Genetics & evolution
Two colepid ciliates, Coleps amphacanthus Ehrenberg, 1833 and Levicoleps biwae jejuensis Chen et al., 2016, were first recorded in China. Their living morphology, infraciliature and small subunit (SSU) rRNA gene sequences were determined using standard methods. The improved diagnosis of Coleps amphacanthus is as follows:cell size about 100×50 μm in vivo, barrel-shaped; 22-28 ciliary rows each composed of about 14-21 monokinetids and two perioral dikinetids; 5-10 caudal cilia; and one terminal contractile vacuole. Levicoleps biwae jejuensis was also investigated, with an improved diagnosis given based on previous and present work. The phylogenetic analyses based on SSU rRNA gene sequences revealed that all Coleps species were grouped together, except for Coleps amphacanthus, which was grouped into a clade of the genus Levicoleps.
Ayu (Plecoglossus altivelis) fish, which are an amphidromous species distributed in East Asia, live in brackish water (BW) during their larval stage and in fresh water (FW) during their adult stage. In this study, we found that FW-acclimated ayu larvae exhibited a slower growth ratio compared with that of BW-acclimated larvae. However, the mechanism underlying FW acclimation on growth suppression is poorly known. We employed transcriptome analysis to investigate the differential gene expression of FW acclimation by RNA sequencing. We identified 158 upregulated and 139 downregulated transcripts in FW-acclimated ayu larvae compared with that in BW-acclimated larvae. As determined by Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway mapping, functional annotation of the genes covered diverse biological functions and processes, and included neuroendocrinology, osmotic regulation, energy metabolism, and the cytoskeleton. Transcriptional expression of several differentially expressed genes in response to FW acclimation was further confirmed by real-time quantitative PCR. In accordance with transcriptome analysis, iodothyronine deiodinase (ID), pro-opiomelanocortin (POMC), betaine-homocysteine S-methyltransferase 1(BHMT), fructose-bisphosphate aldolase B (aldolase B), tyrosine aminotransferase (TAT), and Na+-K+ ATPase (NKA) were upregulated after FW acclimation. Furthermore, the mRNA expressions of b-type natriuretic peptide (BNP) and transgelin were downregulated after FW acclimation. Our data indicate that FW acclimation reduced the growth rate of ayu larvae, which might result from the expression alteration of genes related to endocrine hormones, energy metabolism, and direct osmoregulation.
Carotenoids, which generate yellow, orange, and red colors, are crucial pigments in avian plumage. Investigations into genes associated with carotenoidbased coloration in avian species are important; however, such research is difficult because carotenoids cannot be synthetized in vertebrates as they are only derived from dietary sources. Here, the golden pheasant (Chrysolophus pictus) was used as a model in analysis of candidate gene expression profiles implicated in carotenoid binding and deposition. Using mass and Raman spectrometry to confirm the presence of carotenoids in golden pheasant feathers, we found C40H54O and C40H56O2 in feathers with yellow to red colors, and in the rachis of iridescent feathers. The global gene expression profiles in golden pheasant skins were analyzed by RNA-seq and all six carotenoid binding candidate genes sequenced were studied by realtime PCR. StAR4, GSTA2, Scarb1, and APOD in feather follicles showed different expressions in red breast and orange nape feathers compared with that of iridescent mantle feathers. Further comparison of golden pheasant yellow rump and Lady Amherst's pheasant (Chrysolophus amherstiae) white nape feathers suggested that GSTA2 and APOD played a potential role in carotenoid-based coloration in golden pheasant.
As a novel experimental animal model, tree shrews have received increasing attention in recent years. Despite this, little is known in regards to the time phases of their embryonic development. In this study, surveillance systems were used to record the behavior and timing of copulations; embryos at different post-copulation stages were collected and cultured in vitro; and the developmental characteristics of both early-stage and in vitro cultured embryos were determined. A total of 163 females were collected following effective copulation, and 150 were used in either unilateral or bilateral oviduct embryo collections, with 307 embryos from 111 females obtained (conception rate=74%). Among them, 237 embryos were collected from 78 females, bilaterally, i.e., the average embryo number per female was 3.04; 172 fertilized eggs collected from 55 females, bilaterally, were cultured for 24-108 h in vitro for developmental observations; finally, 65 embryos from 23 bilateral cases and 70 embryos from 33 unilateral cases were used in embryo transplantation.
