Genetic structure of Lake Magadi tilapia populations

The control region of the mitochondrial DNA haplotype frequencies were significantly different among the two separate lagoon populations of Oreochromis alcalicus grahami in Lake Magadi and of O. a. alcalicus from lake Natron, and DNA fingerprint similarity indices were significantly higher for intra-population comparisons of the two Magadi lagoon populations and the Lake Natron population than the inter-population similarity indices among these populations. A modified F _st measure indicated population sub-division and the phylogeographic partitioning of the VNTR fragments observed were unique to specific populations further indicating substantial genetic differentiation. The lagoon populations within Lake Magadi demonstrated the same degree of genetic differentiation as either of these populations did to the outgroup (the Lake Natron population). There appears to be limited gene flow between Lake Magadi tilapia populations and this population structure has important implications for protecting locally adapted populations within this unique ecosystem.     

Functional morphology of the gas-gland cells of the air-bladder of Oreochromis alcalicus grahami (teleostei: cichlidae): an ultrastructural study on a fish adapted to a severe, highly alkaline environment

Oreochromis alcalicus grahami is a small cichlid fish that lives in shallow peripheral lagoons of Lake Magadi, Kenya. The internal surface of the air-bladder is highly vascularized, illustrating possible utilization as an accessory respiratory organ. The wall of the bladder consists of five distinct tissue layers. From the outer to the inner surfaces are: a squamous, undifferentiated epithelial cell; a collagen-elastic tissue space; a smooth muscle tissue band; an inner connective tissue space; and columnar gas-gland cells projecting into the lumen. The cell membrane over the perikarya of the gas-gland cells was sporadically broken. The disruptions were ascribed to possible physical damage by discharging gas-bubbles. Juxtaluminally, the gas-gland cells attached across tight junctions. The cells have highly euchromatic nuclei and conspicuously large intracytoplasmic secretory bodies. On the blood capillary facing (basal) aspect, the cell membrane of the gas-gland cells is highly amplified. The cells insert onto a smooth muscle tissue band. The morphological features and the topographical arrangement of the gas-gland cells in O. a. grahami are indicative of an operative exchange of materials between them and the underlying tissue components especially the blood capillaries. For a fish that subsists in hot, highly saline and alkaline water heavily invested by avian predators and where the partial pressure of oxygen diurnally shifts from virtual anoxia to hyperoxia, development of a versatile air-bladder for efficient buoyancy control conforms to the functional demands placed on it by a unique environment. Illustratively, instead of the gas-gland morphology in O. a. grahami resembling that in the freshwater fishes, the group from which the fish has evolved, it compares more closely to that of the marine fish. This similarity suggests amazing parallel evolutionary adaptation to biophysically corresponding aquatic milieus.     

Scaling of oxygen consumption of Lake Magadi tilapia, a fish living at 37°C

Rates of oxygen consumption were measured in the geothermal, hot spring fish, Oreochromis alcalicus grahami by stopped flow respirometry. At 37° C, routine oxygen consumption followed the allometric relationship: V o₂=0.738 M ^0.75, where V o₂ is ml O₂ h ⁻¹ and M is body mass (g). This represents a routine metabolic rate for a 10 g fish at 37° C of 0.415 ml O₂ g⁻¹ h ⁻¹ (16.4 μmol O₂ g ⁻¹ h ⁻¹). Acutely increasing the temperature from 37 to 42° C significantly elevated the rate of O₂ consumption from 0.739 to 0.970 ml O₂ g ⁻¹ h ⁻¹ ( Q ₁₀=l.72). In the field, O. a. grahami was observed to be 'gulping' air from the surface of the water especially in hot springs that exceeded 40° C. O. a. grahami may utilize aerial respiration when O₂ requirements are high.     

Muscle as the primary site of urea cycle enzyme activity in an alkaline lake-adapted tilapia, Oreochromis alcalicus grahami.

