Exquisite structure of the lateral line system in eyeless cavefish Sinocyclocheilus tianlinensis contrast to eyed Sinocyclocheilus macrophthalmus (Cypriniformes: Cyprinidae)

In this study, the lateral line systems in Chinese cavefish eyeless Sinocyclocheilus tianlinensis and eyed Sinocyclocheilus macrophthalmus were investigated to reveal their morphological changes to survive in harsh environments. Compared with the eyed cavefish S. macrophthalmus (atypical), the lateral line system in the eyeless cavefish S. tianlinensis (typical) has certain features to adapt to the dark cave environments: the superficial lateral line system in the eyeless species possesses a higher number of superficial neuromasts and more hair cells within an individual neuromast, and the trunk lateral line canal system in S. tianlinensis exhibits larger canal pores, higher canal diameter and more pronounced constrictions. Fluid–structure interaction analysis suggested that the trunk lateral line canal system in the eyeless S. tianlinensis should be more sensitive than that in the eyed S. macrophthalmus. These morphological features of the lateral line system in the eyeless S. tianlinensis probably enhance the functioning of the lateral line system and compensate for the lack of eyes. The revelation of the form–function relationship in the cavefish lateral line system provides inspiration for the design of sensitive artificial flow sensors.     

Novel Borrelia genotypes in bats from the Macaregua Cave,Colombia

Bats have been implicated as reservoirs of relapsing fever group spirochaetes since the beginning of the last century. Recently, bat‐associated spirochaetes have been reported as human pathogens. In 1968, a spirochaete was detected in blood of the bat Natalus tumidirostris captured inside the Macaregua cave, Colombia. Data on this microorganism were never published again. The aim of this study was to evaluate the presence of Borrelia DNA in blood from bats of Macaregua cave. We performed molecular analyses using a genus‐specific real‐time PCR targeting the 16S rRNA to detect DNA of Borrelia in blood samples from 46 bats captured in the Macaregua cave. Positive samples were submitted to a battery of PCRs aiming to amply Borrelia 16S rRNA, flaB, glpQ, p66, ospC, clpA, clpX, nifS, pepX, pyrG, recG, rplB and uvrA genes. Seventeen samples were positive for Borrelia after real‐time PCR. With the exception of flaB gene, attempts to amplify further loci were unsuccessful. Nucleotide and amino acid divergences of four flaB haplotypes characterized from blood of Carollia perspicillata showed Borrelia burgdorferi sensu lato (Bbsl) as the most closely related group. A phylogenetic tree including 74 sequences of the genus confirmed this trend, since Borrelia genotypes detected in bats from Macaregua formed a monophyletic group basally positioned to Bbsl. Our results suggest that Borrelia genotypes characterized from bats roosting in the Macaregua cave might constitute a new taxon within the genus. This is the first molecular characterization of a Borrelia sp. in Colombia.     

Partial cloning of CYP2C23a genes and hepatic protein expression in eight representative avian species

Large interspecies differences in avian xenobiotic metabolism have been revealed by microsome‐based studies, but specific enzyme isoforms in different bird species have not yet been compared. We have previously shown that CYP2C23 genes are the most induced CYP isoforms in chicken liver. In this study, we collected partial CYP2C23a gene sequences from eight avian species (ostrich, blue‐eared pheasant, snowy owl, great‐horned owl, Chilean flamingo, peregrin falcon, Humboldt penguin, and black‐crowned night heron) selected to cover the whole avian lineage: Paleognathae, Galloanserae, and Neoaves. Genetic analysis showed that CYP2C23 genes of Galloanserae species (chicken and blue‐eared pheasant) had unique characteristics. We found some duplicated genes (CYP2C23a and CYP2C23b) and two missing amino acid residues in Galloanserae compared to the other two lineages. The genes have lower homology than in other avian lineages, which suggests Galloanserae‐specific rapid evolutionary changes. These genetic features suggested that the Galloanserae are not the most representative avian species, considering that the Neoaves comprise more than 95% of birds. Moreover, we succeeded in synthesizing an antipeptide polyclonal antibody against the region of CYP2C23 protein conserved in avians. However, comparative quantitation of CYP2C23 proteins in livers from six species showed that expression levels of these proteins differed no more than fourfold. Further study is needed to clarify the function of avian CYP2C23 proteins.     