The World Health Organization has declared the present Zika virus epidemic to be a ‘Public Health Emergency of International Concern’. The virus appears to have spread from Thailand to French Polynesia in 2013, and has since infected over a million people in the countries of South and Central America. In most cases the infection is mild and transient, but the virus does appear to be strongly neurotropic and the presumptive cause of both birth defects in fetuses and Guillain-Barré syndrome in some adults. In this paper, the techniques and utilities developed in the study of mitochondrial DNA were applied to the Zika virus. As a result, it is possible to show in a simple manner how a phylogenetic tree may be constructed and how the mutation rate of the virus can be measured. The study showed the mutation rate to vary between 12 and 25 bases a year, in a viral genome of 10272 bases. This rapid mutation rate will enable the geographic spread of the epidemic to be monitored easily and may also prove useful in assisting the identification of preventative measures that are working, and those that are not.
Six main mitochondrial DNA (mtDNA) lineages have been described in minnow (Zacco platypus) samples obtained from northern, western and southern China. Perdices et al. (2004) predicted that further sampling of other tributaries might discover more lineages of this species. In this study, we collected 26 Zacco platypus individuals in the Huangshan area of eastern China and determined the cytochrome b (cytb) sequence variations. Combined with reported data in GenBank, we identified ten matrilines (Zacco A-J) in a total of 169 samples, with relatively high molecular divergence found among them. The Huangshan population had the greatest genetic variation among all sampled regions and hosted six of the ten matrilines. Our results highlight the significance of the Huangshan area for the conservation of Zacco platypus.
Colony-stimulating factor 1 receptor (CSF-1R) is an important regulator of monocytes/macrophages (MO/MΦ). Although several CSF-1R genes have been identified in teleosts, the precise role of CSF- 1R in ayu (Plecoglossus altivelis) remains unclear. In this study, we characterized the CSF-1R homologue from P. altivelis, and named it PaCSF-1R. Multiple sequence alignment and phylogenetic tree analysis showed that PaCSF-1R was most closely related to that of Japanese ricefish (Oryzias latipes). Tissue distribution and expression analysis showed that the PaCSF-1R transcript was mainly expressed in the head kidney-derived MO/MΦ, spleen, and head kidney, and its expression was significantly altered in various tissues upon Vibrio anguillarum infection. After PaCSF-1R neutralization for 48 h, the phagocytic activity of MO/MΦ was significantly decreased, suggesting that PaCSF-1R plays a role in regulating the phagocytic function of ayu MO/MΦ.
Targeted genome editing technology has been widely used in biomedical studies. The CRISPR-associated RNA-guided endonuclease Cas9 has become a versatile genome editing tool. The CRISPR/Cas9 system is useful for studying gene function through efficient knock-out, knock-in or chromatin modification of the targeted gene loci in various cell types and organisms. It can be applied in a number of fields, such as genetic breeding, disease treatment and gene functional investigation. In this review, we introduce the most recent developments and applications, the challenges, and future directions of Cas9 in generating disease animal model. Derived from the CRISPR adaptive immune system of bacteria, the development trend of Cas9 will inevitably fuel the vital applications from basic research to biotechnology and bio-medicine.
Until recently, the agamid species, Japalura flaviceps, was recognized to have the widest geographic distribution among members of the genus occurring in China, from eastern Tibet to Shaanxi Province. However, recent studies restricted the distribution of J. flaviceps to the Dadu River valley only in northwestern Sichuan Province, suggesting that records of J. flaviceps outside the Dadu River valley likely represent undescribed diversity. During two herpetofaunal surveys in 2013 and 2015, eight and 12 specimens of lizards of the genus Japalura were collected from the upper Nujiang (=Salween) Valley in eastern Tibet, China, and upper Lancang (=Mekong) Valley in northwestern Yunnan, China, respectively. These specimens display a unique suite of diagnostic morphological characters. Our robust comparisons of phenotype reveal that these populations can be distinguished readily from J. flaviceps and all other recognized congeners. Herein, we describe the two Japalura lineages as new species, Japalura laeviventris sp. nov. and Japalura iadina sp. nov.. In addition, we provide updated conservation assessments for the new species as well as imperiled congeners according to the IUCN criteria for classification, discuss the importance of color patterns in the diagnosis and description of species in the genus Japalura, and discuss directions for future taxonomic studies of the group.
