2014 Vol. 35, No. 4
2014, 35(4): 249-352.
cDNA clone and expression analysis of α-Tropomyosin during Japanese flounder (Paralichthys olivaceus) metamorphosis
2014, 35(4): 307-312. doi: 10.13918/j.issn.2095-8137.2014.4.307
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.
Molecular identification of Taenia mustelae cysts in subterranean rodent plateau zokors (Eospalax baileyi)
2014, 35(4): 313-318. doi: 10.13918/j.issn.2095-8137.2014.4.313
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.
2014, 35(4): 319-325. doi: 10.13918/j.issn.2095-8137.2014.4.319
Whilethe roles of glutamic acid (Glu), arginine vasopressin (AVP) and their respective receptors in anxiety have been thoroughly investigated, the effects of interactions among Glu, N-methyl-D-aspartic acid (NMDA) receptor, AVP and a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor on anxiety are still unclear. In the present study, the agonist and antagonist of the NMDA receptor and AMPA receptor, as well as the antagonist of AVP V1 receptor (V1aR) were introduced into BALB/cJ mice by intracerebroventricular microinjection, and the anxiety-like behaviors of the mice were evaluated by open field and elevated plus-maze tests. Compared with C57BL/6 mice, BALB/cJ mice displayed higher levels of anxiety-like behavior. Significant anxiolytic effects were found in the NMDA receptor antagonist (MK-801) and the AMPA receptor or V1aR antagonist (SSRI49415), as well as combinations of AVP/MK-801 and SSRI49415/DNQX. These results indicated that anxiety-like behaviors expressed in BALB/CJ mice may be due to a coordination disorder among glutamate, NMDA receptor, AMPA receptor, AVP and V1aR, resulting in the up-regulation of the NMDA receptor and V1aR and down-regulation of the AMPA receptor. However, because the AMPA receptor can execute its anxiolytic function by suppressing AVP and V1aR, we cannot exclude the possibility of the NMDA receptor being activated by AVP acting on V1aR.
2014, 35(4): 326-331. doi: 10.13918/j.issn.2095-8137.2014.4.326
In the current study, 5-nydroxytryptamine (5-HT) and gastrin (GAS) cells in the digestive canals of Rana chensinensis tadpoles at different developmental stages were investigated by immunohistochemistry. Results showed that the 5-HT cells were only detected in the duodenum before metamorphosis began, and were extensively distributed in the stomach, duodenum, small intestine, and rectum thereafter, with the highest counts found in the duodenum and rectum when metamorphosis was completed. The GAS cells were only distributed in the stomach and duodenum, and only rarely detected in the duodenum before metamorphosis began, but increased in the stomach during metamorphosis and showed zonal distribution in the gastric mucosa when metamorphosis was completed. Metamorphosis is a critical period for amphibians, during which structural and functional physiological adaptations are required to transition from aquatic to terrestrial environments. During metamorphosis, the differentiations of 5-HT cells in the gastrointestinal canals of tadpoles could facilitate mucus secretion regulation, improve digestive canal lubrication, and help water-shortage food digestion in terrestrial environments. Conversely, GAS cell differentiations during metamorphosis might contribute to the digestive and absorptive function transition from herbivore to omnivore.
2014, 35(4): 332-337. doi: 10.13918/j.issn.2095-8137.2014.4.332
Though light conditions are known to affect the development and anti-predation strategies of several aquatic species, relatively little is known about how different species react to light, or how light can affect these species during different points in their life-cycle. In this study, we used four sympatric anuran tadpoles (Bufo gargarizans, B. melanostictus, Pelophylax nigromaculatus and Microhyla fissipes) as animal system to examine species-specific activities of the underdoing different light intensity treatments, so as to better understand how they respond to light. We exposed four different species of tadpoles to 1660 and 14 lux light intensity treatments and then measured several parameters including development stage, body length and tail length, and as well as their basic activities. The results of this observation and analysis showed that the activities of tadpoles were significantly greater in B. gargarizans and B. melanostictus than in P. nigromaculatus and M. fissipes; and were also significantly greater during times of high light intensity as compared to during low light intensity. Moreover, the observed relationship between species and light intensity was significant. The activities of B. gargarizans and B. melanostictus tadpoles were greater in high light, while the activity of P. nigromaculatus tadpoles was greater in low light intensity, while M. fissipes tadpoles showed no differences in either low or high intensity light. Furthermore, the activities of B. gargarizans, B. melanostictus and M. fissipes tadpoles in terms of developmental stage, body size or tail length did not seem to differ with light intensity, but during early larval developmental period of P. nigromaculatus, the activity of tadpoles was negatively correlated with development stage, but irrelevant to either body size or tail length in different light intensities. These results lead us to conclude the observed activities of the four sympatric anuran tadpoles are closely correlated with their specific anti-predation strategies.
