Toxicity of diclofenac to Gyps vultures

Three endemic vulture species Gyps bengalensis, Gyps indicus and Gyps tenuirostris are critically endangered following dramatic declines in South Asia resulting from exposure to diclofenac, a veterinary drug present in the livestock carcasses that they scavenge. Diclofenac is widely used globally and could present a risk to Gyps species from other regions. In this study, we test the toxicity of diclofenac to a Eurasian (Gyps fulvus) and an African (Gyps africanus) species, neither of which is threatened. A dose of 0.8 mg kg(-1) of diclofenac was highly toxic to both species, indicating that they are at least as sensitive to diclofenac as G. bengalensis, for which we estimate an LD50 of 0.1-0.2 mg kg(-1). We suggest that diclofenac is likely to be toxic to all eight Gyps species, and that G. africanus, which is phylogenetically close to G. bengalensis, would be a suitable surrogate for the safety testing of alternative drugs to diclofenac.     

The Pied Crow (Corvus albus) is insensitive to diclofenac at concentrations present in carrion

Diclofenac, a nonsteroidal anti-inflammatory drug (NSAID), kills vultures (Gyps spp.) that consume tainted carcasses. As a result, vulture populations in India, Nepal, and Pakistan have been devastated. Studies on meloxicam and ketoprofen demonstrated that the toxicity of the NSAIDs is unpredictable, thereby necessitating individual testing of all available NSAIDs. Because it is no longer practical to use vultures for toxicity testing, we evaluated the Pied Crow (Corvus albus) as a model. Pied Crows (n=6) were exposed to a dose of 0.8 and 10 mg/kg of diclofenac, with no signs of toxicity, and a rapid half-life of elimination. Using primary renal cell and hepatocyte cultures, a high tolerance was demonstrated at the cellular level. Meta-analysis of pharmacokinetic data for the Domestic Chicken (Gallus gallus) and the African White-backed (Gyps africanus), Cape Griffon (Gyps coprotheres), and Turkey Vultures (Cathartes aura) showed a trend toward toxicity when the half-life of elimination increased. We conclude that the crow is not susceptible to diclofenac and, more important, that toxicity in the Gyps species is probably related to zero-order metabolism.     

Establishment of selected baseline blood chemistry and hematologic parameters in captive and wild-caught African white-backed vultures (Gyps africanus)

Despite the devastating collapse of three vulture populations on the Asian subcontinent as a result of their exposure to diclofenac, there is little available information on the normal physiology of many vulture species, including the African White-backed Vulture (Gyps africanus). Such information is needed to fully understand mechanisms for toxicity and to identify and prevent future health problems. The aim of this study was to establish baseline parameters for hematologic and selected serum chemistry parameters for this model species for further studies into the toxicity of diclofenac. Captive nonreleasable and wild African White-backed Vultures were used to determine reference values. For hematology, erythrocyte counts, hemoglobin concentration, hematocrit, packed cell volume, mean corpuscular volume, mean corpuscular hemoglobin concentration, and total and differential leukocyte counts were measured. Chemical analytes measured included sodium, potassium, calcium, albumin, and globulin concentrations, aspartate aminotransferase, creatine kinase, and alanine aminotransferase activities. Uric acid and urea concentrations and the urea:uric acid ratio also were evaluated. Values are presented as means, standard deviations, and reference intervals. The serum chemistry parameters selected may provide a starting point for the evaluation of changes in renal and hepatic function; these organ systems are most severely affected by diclofenac. Results were also compared with values reported for G. africanus nestlings, and from these results it is evident that the clinical pathologic parameters are age related. This indicates that the use of nestling values for the evaluation of clinical pathologic findings in adults may be unreliable and could lead to incorrect assumptions.     