A new species of the genus Amolops Cope, 1865 is described from Nyingchi, southeastern Tibet, China, based on morphological and molecular data. The new species, Amolops nyingchiensis sp. nov. is assigned to the Amolops monticola group based on its skin smooth, dorsolateral fold distinct, lateral side of head black, upper lip stripe white extending to the shoulder. Amolops nyingchiensis sp. nov. is distinguished from all other species of Amolops by the following combination of characters: (1) medium body size, SVL 48.5-58.3 mm in males, and 57.6-70.7 mm in females; (2) tympanum distinct, slightly larger than one third of the eye diameter; (3) a small tooth-like projection on anteromedial edge of mandible; (4) the absence of white spine on dorsal surface of body; (5) the presence of circummarginal groove on all fingers; (6) the presence of vomerine teeth; (7) background coloration of dorsal surface brown, lateral body gray with yellow; (8) the presence of transverse bands on the dorsal limbs; (9) the presence of nuptial pad on the first finger in males; (10) the absence of vocal sac in males. Taxonomic status of the populations that were previously identified to A. monticola from Tibet is also discussed.
A new species of Scutiger Theobald, 1868 is described from Medog, southeastern Tibet, China, based on morphological and molecular data. The new species was previously identified as Scutiger nyingchiensis, but it can be differentiated from the latter and all other congeners by the following combination of characters: (1) medium adult body size, SVL 50.5-55.6 mm in males and 53.8-57.2 mm in females; (2) maxillary teeth absent; (3) web rudimentary between toes; (4) prominent, conical-shaped tubercles on dorsal and lateral surfaces of body and limbs; (5) tubercles covered by black spines in both sexes in breeding condition; (6) a pair of pectoral glands and a pair of axillary glands present and covered by black spines in males in breeding condition, width of axillary gland less than 50% of pectoral gland; (7) nuptial spines present on dorsal surface of first and second fingers, and inner side of third finger in males in breeding condition; (8) spines absent on the abdominal region; (9) vocal sac absent. In addition, the distribution and conservation status of the new species are also discussed.
A new genus and species of threefrog is described from Medog, southeastern Tibet, China based on morphological and phylogenetic data. The new genus can be distinguished from other treefrog genera by the following combination of characters: (1) body size moderate, 45.0 mm in male; (2) snout rounded; (3) canthus rostralis obtuse and raised prominently, forming a ridge from nostril to anterior corner of eyes; (4) web rudimentary on fingers; (5) web moderately developed on toes; (6) phalange "Y" shaped, visible from dorsal side of fingers and toes; (7) skin of dorsal surfaces relatively smooth, scatted with small tubercles; (8) iris with a pale yellow, "X" shaped pattern of pigmentation.
In an effort to study the systematic affinities and specieslevel phylogenetic relationships of the enigmatic anurans variably assigned to the genera Ingerana or Limnonectes (family Dicroglossidae), we collected new molecular sequence data for five species including four Himalayan taxa, Limnonectes xizangensis, Lim. medogensis, Lim. alpine, Ingerana borealis and one southeast Asian species, I. tasanae, and analyzed these together with data from previous studies involving other ostensibly related taxa. Our surprising results demonstrate unequivocally that Lim. xizangensis, Lim. medogensis and Lim. alpine form a strongly supported clade, the sister-group of the family Australasian forest frog family Ceratobatrachidae. This discovery requires an expansion of the definition of Ceratobatrachidae and represents the first record of this family in China. These three species are distinguished from the species of Ingerana and Limnonectes by the: (1) absence of interdigital webbing of the foot, (2) absence of terminal discs on fingers and toes, (3) absence of circumarginal grooves on the fingers and toes, and (4) absence of tarsal folds. Given their phylogenetic and morphological distinctiveness, we assign them to the oldest available generic name for this clade, Liurana Dubois 1987, and transfer Liurana from Dicroglossidae to the family Ceratobatrachidae. In contrast, Ingerana tasanae was found to be clustered with strong support with the recently described genus Alcalus (Ceratobatrachidae), a small clade of otherwise Sundaic species; this constitutes a new record of the family Ceratobatrachidae for Myanmar and Thailand. Finally, Ingerana borealis clustered with the "true" Ingerana (family Dicroglossidae), for which the type species is I. tenasserimensis.