Caprine sex affects skeletal muscle profile and MRFs expression during postnatal development

The important roles of myogenic regulatory factors (MRFs) have been well addressed in the process of mammalian skeletal myogenesis, while limited research has been performed in small ruminants. Furthermore, the effects of gender on the development of skeletal muscle and MRFs expression remain unknown. In this study, we identified the caprine Myf5, Myf6, MyoD and myogenin genes and quantified their expressions at six different postnatal time points by real‐time RT‐PCR. The sex has a marked effect on the expression differences of Myf5, MyoD and myogenin in the five investigated skeletal muscles, while minor influence on the expression difference of Myf6 except for Semitendinosus and Quadriceps femoris tissues (P < 0.001). The histological sections of muscles revealed a constant increase of muscle fiber diameter with aging but non‐significant differences between genders. We provide novel evidence for MRFs expression in age‐ and gender‐dependent manners, which will contribute to prioritizing these genes as potential candidate genes for trait‐associated study and provide a foundation for understanding the molecular control of skeletal muscle growth in goat species.     

RNA-Seq analysis reveals the potential molecular mechanisms of daidzein on adipogenesis in subcutaneous adipose tissue of finishing Xianan beef cattle

Daidzein has been reported to be effective in regulating lipid metabolism in animals. However, the molecular mechanisms of daidzein on adipogenesis in beef cattle are not yet reported and the results of daidzein on affecting lipid metabolism in other species have been conflicting. High-throughput sequencing of mRNA (RNA-Seq) technology was performed to elucidate the underlying molecular mechanisms of daidzein on adipogenesis in subcutaneous adipose tissue of finishing Xianan beef cattle. A total of 893 differentially expressed genes (DEGs) were identified by differential expression analysis, among which 405 genes were upregulated and 488 genes were downregulated. Bioinformatics analysis suggested that these DEGs were significantly enriched to the pathways related to lipid metabolism including ECM–receptor interaction, Glycolysis/Gluconeogenesis and Hedgehog signalling pathway. Daidzein significantly affected the candidate genes (Shh, Pec, Gli, Wnt6, DLK, IGFBP2, ID3 and C/EBPE) related to adipocyte differentiation. Besides, daidzein improved the ability of subcutaneous adipocytes in synthesizing triglycerides by directly using the long-chain fatty acids and enhanced the efficiency of triglyceride synthesis of subcutaneous adipocytes in Xianan steers. In conclusion, daidzein plays a positive role not only in adipogenic differentiation, but also in triglyceride synthesis in subcutaneous adipose tissue of Xianan beef cattle.     

Photokinesis and Djopsin gene expression analysis during the regeneration of planarian eyes

Planarians provide the ideal model for studying eye development, with their simple eye structure and exceptionally rapid regeneration. Here, we observed the eye morphogenesis, photophobic behavior, spectral sensitivity and expression pattern of Djopsin in the freshwater planarian Dugesia japonica. The results showed that: (i) Djopsin encoding the putative protein belonged to the rhabdomeric opsins group and displayed high conservation during animal evolution; (ii) planarians displayed diverse photophobic response to different visible wavelengths and were more sensitive to light blue (495 nm) and yellow (635 nm); (iii) the morphogenesis and functional recovery of eyes were related to the expression pattern of Djopsin during head regeneration; and (iv) Djopsin gene plays a major role in functional recovery during eye regeneration and visual system maintenance in adult planarians.     

Development of gene expression assays measuring immune responses in the spotted hyena (Crocuta crocuta)

As scavengers, spotted hyenas (Crocuta crocuta) are exposed to a wide array of pathogens but exhibit low mortality rates due to infectious disease. This suggests that this species exhibits a unique and robust immune response to pathogens. However, few tools exist to measure cell-mediated immunity (CMI) in hyenas and we aimed to develop a gene expression assay to quantify antigen-specific responses. Whole blood from five Mycobacterium bovis- sensitised hyenas was incubated in Nil and TB antigen tubes of the QuantiFERON®-TB Gold (QFT) system. Using qPCR, the relative expression stability of the reference genes ACTB, GAPDH, YWHAZ and TBP in these samples was determined as well as the mean fold change in the expression of IFNG, CXCL8, CXCL9, CXCL10 and CXCL11 in M. bovis-antigen stimulated blood. The expression of YWHAZ and TBP showed greatest stability, and YWHAZ was selected as a reference for further analysis. The expression of CXCL9 and CXCL11 showed greatest upregulation in antigen-stimulated blood and the assay results for these genes were strongly correlated. The measurement of antigen-induced CXCL9 and CXCL11 expression, relative to that of YWHAZ, can be used to measure CMI responses to infectious diseases in spotted hyenas.     

