrial DNA (mtDNA) control region sequences, and the porpoises reproductive behaviors are studied. Overall 4 full parentage assignments and additional 3 single parentage assignments were determined for the population of 23 individuals. The analysis shows that their estimated reproductive cycle is shorter than that reported previously and there probably exists an overlapping between gestation and lactation period. The Study also shows that the female does not show fidelity to a particular male for breeding and vice versa, the oldest males did not monopolize mating and the dominance rank could not be so strict for the porpoise society. Moreover, the porpoises mating pattern and relatedness among candidate parents are discussed here. These results provide important information for making guidelines of management and conservation for this protected population.

Fecal Bacterial Composition of the Endangered Yangtze Finless Porpoises Living Under Captive and Semi-natural Conditions

Intestinal microbiota is essential to the health and physiology of host animals. We undertook the first microbiological study of the fecal bacterial composition from critically endangered (CR) Yangtze finless porpoises (Neophocaena asiaeorientalis asiaeorientalis; YFPs) living under captive and semi-natural conditions using both high-throughput sequencing method and 16S rRNA gene clone library method. As determined by high-throughput sequencing of V3–V4 regions of the 16S rRNA gene, semi-natural samples harbored 30 and 36 operational taxonomic units (OTUs), which was more than the 22 and 27 OTUs detected from YFPs living in captivity. In captive YFPs Firmicutes was the predominant phylum, whereas this was Proteobacteria for YFPs living in semi-nature conditions. This suggests habitat-specific fecal bacterial composition of YFPs. Plesiomonas spp. and Aeromonas spp., which are potentially pathogenic, were identified in all the feces. Bacterial diversity from one porpoise living in captivity was also determined by constructing a 16S rRNA gene clone library and only 1 phylum was identified. High-throughput sequencing was more effective at determining the bacterial diversity compared to the 16S rRNA gene clone library. This study provides important information for the management and conservation of the CR YFPs.

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.

Physicochemical Evolution and Molecular Adaptation of the Cetacean Osmoregulation-related Gene UT-A2 and Implications for Functional Studies

Cetaceans have an enigmatic evolutionary history of re-invading aquatic habitats. One of their essential adaptabilities that has enabled this process is their homeostatic strategy adjustment. Here, we investigated the physicochemical evolution and molecular adaptation of the cetacean urea transporter UT-A2, which plays an important role in urine concentration and water homeostasis. First, we cloned UT-A2 from the freshwater Yangtze finless porpoise, after which bioinformatics analyses were conducted based on available datasets (including freshwater baiji and marine toothed and baleen whales) using MEGA, PAML, DataMonkey, TreeSAAP and Consurf. Our findings suggest that the UT-A2 protein shows folding similar to that of dvUT and UT-B, whereas some variations occurred in the functional So and Si regions of the selectivity filter. Additionally, several regions of the cetacean UT-A2 protein have experienced molecular adaptations. We suggest that positive-destabilizing selection could contribute to adaptations by influencing its biochemical and conformational character. The conservation of amino acid residues within the selectivity filter of the urea conduction pore is likely to be necessary for urea conduction, whereas the non-conserved amino acid replacements around the entrance and exit of the conduction pore could potentially affect the activity, which could be interesting target sites for future mutagenesis studies.

Isolation of Robinsoniella peoriensis from the fecal material of the endangered Yangtze finless porpoise, Neophocaena asiaeorientalis asiaeorientalis

The aim of this study was to determine the causative agent of diarrhea in an endangered Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis). From the fecal material collected from this porpoise Robinsoniella peoriensis was isolated.

Organization and characteristics of the major histocompatibility complex class II region in the Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis)

Little is known about the major histocompatibility complex (MHC) in the genome of Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) (YFP) or other cetaceans. In this study, a high-quality YFP bacterial artificial chromosome (BAC) library was constructed. We then determined the organization and characterization of YFP MHC class II region by screening the BAC library, followed by sequencing and assembly of positive BAC clones. The YFP MHC class II region consists of two segregated contigs (218,725 bp and 328,435 bp respectively) that include only eight expressed MHC class II genes, three pseudo MHC genes and twelve non-MHC genes. The YFP has fewer MHC class II genes than ruminants, showing locus reduction in DRB, DQA, DQB, and loss of DY. In addition, phylogenic and evolutionary analyses indicated that the DRB, DQA and DQB genes might have undergone birth-and-death evolution, whereas the DQB gene might have evolved under positive selection in cetaceans. These findings provide an essential foundation for future work, such as estimating MHC genetic variation in the YFP or other cetaceans. This work is the first report on the MHC class II region in cetaceans and offers valuable information for understanding the evolution of MHC genome in cetaceans.