Comparative genomics is a powerful approach that comprehensively interprets the genome. Herein, we performed whole genome comparative analysis of 16 Diptera genomes, including four mosquitoes and 12 Drosophilae. We found more than 540 000 constraint elements (CEs) in the Diptera genome, with the majority found in the intergenic, coding and intronic regions. Accelerated elements (AEs) identified in mosquitoes were mostly in the protein-coding regions (>93%), which differs from vertebrates in genomic distribution. Some genes functionally enriched in blood digestion, body temperature regulation and insecticide resistance showed rapid evolution not only in the lineage of the recent common ancestor of mosquitoes (RCAM), but also in some mosquito lineages. This may be associated with lineage-specific traits and/or adaptations in comparison with other insects. Our findings revealed that although universally fast evolution acted on biological systems in RCAM, such as hematophagy, same adaptations also appear to have occurred through distinct degrees of evolution in different mosquito species, enabling them to be successful blood feeders in different environments.
Recent advances in high-throughput sequencing technologies have revolutionized the field of population genetics. Data now routinely contain genomic level polymorphism information, and the low cost of DNA sequencing enables researchers to investigate tens of thousands of subjects at a time. This provides an unprecedented opportunity to address fundamental evolutionary questions, while posing challenges on traditional population genetic theories and methods. This review provides an overview of the recent methodological developments in the field of population genetics, specifically methods used to infer ancient population history and investigate natural selection using large-sample, large-scale genetic data. Several open questions are also discussed at the end of the review.
Venom (toxins) is an important trait evolved along the evolutionary tree of animals. Our knowledges on venoms, such as their origins and loss, the biological relevance and the coevolutionary patterns with other organisms are greatly helpful in understanding many fundamental biological questions, i.e., the environmental adaptation and survival competition, the evolution shaped development and balance of venoms, and the sophisticated correlations among venom, immunity, body power, intelligence, their genetic basis, inherent association, as well as the cost-benefit and trade-offs of biological economy. Lethal animal envenomation can be found worldwide. However, from foe to friend, toxin studies have led lots of important discoveries and exciting avenues in deciphering and fighting human diseases, including the works awarded the Nobel Prize and lots of key clinic therapeutics. According to our survey, so far, only less than 0.1% of the toxins of the venomous animals in China have been explored. We emphasize on the similarities shared by venom and immune systems, as well as the studies of toxin knowledge-based physiological toxin-like proteins/peptides (TLPs). We propose the natural pairing hypothesis. Evolution links toxins with humans. Our mission is to find out the right natural pairings and interactions of our body elements with toxins, and with endogenous toxin-like molecules. Although, in nature, toxins may endanger human lives, but from a philosophical point of view, knowing them well is an effective way to better understand ourselves. So, this is why we study toxins.
Interleukin 1β (IL-1β), the first interleukin to be characterized, plays a key role in regulating the immune response. In this study, we determined the cDNA and genomic DNA sequences of the IL-1β gene from the large yellow croaker, Larimichthys crocea. Phylogenetic analysis indicated that the IL-1β (LcIL-1β) gene was most closely related to that of European seabass (Dicentrarchus labrax), sharing 67.8% amino acid identity. In healthy large yellow croaker, LcIL-1β transcription was detected in all tested tissues, with the highest level found in the head kidney. Upon Vibrio alginolyticus infection, LcIL-1β transcription in all tested tissues was significantly upregulated. Intraperitoneal injection of recombinant LcIL-1β (rLcIL-1β) improved the survival rate and reduced the tissue bacterial load after V. alginolyticus infection. In addition, rLcIL-1β induced monocytes/macrophages (MO/MΦ) chemotaxis and increased phagocytosis and bactericidal activity in vitro. These results suggest that LcIL-1β plays an important role in the large yellow croaker immune response against V. alginolyticus.