Phylogeny and phylogeography of critically endangered Gyps species based on nuclear and mitochondrial markers

Populations of Oriental White-backed Vulture (Gyps bengalensis) and Long-billed Vulture (Gyps indicus) dramatically declined by 95–100% on the Indian subcontinent in mid-1990s. The present study was conducted to discover the phylogeny and phylogeography of Gyps species based on nuclear (recombination activating gene, RAG-1) and mitochondrial (cytochrome b, cytb) markers. Gyps species showed monophyly and no geographic partition was observed within the three groups of Gyps species (G. bengalensis, G. indicus and G. fulvus) despite the large sample size available (n = 149). Our study supports the treatment of G. indicus and G. tenuirostris as separate species. In all analyses, the earliest divergence separated G. bengalensis from all other Gyps taxa while a sister relationship was supported between G. fulvus and G. rueppellii, and these two taxa together were sister group to a clade consisting of G. indicus, G. tenuirostris and G. coprotheres. Molecular clock estimates of both nuclear and mitochondrial (RAG-1, cytb) genes indicated a rapid and recent diversification within the Gyps species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

New methods to identify conserved microsatellite loci and develop primer sets of high cross-species utility - as demonstrated for birds

We have developed a new approach to create microsatellite primer sets that have high utility across a wide range of species. The success of this method was demonstrated using birds. We selected 35 avian EST microsatellite loci that had a high degree of sequence homology between the zebra finch Taeniopygia guttata and the chicken Gallus gallus and designed primer sets in which the primer bind sites were identical in both species. For 33 conserved primer sets, on average, 100% of loci amplified in each of 17 passerine species and 99% of loci in five non-passerine species. The genotyping of four individuals per species revealed that 24-76% (mean 48%) of loci were polymorphic in the passerines and 18-26% (mean 21%) in the non-passerines. When at least 17 individuals were genotyped per species for four Fringillidae finch species, 71-85% of loci were polymorphic, observed heterozygosity was above 0.50 for most loci and no locus deviated significantly from Hardy-Weinberg proportions. This new set of microsatellite markers is of higher cross-species utility than any set previously designed. The loci described are suitable for a range of applications that require polymorphic avian markers, including paternity and population studies. They will facilitate comparisons of bird genome organization, including genome mapping and studies of recombination, and allow comparisons of genetic variability between species whilst avoiding ascertainment bias. The costs and time to develop new loci can now be avoided for many applications in numerous species. Furthermore, our method can be readily used to develop microsatellite markers of high utility across other taxa.     

Rapid population declines of Egyptian vulture (Neophron percnopterus) and red-headed vulture (Sarcogyps calvus) in India

Since the early 1990s, large and rapid population declines of three species of vulture ( Gyps spp.) endemic to south Asia have occurred on the Indian subcontinent and have led to these species being listed by IUCN as critically endangered. Evidence of rates of population decline, cause of death and toxicity is consistent with these declines being caused by poisoning of vultures through the ingestion of tissues from livestock treated with the anti-inflammatory drug diclofenac. In this paper, analysis of repeated surveys in and near protected areas widely spread across India shows that populations of two other vulture species, Egyptian vulture Neophron percnopterus and red-headed vulture Sarcogyps calvus , have also declined markedly and rapidly, but probably with a later onset than Gyps vultures in the same region. The declines continued at least up to 2003. It is recommended that these two species are considered for inclusion in the IUCN Red List and for urgent remedial conservation measures. Research is needed to determine whether or not the principal cause of these declines is diclofenac poisoning and to establish population trends in other scavenging birds in the Indian subcontinent.     

The use of (GACA)4 PCR to sex Old World vultures (Aves: Accipitridae)

A PCR method was developed employing a single primer (GACA)₄ to sex Black vultures ( Aegypius monachus ), Lappet-faced vultures ( Torgos tracheliotus ), and Griffon vultures ( Gyps fulvus ). Using the (GACA)₄ primer several PCR products were generated. One or more PCR products displayed a sex-specific pattern, i.e. they were only present in females (probably corresponding to repetitive DNA on the W chromosome) but absent in males. The sex ratio of 85 Griffon vultures from Spain was almost 1.     

