Auditory sensitivity and tympanic middle ear in a vocal and a non-vocal frog.

The tympanic middle ear is important for anuran hearing on land. However, many species have partly or entirely lost their tympanic apparatus. Previous studies have compared hearing sensitivities in species that possess and lack tympanic membranes capable of sound production and acoustic communication. However, little is known about how these hearing abilities are comparable to those of mutant species. Here, we compared the eardrum and middle ear anatomies of two sympatric sibling species from a noisy stream habitat, namely the "non-vocal" Hainan torrent frog (Amolops hainanensis) and the "vocal" little torrent frog (Amolops torrentis), the latter of which is capable of acoustic communication. Our results showed that the relative (to head size) eardrum diameter of A. hainanensis was smaller than that of A. torrentis, although the absolute size was not smaller. Unlike A. torrentis, the tympanic membrane area of A. hainanensis was not clearly differentiated from the surrounding skin. The middle ear, however, was well-developed in both species. We measured the auditory brainstem responses (ABRs) of A. hainanensis and compared the ABR thresholds and latencies to those previously obtained for A. torrentis. Our results suggested that these two species exhibited significant differences in hearing sensitivity. A. hainanensis (smaller relative eardrum, nonvocal) had higher ABR thresholds and longer initial response times than A. torrentis (larger relative eardrum, vocal) at lower frequencies. Neurophysiological responses from the brain were obtained for tone pips between 800 Hz and 7,000 Hz, with peak sensitivities found at 3,000 Hz (73 dB SPL) for A. hainanensis, and at 1,800 Hz (61 dB SPL) for A. torrentis. Our results suggest that the non-vocal A. hainanensis has lower hearing sensitivity than its vocal sister species (i.e., A. torrentis), which may be related to differences in tympanic or inner ear structure and morphology.

Geographical variation of the acoustic signals in the spot-legged treefrog (Polypedates megacephalus) of Hainan Island.

Geographical divergence in acoustic signals is often affected by multiple selective pressures. Environmental factors are expected to shape signals by adjusting for greater propagation efficiency. Nevertheless, such habitat-driven hypotheses have not been well-examined with multi-signal and multi-habitat types included simultaneously. In this study, we evaluated call similarity of the spot-legged treefrog (Polypedates megacephalus), characterized by multiple call types, from 11 geographic populations across Hainan Island. We also examined multiple factors (i.e. body mass, temperature, altitude, and vegetation) among different acoustical geographic clusters, and tested whether the variation in acoustic length and frequency among different habitats was in accordance with the prediction derived from the acoustic adaptation hypothesis. We showed that P. megacephalus from Hainan Island included 3 acoustic geographic clusters: the northeastern flatlands, the southwest mountains, and the hills around the mountains. We also showed that environmental factors (i.e. altitude and vegetation) varied across these 3 acoustically distinct areas. Moreover, the duration of the different call types supported the hypothesis that they should have optimal transmission in their own habitats. Thus, our results reveal the roles of ecological selection in the geographical divergence of anuran acoustic signals.

Avian Alarm Calls Do Not Induce Anti-Predator Response in Three Anuran Species.

Many species produce alarm calls in response to predators, and the anti-predator signals are often used by other species. Eavesdropping on heterospecific alarm calls has been widely found in bird and mammal species. Other taxa, such as reptiles and amphibians, however, receive limited attention at present. Here, we selected three types of alarm calls of Japanese Tits (Parus minor) that were evoked by the Siberian Chipmunk (Eutamias sibiricus), Eurasian Sparrow Hawk (Accipiter nisus), and model snake (Elaphe spp.), respectively, and then carried out playback experiments to test whether three frog species changed their behaviors in response to the three treatments of Japanese Tit calls while the tit's territory song was used as a control. The results showed that Little Torrent Frogs (Amolops torrentis), Ornamented Pygmy Frogs (Microhyla fissipes) and Spot-legged Treefrogs (Polypedates megacephalus) did not jump off their positions in response to the same four acoustic signals. They also did not change their calling behaviors in response to the alarm calls of Japanese Tits. This study found no evidence that these anuran species can eavesdrop on heterospecific tits' alarm signals.

Genetic variants in SCNN1B and AHCYL1 are associated with eggshell quality in Chinese domestic laying ducks (Anas platyrhynchos).

1. The objective of this study was to investigate the evolution of SCNN1B and AHCYL1 proteins among 10 domestic avian and mammalian animal species, to uncover the expression patterns of SCNN1B and AHCYL1 genes in ducks, identify the genetic variants of the SCNN1B and AHCYL1 genes and analyse their effects on eggshell quality.2. Expression profiles of the SCNN1B and AHCYL1 genes in Sansui female ducks were determined using real-time fluorescence quantitative PCR to identify SNPs. The duck SCNN1B and AHCYL1 genes were amplified to identify SNPs. A total of 502 Sansui female ducks were genotyped by sequencing, and the associations between the mRNA expression/SNP genotypes and 6 eggshell quality indices were analysed using PASW Statistics 18.0.3. The results showed that the SCNN1B and AHCYL1 proteins are highly conserved in different mammalian or domestic animals, especially the AHCYL1 protein. The SCNN1B and AHCYL1 genes were widely expressed in different tissues of male and female ducks, and expression level in the uterus was greater than in other tissues. The expression of SCNN1B and AHCYL1 during the oviposition cycle indicated that expression levels were related to the eggshell mineralisation stage.4. The mRNA expression levels of the uterine SCNN1B and AHCYL1 genes were positively correlated with eggshell strength (ESS), percentage (ESP) and weight (ESW) (P < 0.05), respectively. Ten novel SNPs in SCNN1B and AHCYL1 genes from Chinese domestic laying ducks were identified through PCR amplicon sequencing.5. Genetic association analysis indicated g.797509 C > T, g.797573 C > T and g.797834 C > T in SCNN1B gene and g.169244 T > A, g.169265 T > C and g.175311 T > C in AHCYL1 gene had a significant effect on eggshell quality. Correlation analysis between the SNP genotype and SCNN1B and AHCYL1 genes expression in the uterus showed that the genotypes of g.797509 C > T, g.797573 C > T, g.797834 C > T, g.169244 T > A and g.175311 T > C sites affected the expression of SCNN1B and AHCYL1 genes in utero (P < 0.05).6. The study indicated SCNN1B and AHCYL1 as candidate genes to improve eggshell traits in ducks.