Many ecological studies and conservation management plans employ noninvasive scat sampling based on the assumption that species' scats can be correctly identified in the field. However, in habitats with sympatric similarly sized carnivores, misidentification of scats is frequent and can lead to bias in research results. To address the scat identification dilemma, molecular scatology techniques have been developed to extract DNA from the donor cells present on the outer lining of the scat samples. A total of 100 samples were collected in the winter of 2009 and 2011 in Taxkorgan region of Xinjiang, China. DNA was extracted successfully from 88% of samples and genetic species identification showed that more than half the scats identified in the field as snow leopard (Panthera uncia) actually belonged to fox (Vulpes vulpes). Correlation between scat characteristics and species were investigated, showing that diameter and dry weight of the scat were significantly different between the species. However it was not possible to define a precise range of values for each species because of extensive overlap between the morphological values. This preliminary study confirms that identification of snow leopard feces in the field is misleading. Research that relies upon scat samples to assess distribution or diet of the snow leopard should therefore employ molecular scatology techniques. These methods are financially accessible and employ relatively simple laboratory procedures that can give an indisputable response to species identification from scats.
To explore the neural mechanisms mediating aging-related visual function declines, we compared the expressions of brain-derived neurotrophic factor (BDNF) and its high affinity receptor-tyrosine kinase B (TrkB) between young and old adult cats. Nissl staining was used to display neurons in each layer of the lateral geniculate nucleus (LGN). The BDNF- and TrkB receptor-immunoreactive neurons were labeled immunohistochemically, observed under optical microscope and photographed. Their neuronal density and immunoreactive intensity were measured. Results showed that the mean density of the Nissl stained neurons in each LGN layer were comparable between old and young adult cats, and their BDNF and TrkB proteins were widely expressed in all LGN layers. However, compared with young adult cats, both the density and optical absorbance intensity of BDNF- and TrkB-immunoreactive cells in each LGN layer in old cats were significantly decreased. These findings indicate that the decreased expressions of BDNF and TrkB proteins in the LGN may be an important factor inducing the compromised inhibition in the central visual nucleus and the functional visual decline in senescent individuals.
Multidrug resistant (MDR) pathogen infections are serious threats to hospitalized patients because of the limited therapeutic options. A novel group of antibiotic candidates, antimicrobial peptides (AMPs), have recently shown powerful activities against both Gram-negative and Gram-positive bacteria. Unfortunately, the viability of using these AMPs in clinical settings remains to be seen, since most still need to be evaluated prior to clinical trials and not all of AMPs are potent against MDR clinical isolates. To find a connection between the characteristics of several of these AMPs and their effects against MDR pathogens, we selected 14 AMPs of animal origin with typical structures and evaluated their in vitro activities against clinical strains of extensive drug-resistant Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus, extended spectrum β-lactamase-producing Pseudomonas aeruginosa and extended spectrum β-lactamase-producing Escherichia coli. Our results showed that these peptides' hydrophilic/hydrophobic characteristics, rather than their secondary structures, may explain their antibacterial effects on these clinical isolates. Peptides that are amphipathic along the longitudinal direction seemed to be effective against Gram-negative pathogens, while peptides with hydrophilic terminals separated by a hydrophobic intermediate section appeared to be effective against both Gram-negative and Gram-positive pathogens. Among these, cathelicidin-BF was found to inhibit all of the Gram-negative pathogens tested at dosages of no more than 16 mg/L, killing a pandrug-resistant A. baumannii strain within 2 h at 4×MICs and 4 h at 2×MICs. Tachyplesin III was also found capable of inhibiting all Gram-negative and Gram-positive pathogens tested at no more than 16 mg/L, and similarly killed the same A. baumannii strain within 4 h at 4×MICs and 2×MICs. These results suggest that both cathelicidin-BF and tachyplesin III are likely viable targets for the development of AMPs for clinical uses.