The tilapia fish Oreochromis alcalicus grahami from Kenya has adapted to living in waters at pH 10.5 by excreting the end product of nitrogen metabolism as urea rather than as ammonia directly across the gills as occurs in most fish. The level of activity in liver of the first enzyme in the urea cycle pathway, carbamoyl-phosphate synthetase III (CPSase III), is too low to account for the observed high rates of urea excretion. We report here the surprising finding that CPSase III and all other urea cycle enzyme activities are present in muscle of this species at levels more than sufficient to account for the rate of urea excretion; in addition, the basic kinetic properties of the CPSase III appear to be different from those of other known type III CPSases. The sequence of the CPSase III cDNA is reported as well as the finding that glutamine synthetase activity is present in liver but not in muscle. This unusual form of adaptation may have occurred because of the apparent impossibility of packaging the needed amount of urea cycle enzymes in liver.     

Age Structure of Two Species of Odorous Frogs(Odorrana margaretae and Odorrana grahami)

Variation in age structure and body size benefits are identified to understand the evolution of life history. Here, we estimated the age structure and body size of two species of odorous frogs(Odorrana margaretae and Odorrana grahami) by using skeletochronology. The ages at sexual maturity of O. grahami and O. margaretae in both sexes were 1 and 2 years, respectively. For both sexes, the maximum age observed in O. margaretae was six years. For O. grahami, the maximum age observed in males and females were 4 and 5 years, respectively. Males and females did not differ in mean age in the two species.The average body size of both species considerably differed between sexes, with females being larger than males. The body size of females was also larger than that of males when the effect of age was removed. We also found positive correlations between body size and age within each sex in O. margaretae, but only for female in O. grahami. The female-biased sexual size dimorphism of the two species suggested that fecundity selection for larger female size may increase the reproductive output.     

Discordance between genetic structure and morphological, ecological, and physiological adaptation in Lake Magadi tilapia

The Magadi tilapia (Alcolapia grahami, formerly Oreochromis alcalicus grahami) is a remarkable example of teleost life in an extreme environment. Typical conditions include water pH=10, titration alkalinity>300 mM, osmolality=525 mOsm, temperatures ranging from 23 degrees to 42 degrees C, and O(2) levels fluctuating diurnally between extreme hyperoxia and anoxia. A number of relatively small tilapia populations are present in various thermal spring lagoons around the margin of the lake separated by kilometers of solid trona crust (floating Na(2)CO(3)) underlain by anoxic water. Despite the apparent isolation of different populations, annual floods may provide opportunities for exchange of fish across the surface of the trona and subsequent gene flow. To assess the question of isolation among Lake Magadi populations, we analyzed the variable control region of the mitochondrial DNA (mtDNA) from six lagoons. A total of seven mtDNA haplotypes, including three common haplotypes, were observed in all six populations. Several of the Lake Magadi populations showed haplotype frequencies indicative of differentiation, while others showed very little. However, differentiation among lagoon populations was discordant with their geographical distribution along the shoreline. All populations exhibited the unusual trait of 100% ureotelism but specialized morphological and physiological characteristics were observed among several of the lagoon systems. In addition, distinct differences were observed in the osmolality among the lagoons with levels as high as 1,400-1,700 mOsm kg(-1), with corresponding differences in the natural levels of whole-body urea. These levels of osmotic pressure proved fatal to fish from less alkaline systems but remarkably were also fatal to the fish that inhabited lagoons with this water chemistry. Upon more detailed inspection, specific adaptations to differential conditions in the lagoon habitat were identified that allowed survival of these cichlids. Additional evidence against potential for gene flow among lagoons despite the sharing of common mtDNA haplotypes was that the osmolality of floodwaters following a heavy rain showed lethal levels exceeding 1,700 mOsm kg(-1). In isolation, different mtDNA haplotypes would be predicted to go to fixation in different populations due to rapid generation times and the small effective population sizes in a number of lagoons. We propose a model of balancing selection to maintain common mtDNA sequences through a common selection pressure among lagoons that is based on microhabitats utilized by the tilapia.     