A simplified one-step nuclear transfer procedure alters the gene expression patterns and developmental potential of cloned porcine embryos

Various somatic cell nuclear transfer (SCNT) techniques for mammalian species have been developed to adjust species-specific procedures to oocyte-associated differences among species. Species-specific SCNT protocols may result in different expression levels of developmentally important genes that may affect embryonic development and pregnancy. In the present study, porcine oocytes were treated with demecolcine that facilitated enucleation with protruding genetic material. Enucleation and donor cell injection were performed either simultaneously with a single pipette (simplified one-step SCNT; SONT) or separately with different pipettes (conventional two-step SCNT; CTNT) as the control procedure. After blastocysts from both groups were cultured in vitro, the expression levels of developmentally important genes (OCT4, NANOG, EOMES, CDX2, GLUT-1, PolyA, and HSP70) were analyzed by real-time quantitative polymerase chain reaction. Both the developmental rate according to blastocyst stage as well as the expression levels CDX2, EOMES, and HSP70 were elevated with SONT compared to CTNT. The genes with elevated expression are known to influence trophectoderm formation and heat stress-induced arrest. These results showed that our SONT technique improved the development of SCNT porcine embryos, and increased the expression of genes that are important for placental formation and stress-induced arrest.     

Lineage‐specific evolution of bitter taste receptor genes in the giant and red pandas implies dietary adaptation

Taste 2 receptors (TAS2R) mediate bitterness perception in mammals, thus are called bitter taste receptors. It is believed that these genes evolved in response to species‐specific diets. The giant panda (Ailuropoda melanoleuca) and red panda (Ailurus fulgens styani) in the order Carnivora are specialized herbivores with an almost exclusive bamboo diet (>90% bamboo). Because bamboo is full of bitter tasting compounds, we hypothesized that adaptive evolution has occurred at TAS2R genes in giant and red pandas throughout the course of their dietary shift. Here, we characterized 195 TAS2R genes in 9 Carnivora species and examined selective pressures on these genes. We found that both pandas harbor more putative functional TAS2R genes than other carnivores, and pseudogenized TAS2R genes in the giant panda are different from the red panda. The purifying selection on TAS2R1, TAS2R9 and TAS2R38 in the giant panda, and TAS2R62 in the red panda, has been strengthened throughout the course of adaptation to bamboo diet, while selective constraint on TAS2R4 and TAS2R38 in the red panda is relaxed. Remarkably, a few positively selected sites on TAS2R42 have been specifically detected in the giant panda. These results suggest an adaptive response in both pandas to a dietary shift from carnivory to herbivory, and TAS2R genes evolved independently in the 2 pandas. Our findings provide new insight into the molecular basis of mammalian sensory evolution and the process of adaptation to new ecological niches.     

Scanning 17 candidate genes for association with primary cataracts in the wire-haired Dachshund

Primary cataracts are breed-related eye diseases and are common in many dog breeds. In this study, 17 genes (BFSP2, EYA1, FOXE3, FTL, GCNT2, GJA3, GJA8, HSF4, MAF, MIP, PAX6, PITX3, SIX5, SORD, SOX1, SPARC, TRNT1) were evaluated as candidates for primary non-congenital cataracts (CAT) in the Dachshund using microsatellites adjacent to the candidate genes. Linkage and association with CAT was tested in 15 affected and six unaffected wire-haired Dachshunds. Non-parametric linkage analysis and association tests did not reveal significant linkage or association for the candidate gene flanking microsatellites tested. Thus, it is unlikely that the 17 investigated candidate genes harbour a causative mutation for CAT in these Dachshunds.     