Lamprotula leai is one of the most commercially important freshwater pearl mussels in China, but there is limited data on its genetic diversity and population structure. In the present study, 119 individuals from four major geographical populations were investigated using 15 microsatellite loci identified via cross-species amplification. A total of 114 alleles were detected, with an average of 7.6 alleles per locus (range: 2 to 21). Among the four stocks, those from Hung-tse Lake and Poyang Lake had the lowest (0.412) and highest (0.455) observed heterozygosity respectively. The polymorphism information content (PIC) ranged from 0.374 to 0.927 (mean: 0.907). AMOVA showed that 12.56% and 44.68% genetic variances were among populations and within individuals, respectively. Pairwise Fst ranged from 0.073 to 0.146, indicating medium genetic differentiation among the populations. In aggregate, our results suggest that inbreeding is a crucial factor accounting for deviations from Hardy–Weinberg equilibrium at 12 loci. Moreover, the genetic distance among four stocks ranged from 0.192 to 0.890. Poyang Lake and Hung-tse Lake were clustered together, joined with Dongting Lake and Anqing Lake. Given that specimens from Hung-tse Lake showed the highest average allele richness, expected heterozygosity and PIC, this location may be the source of the highest quality germplasm resources and the stock from this area may be the best for future breeding efforts.
Ruminant stomach lysozyme is a long established model of adaptive gene evolution. Evolution of stomach lysozyme function required changes in the site of expression of the lysozyme c gene and changes in the enzymatic properties of the enzyme. In ruminant mammals, these changes were associated with a change in the size of the lysozyme c gene family. The recent release of near complete genome sequences from several ruminant species allows a more complete examination of the evolution and diversification of the lysozyme c gene family. Here we characterize the size of the lysozyme c gene family in extant ruminants and demonstrate that their pecoran ruminant ancestor had a family of at least 10 lysozyme c genes, which included at least two pseudogenes. Evolutionary analysis of the ruminant lysozyme c gene sequences demonstrate that each of the four exons of the lysozyme c gene has a unique evolutionary history, indicating that they participated independently in concerted evolution. These analyses also show that episodic changes in the evolutionary constraints on the protein sequences occurred, with lysozyme c genes expressed in the abomasum of the stomach of extant ruminant species showing the greatest levels of selective constraints.
The yellow meal worm (Tenebrio molitor L.) is an important resource insect typically used as animal feed additive. It is also widely used for biological research. The first complete mitochondrial genome of T. molitor was determined for the first time by long PCR and conserved primer walking approaches. The results showed that the entire mitogenome of T. molitor was 15 785 bp long, with 72.35% A+T content [deposited in GenBank with accession number KF418153]. The gene order and orientation were the same as the most common type suggested as ancestral for insects. Two protein-coding genes used atypical start codons (CTA in ND2 and AAT in COX1), and the remaining 11 protein-coding genes started with a typical insect initiation codon ATN. All tRNAs showed standard clover-leaf structure, except for tRNASer (AGN), which lacked a dihydrouridine (DHU) arm. The newly added T. molitor mitogenome could provide information for future studies on yellow meal worm.
Cervus sichuanicus is a species of sika deer (Cervus nippon Group). To date, research has mainly focused on quantity surveying and behavior studies, with genetic information on this species currently deficient. To provide scientific evidence to assist in the protection of this species, we collected Sichuan sika deer fecal samples from the Sichuan Tiebu Nature Reserve (TNR) and extracted DNA from those samples. Microsatellite loci of bovine were used for PCR amplification. After GeneScan, the genotype data were used to analyze the genetic diversity and population structure of the Sichuan sika deer in TNR. Results showed that the average expected heterozygosity of the Sichuan sika deer population in TNR was 0.562, equivalent to the average expected heterozygosity of endangered animals, such as Procapra przewalskii. Furthermore, 8 of 9 microsatellite loci significantly deviated from the Hardy-Weinberg equilibrium and two groups existed within the Sichuan sika deer TNR population. This genetic structure may be caused by a group of Manchurian sika deer (Cervus hortulorum) released in TNR.