An antimicrobial peptide with antimicrobial activity against Helicobacter pylori

An antimicrobial peptide named odorranain-HP was identified from skin secretions of the diskless odorous frog, Odorrana grahami. It is composed of 23 amino acids with an amino acid sequence of GLLRASSVWGRKYYVDLAGCAKA. By BLAST search, odorranain-HP had similarity to antimicrobial peptide odorranain-W1 but it has a different GLLR N-terminus. The cDNA encoding odorranain-HP was cloned from the cDNA library of the skin of O. grahami. This peptide showed antimicrobial activities against tested microorganisms. Interestingly, odorranain-HP could exert antimicrobial capability against Helicobacter pylori, along with its antimicrobial activities similar to odorranain-W1. This is the first report of naturally occurring peptide with anti-H. pylori activity from amphibian skins.     

Molecular phylogenetic perspective on evolution of lizards of the Anolis grahami series

We report the results of phylogenetic analyses of 1447 bases of mitochondrial DNA sequence for 21 populations representing seven species of the Anolis grahami series (A. conspersus, A. garmani, A. grahami, A. lineatopus, A. opalinus, A. reconditus, and A. valencienni), six of which occur on Jamaica. These data include 705 characters that are phylogenetically informative according to parsimony. A parsimony analysis of these data combined with previously published allozymic data yields a single most parsimonious tree with strong support for monophyly of the A. grahami series, the sister-group relationship between Anolis lineatopus and A. reconditus and a clade composed of Anolis garmani, A. grahami, and A. opalinus. Based on DNA data alone, A. conspersus is nested within A. grahami. Haplotypes sampled from geographic populations of A. grahami, A. lineatopus, and A. opalinus are highly divergent (approximately 12-15% sequence difference on average for each species) and show similar phylogeographic patterns, suggesting that each of these currently recognized species may be a complex of species. Anolis valencienni also shows high sequence divergence among haplotypes from different geographic populations (approximately 8% sequence difference) and may contain cryptic species. Divergence among haplotypes within A. garmani is substantially lower (approximately 3% sequence difference), and phylogeographic patterns are significantly different from those observed in A. grahami, A. lineatopus and A. opalinus.     

The effects of temperature on muscle pH, adenylate and phosphogen concentrations in Oreochromis alcalicus grahami, a fish adapted to an alkaline hot-spring

The concentrations of phosphorylcreatine (PCr), adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), inorganic phosphate (Pi), pyruvate and lactate were determined in freeze-clamped fast muscle samples from Oreochromis alcalicus grahami a fish adapted to extreme alkalinity (∼ pH 10·0) and high temperatures (Lake Magadi, Kenya). Specimens were analysed from both geothermally heated hotsprings (35–37°C) and from isolated cool pools (28°C) and from stocks acclimated to 20°C in the laboratory. The ratios of (ATP)/(ADP) and (ATP)/(ADP) (Pi) decreased with increasing body temperature consistent with an increase in glycolysis and tissue respiration rates, respectively. The apparent equilibrium constant of creatine kinase (K_CK), (creatine) (ATP)/(phosphorylcreatine) (ADP) was found to decrease with increasing temperature: 20·2 (20°C), 13·9 (28°C), 8·0 (37°C). A near constant muscle and blood pH (or slight increase in alkalinity with higher temperatures) was found regardless of body temperature (Blood pH 7·64, 7·74, muscle pH 7·27, 7·51 at 20°C and 35°C, respectively). These results are consistent with an unusual pattern of acid-base regulation in this species.     