[Effects of the ITPR1 gene overexpression on Ca²âº concentration, lipid content and calcium transport-related genes in duck uterine epithelial cells].

Inositol 1,4,5-trisphosphate receptor 1 (ITPR1) is an important intracellular channel for releasing Ca²âº. In order to investigate the effects of the ITPR1 overexpression on Ca²âº concentration and lipid content in duck uterine epithelial cells and its effects on calcium transport-related genes, the structural domain of ITPR1 gene of duck was cloned into an eukaryotic expression vector and transfected into duck uterine epithelial cells. The overexpression of the ITPR1 gene, the concentration of Ca²âº, the lipid content, and the expression of other 6 calcium transport-related genes was determined. The results showed that the concentration of Ca²âº in uterine epithelial cells was significantly reduced after transfection (P<0.05), the triglyceride content was significantly increased (P<0.01), and the high-density lipoprotein content was significantly decreased (P<0.01). The correlation analysis results showed that the overexpression of the C-terminal half of the ITPR1 gene was significantly positively correlated with the total cholesterol content (P<0.01), which was significantly positively correlated with the low-density lipoprotein content (P<0.05). The overexpression of the N-terminal half of the ITPR1 gene was significantly positively correlated with the triglyceride content (P<0.01), which was significantly negatively correlated with the concentration of Ca²âº (P<0.05). RT-qPCR results of 6 calcium transport-related genes showed that the overexpression of the C-terminal half of the ITPR1 gene significantly inhibited the expression of the IP3R2, VDAC2 and CAV1 genes, and the overexpression of the N-terminal half of the ITPR1 gene significantly promoted the expression of the IP3R3 and CACNA2D1 genes. In conclusion, the ITPR1 gene overexpression can promote Ca²âº release in duck uterus epithelial cells, promote the synthesis of triglyceride, low-density lipoprotein and cholesterol, and inhibit the production of high-density lipoprotein, and the ITPR1 gene overexpression affected the expression of all 6 calcium transport-related genes.

[Effects of SCD-1 gene overexpression on the content of calcium ion and lipids in duck uterine epithelial cells].

Stearoyl-CoAdesaturase-1 (SCD-1) is a key regulator of monounsaturated fatty acid synthesis. It plays a vital role in lipid synthesis and metabolism. Ca²âº is an important cation in the body and plays an important role in the organism. The aims of this study were to investigate the correlation of SCD-1 gene overexpression with lipid indexes and calcium ion level. The pcDNA3.1 (+) + SCD-1 +Flag eukaryotic expression vector and cultured duck uterine epithelial cells were co-transfected. The overexpression of SCD-1 gene was measured using the Flag Label Detection Kit. Ca ions and lipid contents were detected through Fluo-3/AM Calcium Ion Fluorescence Labeling method and Lipid Measuring Kit, respectively. SCD-1 gene overexpression was negatively correlated with triglyceride (TG) and high-density lipoprotein cholesterol (HDL-C), and positively correlated with Ca ion, total cholesterol (TC), very low-density lipoprotein cholesterol (VLDL-C) and low density lipoprotein cholesterol (LDL-C) levels. Meanwhile, Ca ion was positively correlated with TG, LDL-C and HDL-C contents, and negatively correlated with TC and VLDL-C levels. Overexpression of SCD-1 gene could regulate Ca ion secretion, as well as lipid synthesis and transport in duck uterine epithelial cells.

Association of three SNPs in adiponectin gene with lipid traits of Tianzhu Black Muscovy (Cairina moschata).

Adiponectin plays a critically biological role in atherosclerosis, glucose utilization, lipid and carbohydrate metabolism, and triglyceride synthesis in animals and humans. However, little is known about the effect of adiponectin on lipid metabolism of the avian species. The aim of the preset study was to investigate the potential associations between adiponectin gene single nucleotide polymorphisms (SNPs) and the lipid traits in 348 females of Tianzhu Black Muscovy. Three novel SNPs (167G>A, 290T>C and 711G>A) were detected in adiponectin gene. 167G>A and 290T>C has linked very closely, and then 711G>A with 167G>A and 290T>C has no strong linkage disequilibrium, respectively. The Chi square test showed that allelic frequency and genotype frequency of two SNPs (167G>A and 711G>A) didn't agree with the Hardy-Weinberg equilibrium (P>0.05). Four haplotypes and nine diplotypes were formed on the three SNPs of adiponectin gene. Association analysis indicated that the 167G>A genotypes were strongly associated with intramuscular fat (IMF) of chest muscle and serum total cholesterol (TC) (P < 0.01); the 290T>C genotypes were strongly associated with IMF, TC, and serum triglyceride (TG) (P < 0.01); furthermore, the 711G>A genotypes were significantly associated with TG and TC (P < 0.05); the diplotypes were strongly associated with IMF, TC, and TG (P < 0.01). Therefore, three SNPs in adiponectin were potential markers for improving IMF in Muscovy ducks.