The cheer pheasant Catreus wallichi is a globally threatened species that inhabits the western Himalayas. Though it is well established that the species is threatened and its numbers declining, updated definitive estimates are lacking, so in 2011, we conducted a survey to assess the density, population size, and threats to the species in Jhelum valley, Azad Kashmir, which holds the largest known population of cheer pheasants in Pakistan. We conducted dawn call count surveys at 17 points clustered in three survey zones of the valley, 11 of which had earlier been used for a 2002-2003 survey of the birds. Over the course of our survey, 113 birds were recorded. Mean density of cheer pheasant in the valley was estimated at 11.8±6.47 pairs per km2, with significant differences in terms of both counts and estimated density of cheer were significantly different across the three survey zones, with the highest in the Chinari region and the lowest, that is the area with no recorded sightings of the pheasants, in Gari Doppata. The total breeding population of cheer pheasants is estimated to be some 2 490 pairs, though this does not consider the actual area of occupancy in the study area. On the whole, more cheer pheasants were recorded in this survey than from the same points in 2002-2003, indicating some success in population growth. Unfortunately, increasing human settlement, fires, livestock grazing, hunting, and the collection of non-timber forest products continue to threaten the population of cheer in the Jhelum valley. To mitigate these potential impacts, some degree of site protection should be required for the conservation of cheer pheasants in Pakistan, and more effective monitoring of the species is clearly needed.
2014, 35(4): 346-349. doi: 10.13918/j.issn.2095-8137.2014.4.346
In this brief report, we analyzed ecological correlates of risk of extinction for mammals endemic to China using phylogenetic eigenvector methods to control for the effect of phylogenetic inertia. Extinction risks were based on the International Union for Conservation of Nature (IUCN) Red List and ecological explanatory attributes that include range size and climatic variables. When the effect of phylogenetic inertia were controlled, climate became the best predictor for quantifying and evaluating extinction risks of endemic mammals in China, accounting for 13% of the total variation. Range size seems to play a trivial role, explaining ~1% of total variation; however, when non-phylogenetic variation partitioning analysis was done, the role of range size then explained 7.4% of total variation. Consequently, phylogenetic inertia plays a substantial role in increasing the explanatory power of range size on the extinction risks of mammals endemic to China. Limitations of the present study are discussed, with a focus on under-represented sampling of endemic mammalian species.
2014, 35(4): 249-255. doi: 10.13918/j.issn.2095-8137.2014.4.249
In the current study, the alopecia areata gene was introduced into the C57BL/6 (B6) mouse through repeated backcrossing/intercrossing, and the allelic homozygosity of congenic AAtjmice (named B6.KM-AA) was verified using microsatellites. The gross appearance, growth characteristics, pathological changes in skin, and major organs of B6.KM-AA mice were observed. Counts and proportions of CD4+ and CD8+ T lymphocytes in peripheral blood were determined by flow cytometry. Results show that congenic B6.KM-AA mice were obtained after 10 generations of backcrossing/intercrossing. B6.KM-AA mice grew slower than B6 control mice and AA skin lesions were developed by four weeks of age. The number of hair follicles was reduced, but hair structures were normal. Loss of hair during disease progression was associated with CD4+ and CD8+ T lymphocytes infiltration peri-and intra-hair follicles. No pathological changes were found in other organs except for the skin. In the peripheral blood of B6.KM-AA mice, the percentage of CD4+ T cells was lower and percentage of CD8+ T cells higher than in control mice. These findings indicate that B6.KM-AA mice are characterized by a dysfunctional immune system, retarded development and T-cell infiltration mediated hair loss, making them a promising new animal model for human alopecia areata.