Identification of Stably Expressed Reference Genes for RT‐qPCR Data Normalization in Defined Localizations of Cyclic Bovine Ovaries

Ovaries are highly complex organs displaying morphological, molecular and functional differences between their cortical zona parenchymatosa and medullary zona vasculosa, and also between the different cyclic luteal stages. Objective of the present study was to validate expression stability of twelve putative reference genes (RGs) in bovine ovaries, considering the intrinsic heterogeneity of bovine ovarian tissue with regard to different luteal stages and intra‐ovarian localizations. The focus was on identifying RGs, which are suitable to normalize RT‐qPCR results of ovaries collected from clinical healthy cattle, irrespective of localization and the hormonal stage. Expression profiles of twelve potential reference genes (GAPDH, ACTB, YWHAZ, HPRT1, SDHA, UBA52, POLR2C, RPS9, ACTG2, H3F3B, RPS18 and RPL19) were analysed. Evaluation of gene expression differences was performed using genorm , normfinder , and bestkeeper software. The most stably expressed genes according to genorm , normfinder and bestkeeper approaches contained the candidates H3F3B, RPS9, YWHAZ, RPS18, POLR2C and UBA52. Of this group, the genes YWHAZ, H3F3B and RPS9 could be recommended as best‐suited RGs for normalization purposes on healthy bovine ovaries irrespective of the luteal stage or intra‐ovarian localization.     

N -ethyl- N -nitrosourea induces retinal photoreceptor damage in adult rats

Seven-week-old male Lewis rats received a single intraperitoneal injection of N-ethyl-N-nitrosourea (ENU) (100, 200, 400 or 600 mg/kg), and retinal damage was evaluated 7 days after the treatment. Sequential morphological features of the retina and retinal DNA damage, as determined by a TUNEL assay and phospho-histone H2A.X (γ-H2AX), were analyzed 3, 6, 12, 24 and 72 hr, 7 days, and/or 30 days after 400 mg/kg ENU treatment. Activation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP) was analyzed immunohistochemically by poly (ADP-ribose) (PAR) expression in response to DNA damage of the retina. All rats that received ≥ 400 mg/kg of ENU developed retinal degeneration characterized by the loss of photoreceptor cells in both the central and peripheral retina within 7 days. In the 400 mg/kg ENU-treated rats, TUNEL-positive signals were only located in the photoreceptor cells and peaked 24 hr after ENU treatment. The γ-H2AX signals in inner retinal cells appeared at 24 hr and peaked at 72 hr after ENU treatment, and the PAR signals selectively located in the photoreceptor cell nuclei appeared at 12 hr and peaked at 24 hr after ENU treatment. However, degeneration was restricted to photoreceptor cells, and no degenerative changes in inner retinal cells were seen at any time points. Retinal thickness and the photoreceptor cell ratio in the central and peripheral retina were significantly decreased, and the retinal damage ratio was significantly increased 7 days after ENU treatment. In conclusion, ENU induced retinal degeneration in adult rats that was characterized by photoreceptor cell apoptosis through PARP activity.     

Effect of chicken leptin recptor short hairpin RNA on expression of JAK2, STAT3, SOCS3 and CPT1 genes in chicken preadipocytes

In this study, we detect depressive effect on leptin receptor (LEPR) by LEPR‐specific short hairpin RNA (shRNA) expression plasmids in chicken preadipocytes, and effect on messenger RNA (mRNA) expression levels of genes related to signal transduction, including JAK2, STAT3, SOCS3 as well as CPT1, which is associated with fatty acid metabolism. shRNA expression vectors targeting LEPR were constructed and transfected into chicken preadipocytes. The transfection efficiency was evaluated by fluorescence microscopy. Real‐time PCR was used to detect its effect on mRNA expression levels of JAK2, STAT3, SOCS3 and CPT1. Results showed that LEPR mRNA was knocked down by 99% (P < 0.01) after transfection for 72 h. In the knockdown preadipocytes, the mRNA levels of JAK2 and CPT1 were down‐regulated by 47.56% (P < 0.01) and 42.26% (P < 0.05), respectively; while expression of STAT3 and SOCS3 increased 7.72‐fold (P < 0.01), 1.71‐fold (P < 0.01), respectively. It is concluded that knockdown of LEPR influences mRNA expression of its down‐stream genes, suggesting that chicken LEPR play a certain role in regulating genes in the complicated gene network of preadipocytes.