Escape tactics and effects of perch height and habituation on flight initiation distance in two Jamaican anoles (Squamata: polychrotidae)

Escape by Anolis lizards is influenced by microhabitats and fight initiation distance increases with predation risk. Differences in microhabitat use among ecomorphs affect escape behavior, but only two studies have reported ecomorphological differences in flight initiation distance among Greater Antillean species. I studied effects of predation risk and microhabitats on escape behavior by conducting field experiments using two species of anoles, Anolis lineatopus and A. grahami, on the campus of the University of the West Indies at Mona, Jamaica. Because ecomorphological variation of anoles has evolved independently within each island of the Greater Antilles, but relationships between ecomorphs and escape behaviors are poorly known, I characterized microhabitat use and escape tactics, and determined relationships between flight initiation distance and two risk factors, habituation to human presence and perch height, in Anolis lineatopus, a trunk-ground anole and A. grahami, a trunk-crown anole. Sample sizes for A. lineatopus and A. grahami were 214 and 93, for microhabitat use and escape destinations, 74 and 34 for human presence and 125 and 34 for perch height. The two species occurred in similar microhabitats and exhibited similar escape tactics, but exhibited key differences expected for their ecomorphs. Both species were sighted frequently on the ground and on trees, but A. lineatopus were more frequently on ground and were perched lower than A. grahami. Both species escaped from ground to trees and when on trees hid on far sides and escaped without changing climbing direction with equal frequency. The frequency of fleeing upward was greater for A. grahami than A. lineatopus. Both species exhibited habituation by having shorter flight initiation distances in areas with more frequent exposure to people. In both species flight initiation distance increased as perch height decreased because, lizards had to climb farther to be out of reach when perched lower. The relationship between flight initiation distance and perch height may apply to other anole ecomorphs that flee upward when low perched on trees.     

A novel antimicrobial peptide from amphibian skin secretions of Odorrana grahami

A novel antimicrobial peptide named odorranain-NR was identified from skin secretions of the diskless odorous frog, Odorrana grahami. It is composed of 23 amino acids with an amino acid sequence of GLLSGILGAGKHIVCGLTGCAKA. Odorranain-NR was classified into a novel family of antimicrobial peptide although it shared similarity with amphibian antimicrobial peptide family of nigrocin. Odorranain-NR has an unusual intramolecular disulfide-bridged hexapeptide segment that is different from the intramolecular disulfide-bridged heptapeptide segment at the C-terminal end of nigrocins. Furthermore, the -AKA fragment at the C-terminal of odorranain-NR is also different from nigrocins. Three different cDNAs encoding two odorranain-NR precursors and only one mature odorranain-NR was cloned from the cDNA library of the skin of O. grahami. This peptide showed antimicrobial activities against tested microorganisms except Escherichia coli (ATCC25922). Its antimicrobial mechanisms were investigated by transmission electron microcopy. Odorranain-NR exerted its antimicrobial functions by various means depending on different microorganisms.     

Amphibian tachykinin precursor

The precursor of amphibian tachykinin has not been found although more than 30 tachykinins have been isolated from amphibians since 1964. In this report, two tachykinin-like peptides are identified from the skin secretions of the frog, Odorrana grahami. Their amino acid sequences are DDTEDLANKFIGLM-NH(2) (named tachykinin OG1) and DDASDRAKKFYGLM-NH(2) that is the same with ranamargarin found in Rana margaretae, respectively, with a conserved FXGLM-NH(2) C-terminal consensus motif. By cDNA cloning, their precursors were screened from the skin cDNA library of O. grahami. The precursors are composed of 61 amino acid (aa) residues including a signal peptide followed by an acidic spacer peptide and one copy of mature tachykinin-like peptide. Their overall structure is different from structures of other tachykinin precursors such as human protachykinin 1 precursor containing 143 aa including one copy of substance P (SP) and neurokinin A (NKA), and ascidian tachykinin 1 precursor containing 164 aa including two copies of tachykinin-like peptides. The current results demonstrate that the biosynthesis mode of tachykinins in amphibians is different from other animals.     