2014, 35(4): 256-261. doi: 10.13918/j.issn.2095-8137.2014.4.256
The social transmission of food preferences (STFP) is a behavioural task of olfactory memory, in which an observer rat learns safe food odours from a demonstrator rat, and shows preference for this odour in a subsequent choice test. However, previous studies have failed to detect the transmission of information about food of potential danger and food aversion using STFP test. In this study, we tested how demonstrators' health affects the exchange of odour information and whether observers can learn danger information from an unhealthy demonstrator. As expected, the observer rat formed an odour preference after interacting with a demonstrator rat that had just eaten food containing a new odour, however, odour preference rather than aversion was also formed after interacting with a demonstrator rat injected with LiCl (used to induce gastric malaise). Furthermore, anaesthetized demonstrator rats and half-anaesthetized demonstrator rats, which showed obvious motor deficits suggesting an unhealthy state, also socially transmitted food preferences to observers. These results suggest that the social transmission of food preferences task is independent of a demonstrators' health, and that information about dangerous foods cannot be transmitted using this behavioural task.
2014, 35(4): 262-271. doi: 10.13918/j.issn.2095-8137.2014.4.262
Food restriction (FR) and refeeding (Re) have been suggested to impair body mass regulation and thereby making it easier to regain the lost weight and develop over-weight when FR ends. However, it is unclear if this is the case in small mammals showing seasonal forging behaviors. In the present study, energy budget, body fat and serum leptin level were measured in striped hamsters that were exposed to FR-Re. The effects of leptin on food intake, body fat and genes expressions of several hypothalamus neuropeptides were determined. Body mass, fat content and serum leptin level decreased during FR and then increased during Re. Leptin supplement significantly attenuated the increase in food intake during Re, decreased genes expressions of neuropepetide Y (NPY) and agouti-related protein (AgRP) of hypothalamus and leptin of white adipose tissue (WAT). Hormone-sensitive lipase (HSL) gene expression of WAT increased in leptin-treated hamsters that were fed ad libitum, but decreased in FR-Re hamsters. This indicates that the adaptive regulation of WAT HSL gene expression may be involved in the mobilization of fat storage during Re, which partly contributes to the resistance to FR-Re-induced overweight. Leptin may be involved in the down regulations of hypothalamus orexigenic peptides gene expression and consequently plays a crucial role in controlling food intake when FR ends.
Acute and joint toxicity of three agrochemicals to Chinese tiger frog (Hoplobatrachus chinensis) tadpoles
2014, 35(4): 272-279. doi: 10.13918/j.issn.2095-8137.2014.4.272
We studied acute and joint toxicity of three different agrochemicals (chlorantraniliprole, flubendiamide-abamectin and penoxsulam) to Chinese tiger frog (Hoplobatrachus chinensis) tadpoles with the method of stability water tests. Results showed that the three agrochemicals increased tadpole mortality. For acute toxicity, the LC50 values after 24, 48 and 72 h of chlorantraniliprole, flubendiamide-abamectin and penoxsulam exposure were 5.37, 4.90 and 4.68 mg/L; 0.035, 0.025 and 0.021 mg/L; 1.74, 1.45 and 1.29 mg/L, respectively. The safety concentrations (SC) of chlorantraniliprole, flubendiamide-abamectin and penoxsulam to the tadpoles were 1.23, 0.30 and 0.003 mg/L, respectively. Based on these findings, chlorantraniliprole and penoxsulam were moderately toxic, while flubendiamide-abamectin was highly toxic. All pairwise joint toxicity tests showed moderate toxicity. The LC50 values after 24, 48 and 72 h of exposure were 7.08, 6.61 and 6.03 mg/L for chlorantraniliprole+penoxsulam, with corresponding values of 2.455, 2.328 and 2.183 mg/L for chlorantraniliprole+flubendiamide-abamectin, and 1.132, 1.084 and 1.050 mg/L for penoxsulam+flubendiamide-abamectin, with safe concentrations of 1.73, 0.63 and 0.30 mg/L, respectively. For toxic evaluations of pairwise combinations of the three agrochemicals, only the joint toxicity of chlorantraniliprole and flubendiamide-abamectin after 24 h was found to be synergistic, whereas all other tests were antagonistic. Our findings provide valuable information on the toxic effects of agrochemicals on amphibians and how various types of agrochemicals can be reasonably used in agricultural areas.