Isolation and characterization of 15 microsatellite loci in four endangered Amentotaxus species (Taxaceae)

? Premise of the study: Fifteen microsatellite loci were developed in an endangered species, Amentotaxus formosana, and were tested in an additional three species, A. argotaenia, A. yunnanensis, and A. poilanei, to evaluate the population structure for conservation efforts and reconstruct the phylogeographic patterns of this ancient lineage. ? Methods and Results: Polymorphic primer sets were developed from A. formosana; the number of alleles ranged from two to 10, with an observed heterozygosity ranging from 0 to 0.60. All of the loci were found to be interspecifically amplifiable. ? Conclusions: These polymorphic and transferable loci will be potentially useful for future studies that will focus on identifying distinct genetic units within species and establishing the phylogeographic patterns and the process of speciation among closely related species.     

Isolation and characterization of chloroplast microsatellite markers in four mangrove species,Aegiceras corniculatum,Avicennia marina,Acanthus ilicifolius and Lumnitzera racemosa

One, three, seven, and six of polymorphic chloroplast microsatellite (cpSSR) markers were developed from four mangrove species, Acanthus ilicifolius, Aegiceras corniculatum, Avicennia marina, and Lumnitzera racemosa, respectively. Characterization of 229, 509, 369, and 216 individuals of A. ilicifolius, A. corniculatum, A. marina, and L. racemosa, collected from different natural mangrove populations (A. ilicifolius, 6; A. corniculatum, 14; A. marina, 10; L. racemosa, 6) in the southern coastline of China showed that these loci provide cpSSR markers with polymorphisms ranging from two to four alleles per locus and gene diversity between 0.005 and 0.675. Combining the polymorphic cpSSR loci of each species, 3, 5, 11, and 4 of cpSSR haplotypes were separately detected in populations of A. ilicifolius, A. corniculatum, A. marina, and L. racemosa in the southern coastline of China. These cpSSR markers will be useful for analyzing the maternal lineage distributions and population genetic structures of these four species.     

Isolation and characterization of thirteen polymorphic microsatellite loci in the A-genome perennial group of the legume genus Glycine

Thirteen polymorphic microsatellite loci were developed for the closely related and reproductively compatible species comprising the A-genome perennial group of the legume genus Glycine. Primers developed from the widespread and isozymically differentiated G. canescens amplified successfully across G. clandestina and four other species within the complex. Species were highly polymorphic, and observed heterozygosities were extremely low for all loci, as expected for these predominantly autogamous taxa. These markers will be useful in studying genetic variation, population structure, gene flow, and polyploidy within the A-genome group.     

Isolation and characterization of polymorphic microsatellite markers in Anthyllis vulneraria

To study the reproductive system of the putative autogamous species Anthyllis vulneraria in Belgian calcareous grasslands, eight polymorphic microsatellite loci were developed using the sequence‐tagged microsatellite profiling technique and 35 plants were typed for all loci. No linkage disequilibrium was found between any pair of loci. Over the loci, the number of alleles ranged from two to five. Observed and expected heterozygosities ranged from 0.053 to 0.688 and from 0.053 to 0.631, respectively. The estimated F_IS values (F_IS = 0.031 and 0.122) are not in accordance with a predominantly selfing reproductive system.     

New microsatellite loci isolated from the field cricket Gryllus bimaculatus characterized in two cricket species, Gryllus bimaculatus and Gryllus campestris

We have developed a new set of 27 polymorphic markers for each of two cricket species, Gryllus bimaculatus and Gryllus campestris. Initially, 14 published G. bimaculatus loci were tested in G. campestris; however, only five loci were polymorphic. Therefore, we isolated an additional 50 new microsatellite loci from G. bimaculatus and tested these in both species. In a minimum of 20 individuals, 27 of the new loci were polymorphic in G. bimaculatus and 25 in G. campestris.     

The digestive tract of the whiteback griffon vulture and its role in disease transmission among wild ungulates.

The digestive tract of the whiteback vulture (Gyps africanus) is described. Some disease organisms were fed to a captive bird to discover if they could survive passage through the tract, and the role of these scavenging birds in the spread of diseases among wild ungulates is discussed.     