滇池金线鲃亲鱼培育、繁殖力以及卵径大小与胚胎存活率的关系

2007—2010年,对滇池金线鲃(Sinocyclocheilus grahami)进行了亲鱼培育、繁殖力和卵径(egg diameter,ED)大小的研究。滇池金线鲃的催产率、核偏位率和孵化率分别由2007年的25.2%、61.5%和30.4%到2010年的91.3%、85.2%和44.5%。四年间这3项指标均有不同程度提高,其中,催产率提高的幅度最大。提供适合滇池金线鲃营养全面的饵料可以保证亲鱼培育的最佳效果,获得高质量的鱼卵和鱼苗。滇池金线鲃绝对怀卵量与体长的关系是F=0.0004826SL3.166(R2=0.6424,P<0.05)。四年来滇池金线鲃的平均产卵量为(2118.4±899.1)粒,平均绝对怀卵量为(2402.9±881.9)粒,平均相对怀卵量为(70.4±20.8)粒。产卵量、绝对怀卵量和相对怀卵量都有随着体长增加而增加的趋势。卵的大小与胚胎存活相关,在胚胎发育的前两天,不同批次胚胎的死亡呈现一种稳定的或低的死亡率;而后5～7d不同大小的卵呈现不同的死亡率,即小的卵具有更高的死亡率;而卵径>2.0mm的胚胎死亡率趋于稳定;大的卵有更高的生存潜力,能保证仔鱼开口前继续完善身体器官的形成或发生所需要的能量。     

用巨尾阿丽蝇蛹期发育形态来推断死者死亡时间

为了明确对于凶杀案件中死亡时间判断有重要意义的蝇蛹的形态随时间的变化规律,在16、20、24、28、32℃饲养了巨尾阿丽蝇并定期取样、解剖,显微镜下观察其体表形态变化。结果表明：巨尾阿丽蝇蛹壳内蛹呈现出规律性的变化,这些规律性的变化可指示出蛹的发育进程,进而可指示该昆虫在尸体上出现的时间,从而对死者的死亡时间做出判断。根据这些规律性的变化可将蛹期划分为7个阶段。不同恒温下达到以上阶段所需时间不同,高温下短而低温下长。     

云南滇池濒危特有种滇池金线鲃繁殖初报

滇池金线鲃(Sinocyclocheilus grahami)是滇池湖泊生态系统的指示物种和特有种。因水质污染和外来物种入侵等因素的影响,该鱼类自1986年起就从湖体中消失,仅有湖周围的少数龙潭中尚保存有少量滇池金线鲃。至2007年3月初,有两次繁殖试验取得了成功。先后试验了5尾雌鱼和6尾雄鱼,获得鱼卵约1600粒,并实施干法受精。约有1320粒受精,平均受精率为73%。孵化出鱼苗约480尾,平均孵化率为36%。经10天饲养,约有95%以上的鱼苗存活,体长达8-12mm。滇池金线鲃人工繁殖成功的意义有三点:能有效保护该物种免于灭绝;繁殖的鱼苗放归湖泊合适水域,将有助于恢复滇池的土著生物多样性;有助于推动当地水产养殖业从非土著鱼类养殖向土著鱼类养殖的方向转变。     

昆明裂腹鱼骨骼系统解剖

常规方法和透明骨法制备昆明裂腹鱼的骨骼标本,记录观测结果并拍照,对其骨骼系统进行描述。其特征为脊椎数目在46～47之间,肋骨在18～19之间,咽骨上有三排咽齿,齿式呈2:3:5式。骨骼系统的形状和变化可以为裂腹鱼亚属的分类及演化提供分类方面的基础资料。     