The grass carp (Ctenopharyngodon idella) is one of the most important cultivated fish species in China. Mounting evidences suggests that microRNAs (miRNAs) may be key regulators of skeletal muscle among the grass carp, but the knowledge of the identity of myogenic miRNAs and role of miRNAs during skeletal muscle anabolic state remains limited. In the present study, we choose 8 miRNAs previously reported to act as muscle growth-related miRNAs for fasting-refeeding research. We investigated postprandial changes in the expression of 8 miRNAs following a single satiating meal in grass carp juveniles who had been fasting for one week and found that 7 miRNAs were sharply up-regulated within 1 or 3 h after refeeding, suggesting that they may be promising candidate miRNAs involved in a fast-response signaling system that regulates fish skeletal muscle growth.
In this study, to clarify the bioactive polypeptides included in the skins and secretions of Bufo, we screened the Japanese toad (Bufo japonicus formosus) skin cDNA library by colony polymerase chain reaction (PCR), and obtained a transcript of 1 075 bp consisting of 1 37 bp 5' untranslated region (UTR), 515 bp 3' UTR and a 423 bp open reading frame (ORF) encoding a polypeptide of 140 amino acid residues (GenBank accession number: KF359945). Homolog analysis showed a 70%-96% homology with sterol carrier protein-2 (SCP-2) present in other animals, which is implicated in lipid metabolism of other organisms. The gene SCP-2 of Chinese toad (B. gargarizans) was cloned from a first strand cDNA of Bufo skin (GenBank accession number: KF381341) via PCR, whose encoding polypeptide has only one amino acid difference from that of Japanese toad. Tissue distribution analysis showed that SCP-2 expressed in all organs tested, though in the liver and spleen it manifested lower expression than in other organs. These findings might indicate SCP-2 being one of the active ingredients in toad skin. These findings may in turn have implications for further drug development from traditional Chinese medicine sources.
The B cells translocation gene 1 (BTG1) is a member of the BTG/TOB family of anti-proliferative genes, which have recently emerged as important regulators of cell growth and differentiation among vertebrates. Here, for the first time we cloned the full-length cDNA sequence of Hyriopsis schlegelii (Hs-BTG1), an economically important freshwater shellfish and potential indicator of environmental heavy metal pollution, for the first time. Using rapid amplification of cDNA ends (RACE) together with splicing the EST sequence from a haemocyte cDNA library, we found that Hs-BTG1 contains a 525 bp open reading frame (ORF) encoding a 174 amino-acid polypeptide, a 306 bp 5' untranslated region (5' UTR), and a 571 bp 3' UTR with a Poly(A) tail as well as a transcription termination signal (AATAAA). Homologue searching against GenBank revealed that Hs-BTG1 was closest to Crassostrea gigas BTG1, sharing 50.57% of protein identities. Hs-BTG1 also shares some typical features of the BTG/TOB family, possessing two well-conserved A and B boxes. Clustering analysis of Hs-BTG1 and other known BTGs showed that Hs-BTG1 was also closely related to BTG1 of C. gigas from the invertebrate BTG1 clade. Function prediction via homology modeling showed that both Hs-BTG1 and C. gigas BTG1 share a similar three-dimensional structure with Homo sapiens BTG1. Tissue-specific expression analysis of the Hs-BTG1 via real-time PCR showed that the transcripts were constitutively expressed, with the highest levels in the hepatopancreas and gills, and the lowest in both haemocyte and muscle tissue. Expression levels of Hs-BTG1 in hepatopancreas (2.03-fold), mantle (2.07-fold), kidney (2.2-fold) and haemocyte (2.5-fold) were enhanced by cadmium (Cd2+) stress, suggesting that Hs-BTG1 may have played a significant role in H. schlegelii adaptation to adverse environmental conditions.