2014, 35(4): 280-286. doi: 10.13918/j.issn.2095-8137.2014.4.280
To better understand the physiological characteristics of the silky starling (Sturnus sericeus), its body temperature (Tb), basal metabolic rate (BMR), evaporative water loss (EWL) and thermal conductance (C) elicited by different ambient temperatures (Ta) (5-30℃) were determined in the present study. Our results showed that they have a high Tb (41.6±0.1℃), a wide thermal neutral zone (TNZ) (20-27.5℃) and a relatively low BMR within the TNZ (3.37±0.17 mL O2/g·h). The EWL was nearly stable below the TNZ (0.91±0.07 mg H2O/g·h) but increased remarkably within and above the TNZ. The C was constant below the TNZ, with a minimum value of 0.14±0.01 mL O2/g·h·℃. These findings indicate that the BMR, Tb and EWL of the silky starling were all affected by Ta, especially when Ta was below 20℃ and the EWL plays an important role in thermal regulation.
Postnatal ontogenetic size and shape changes in the craniums of plateau pika and woolly hare (Mammalia：Lagomorpha)
2014, 35(4): 287-293. doi: 10.13918/j.issn.2095-8137.2014.4.287
In the present study, postnatal ontogenetic size and shape changes in the cranium of two lagomorph species, the plateau pika (Ochotona curzoniae) and woolly hare (Lepus oiostolus), were investigated by geometric morphometrics. The ontogenetic size and shape changes of their cranium exhibited different growth patterns in response to similar environmental pressures on the Qinghai-Tibetan Plateau. The overall size change in the cranium of the plateau pika was slower than that of the woolly hare. The percentage of ontogenetic shape variance explained by size in the woolly hare was greater than that in the plateau pika. The overall shape of the cranium was narrowed in both species, and morphological components in relation to neural maturity showed negative allometry, while those responsible for muscular development showed isometric or positive allometry. The most remarkable shape variations in the plateau pika were associated with food acquisition (temporalis development), though other remarkable shape variations in the incisive and palatal foramen in the ventral view were also observed. The most important shape change in the woolly hare was demonstrated by the elongation of the nasal bones, expansion of the supra-orbital process and shape variation of the neurocranium.
2014, 35(4): 294-299. doi: 10.13918/j.issn.2095-8137.2014.4.294
Cathepsin D (CTSD) is a lysosomal acidic endoproteinase that plays an important role in immune response. In this study, we obtained sweetfish (Plecoglossus altivelis) CTSD (PaCTSD) via de-novo transcriptome sequencing of sweetfish macrophages. The full length cDNA sequence of PaCTSD was 1 955 bp encoding a propeptide of 397 amino acids. The deduced protein had a calculated molecular weight of 43.17×103. Multiple alignment with other known CTSD amino acid sequences revealed amino acid conservation through the teleosts. Phylogenetic tree analysis showed that PaCTSD grouped tightly with other fish CTSD, and was close to that of Atlantic salmon and rainbow trout. Subsequently, PaCTSD was prokaryotically expressed and refolded by the urea gradient method on a nickel-nitrilotriacetic acid column. Enzyme activity analysis showed that PaCTSD exhibited pH-dependent proteolytic activity. Quantitative real-time PCR showed that PaCTSD mRNA was expressed in all detected tissues in healthy sweetfish. The highest expression was observed in the spleen and white blood cells, followed by liver, head-kidney, kidney, intestine, gill, and muscle. After Listonella anguillarum infection, PaCTSD transcripts were up-regulated significantly in liver, spleen, white blood cells, and head-kidney of sweetfish. In summary, PaCTSD has proteolytic activity and is closely involved in the immune response of sweetfish.
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.
2014, 35(4): 350-352. doi: 10.13918/j.issn.2095-8137.2014.4.350
Three Asian green pit vipers were collected in August 2013 during a field trip in Fanjin Mt. National Conservation Area, Guizhou. These specimens were identified as Sinovipera sichuanensis, based on subsequent examination and comparison. This is a new record of the genus Sinovipera and S. sichuanensis in Guizhou, and the first time that male specimens have been collected in the field.