白鱼线粒体DNA控制区结构和种群遗传多样性分析

用特异性引物对白鱼(Anabarilius grahami)DNA进行PCR扩增,获得了白鱼线粒体DNA控制区基因全序列(930bp)。控制区T、C、A和G碱基组成为29.8%、22.5%、33.0%和14.7%。对照其他已报道的鱼类控制区结构,对白鱼控制区结构进行了分析,识别了其终止序列区、中央保守区和保守序列区,找到了终止相关的序列TAS以及保守序列(CSB-F、CSB-D、CSB-1、CSB-2、CSB-3)。同时运用DNA分析软件对白鱼一个驯养种群(中国科学院昆明动物研究所珍稀鱼类繁育中心)及两个自然地理种群(江川县明星鱼洞、江川县牛摩村)进行了遗传多样性分析。结果显示:两个自然种群存在较强基因交流,未出现遗传分化;人工驯养种群遗传多样性最高,种群复壮程度较好。     

Anti-infection peptidomics of amphibian skin

Peptidomics and genomics analyses were used to study an anti-infection array of peptides of amphibian skin. 372 cDNA sequences of antimicrobial peptides were characterized from a single individual skin of the frog Odorrana grahami that encode 107 novel antimicrobial peptides. This contribution almost triples the number of currently reported amphibian antimicrobial peptides. The peptides could be organized into 30 divergent groups, including 24 novel groups. The diversity in peptide coding cDNA sequences is, to our knowledge, the most extreme yet described for any animal. The patterns of diversification suggest that point mutations as well as insertion, deletion, and "shuffling" of oligonucleotide sequences were responsible for the diversity. The diversity of antimicrobial peptides may have resulted from the diversity of microorganisms. These diverse peptides exhibited both diverse secondary structure and "host defense" properties. Such extreme antimicrobial peptide diversity in a single amphibian species is amazing. This has led us to reconsider the strong capability of innate immunity and molecular genetics of amphibian ecological diversification and doubt the general opinion that 20-30 different antimicrobial peptides can protect an animal because of the relatively wide specificity of the peptide antibiotics. The antimicrobial mechanisms of O. grahami peptides were investigated. They exerted their antimicrobial functions by various means, including forming lamellar mesosome-like structures, peeling off the cell walls, forming pores, and inducing DNA condensation. With respect to the development of antibiotics, these peptides provide potential new templates to explore further.     

云南抚仙湖鱇(鱼良)白鱼若干生物学特性的形成和演化及其与湖泊环境演变的关系

根据1988年11月至1991年3月间野外收集的资料,本文记述了云南抚仙湖鱇(鱼良)白鱼的空间分布、食性和繁殖生物学特性。在此基础上,本文首次尝试从进化的观点出发,结合湖泊环境的演变历史深入一步分析了鱇(鱼良)白鱼生物学特性的形成和演化。研究结果表明,鱇(鱼良)白鱼喜在山泉水口等流水环境产卵及幼体喜居湖泊沿岸浅水区等生活习性属较原始的性状,提示了鱇(鱼良)白鱼的祖先是营溪流生活的鱼类。成体喜居湖泊敞水区中上层、营滤食为主、繁殖季节较长、各产卵群体的产卵时间间隔有明显规律性、繁殖力较低、性比较悬殊等特性则属较特化的性状,这些较特化性状的形成与湖泊环境的总体演变过程密切相关。在性状分析的基础上还进一步探讨了生物学特性的适应意义。     

Fine structure of the eggs of blowflies Aldrichina grahami and Chrysomya pacifica (Diptera: Calliphoridae)

We report here the fine structure of the eggs of blowflies Aldrichina grahami (Aldrich) and Chrysomya pacifica Kurahashi. For A. grahami, the plastron is wide and extends to almost the entire length of the eggs. The plastron near the micropyle is truncated. The polygonal patterns of chorionic sculpture bear a distinct swollen boundary. Regarding C. pacifica, the plastron is narrow and extends to almost the entire length of the eggs. The plastron near the micropyle bifurcates to a Y-shape, but the arms of the 'Y' are short. Information presented herein allows some distinctive features to differentiate among eggs of blowfly species.