The Asian swamp eel (Monopterus albus) is one of the most economically important freshwater fish in East Asia, but data on the immune genes of M. albus are scarce compared to other commercially important fish. A better understanding of the eel's immune responses may help in developing strategies for disease management, potentially improving yields and mitigating losses. In mammals, interferon regulatory factors (IRFs) play a vital role in both the innate and adaptive immune system; though among teleosts IRF4 and IRF10 have seldom been studied. In this study, we characterized IRF4 and IRF10 from M. albus (maIRF4 and maIRF10) and found that maIRF4 cDNA consists of 1 716 nucleotides encoding a 451 amino acid (aa) protein, while maIRF10 consists of 1 744 nucleotides including an open reading frame (ORF) of 1 236 nt encoding 411 aa. The maIRF10 gene was constitutively expressed at high levels in a variety of tissues, while maIRF4 showed a very limited expression pattern. Expression of maIRF4 and maIRF10 in head kidney, and spleen tissues was significantly up-regulated from 12 h to 48 h post-stimulation with polyinosinic: polycytidylic acid (poly I:C), lipopolysaccharide (LPS) and a common pathogenic bacteria Aeromonas hydrophila. These results suggest that IRF4 and IRF10 play roles in immune responses to both viral and bacterial infections in M. albus.
Cestode larvae spend one phase of their two-phase life cycle in the viscera of rodents, but cases of cestodes infecting subterranean rodents have only been rarely observed. To experimentally gain some insight into this phenomenon, we captured approximately 300 plateau zokors (Eospalax baileyi), a typical subterranean rodent inhabiting the Qinghai-Tibet Plateau, and examined their livers for the presence of cysts. Totally, we collected five cysts, and using a mitochondrial gene (cox1) and two nuclear genes (pepck and pold) as genetic markers, we were able to analyze the taxonomy of the cysts. Both the maximum likelihood and Bayesian methods showed that the cysts share a monophyly with Taenia mustelae, while Kimura 2-parameter distances and number of different sites between our sequences and T. mustelae were far less than those found between the examined sequences and other Taeniidae species. These results, alongside supporting paraffin section histology, imply that the cysts found in plateau zokors can be regarded as larvae of T. mustelae, illustrating that zokors are a newly discovered intermediate host record of this parasite.
Tropomyosin (TM) plays a critical role in skeletal and cardiac muscle development and function. To assess the functional significance of α-TM in Japanese flounder (Paralichthys olivaceus) development and metamorphosis, cDNA from Japanese flounder was cloned and α-TM mRNA measured during development and metamorphosis. The full-length cDNA is 1 191 bp, including a 5'-untranslated region of 114 bp, a 3'-UTR of 222 bp, and an open reading frame of 855 bp encoding a polypeptide of 284 amino acids. Real-time quantitative PCR revealed that α-TM mRNA is initially expressed in unfertilized ovum, indicating the α-TM gene is maternal. Relatively low mRNA levels were observed in different embryonic stages. A higher level of α-TM mRNA was detected 3 days post hatching (dph), while the highest level was measured at 29 dph (metamorphic climax) after which it declined towards the end of metamorphosis. The expression of α-TM mRNA was up-regulated in thyroid hormone-treated larvae at 36 dph, but there was no marked difference at other stages when compared to control animals. After thiourea treatment, the expression of α-TM mRNA declined slightly. These data provide basic information that can be utilized in further studies into the role of α-TM in P. olivaceus development and metamorphosis.
Insulin-like growth factor-binding protein 1 (IGFBP-1), a hypoxia-induced protein, is a member of the IGFBP family that regulates vertebrate growth and development. In this study, full-length IGFBP-1a cDNA was cloned from a hypoxia-sensitive Cyprinidae fish species, the blunt snout bream (Megalobrama amblycephala). IGFBP-1a was expressed in various organs of adult blunt snout bream, including strongly in the liver and weakly in the gonads. Under hypoxia, IGFBP-1a mRNA levels increased sharply in the skin, liver, kidney, spleen, intestine and heart tissues of juvenile blunt snout bream, but recovered to normal levels after 24-hour exposure to normal dissolved oxygen. In blunt snout bream embryos, IGFBP-1a mRNA was expressed at very low levels at both four and eight hours post-fertilization, and strongly at later stages. Embryonic growth and development rates decreased significantly in embryos injected with IGFBP-1a mRNA. The average body length of IGFBP-1a-overexpressed embryos was 82.4% of that of the control group, and somite numbers decreased to 85.2%. These findings suggest that hypoxia-induced IGFBP-1a may inhibit growth in this species under hypoxic conditions.
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