Genetic characterization in four sciaenid species from the Arabian Sea

Four sciaenid species Johnieops dussumieri, Kathala axillaris, Pennahia macropthalmus and Otolithes ruber were analysed electrophoretically for genetic variation at 18 loci (16 in P. macropthalmus and O. ruber ). Twelve loci were polymorphic in J. dussumieri , 10 in K. axillaris , three in P. macropthalmus and 12 in O. ruber ( P <0·99). Average heterozygosity ranged from 0·033 ± 0·100 to 0·070 ± 0·122. The allele frequencies of 14 loci were used to estimate Nei's genetic distance (). The values ranged from 0·334 to 0·612. Three isozyme loci ( LDH-B*, MDH-2* and G3PDH-1* ) were found to be the most reliable species-specific markers.     

Chloroplast DNA Diversity among Trees, Populations and Species in the California Closed-Cone Pines (Pinus Radiata, Pinus Muricata and Pinus Attenuata)

The amount, distribution and mutational nature of chloroplast DNA polymorphisms were studied via analysis of restriction fragment length polymorphisms in three closely related species of conifers, the California closed-cone pines-knobcone pine: Pinus attenuata Lemm.; bishop pine: Pinus muricata D. Don; and Monterey pine: Pinus radiata D. Don. Genomic DNA from 384 trees representing 19 populations were digested with 9-20 restriction enzymes and probed with cloned cpDNA fragments from Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] that comprise 82% of the chloroplast genome. Up to 313 restriction sites were surveyed, and 25 of these were observed to be polymorphic among or within species. Differences among species accounted for the majority of genetic (haplotypic) diversity observed [G(st) = 84(+/-13)%]; nucleotide diversity among species was estimated to be 0.3(+/-0.1)%. Knobcone pine and Monterey pine displayed almost no genetic variation within or among populations. Bishop pine also showed little variability within populations, but did display strong population differences [G(st) = 87(+/-8)%] that were a result of three distinct geographic groups. Mean nucleotide diversity within populations was 0.003(+/-0.002)%; intrapopulation polymorphisms were found in only five populations. This pattern of genetic variation contrasts strongly with findings from study of nuclear genes (allozymes) in the group, where most genetic diversity resides within populations rather than among populations or species. Regions of the genome subject to frequent length mutations were identified; estimates of subdivision based on length variant frequencies in one region differed strikingly from those based on site mutations or allozymes. Two trees were identified with a major chloroplast DNA inversion that closely resembled one documented between Pinus and Pseudotsuga.     

药用植物天麻（Gastrodia elata Bl.）的等位酶遗传变异及其变型间的遗传关系

采用超薄平板聚丙烯酰胺等电聚焦电泳,对湖北西部药用植物天麻（Gastrodia elata Bl.）的4种变型进行了等位酶遗传变异的初步分析。在6个酶系统共检测到17个清晰位点和37个等位基因,其中多态位点11个,位点最大等位基因数为4,位点Acp-4和Prx-2的等位基因表现出变型特异性。遗传多样性分析结果表明：天麻在物种水平维持着较高遗传多样性,平均多态位点比率P=56.3%,每位点平均等位基因数A=2.1,平均预期杂合度He=0.222;但其变型水平遗传多样性较低（P=42.2%,A=1.7,He=0.146）,其中乌天麻遗传多样性最高,药天麻最低。天麻变型间遗传分化系数（GST）达0.3595,平均基因流（Nm）仅为0.4450,说明天麻变型间遗传分化较大,缺乏基因交流。聚类分析结果表明,天麻居群以变型为单元优先聚类而非地域优先聚类,揭示各天麻变型在遗传上是相对独立的进化显著单元,支持天麻种内变型的划分。红天麻与乌天麻两变型最为近缘,火天麻与药天麻两类群有着较近的遗传关系。     

Identification and properties of microsatellite markers in tsetse flies Glossina morsitans sensu lato (Diptera: Glossinidae)

Genomic libraries enriched for simple sequence repeats were constructed for Glossina morsitans morsitans, G. m. submorsitans, and G. m. centralis. Sixteen microsatellite markers were isolated from the libraries and evaluated on flies from natural G. m. morsitans populations and other Glossina species in the Morsitans and Palpalis species groups. The primers amplified appropriate sized DNA fragments in the Morsitans and Palpalis groups. In G. morsitans s.l., eight of 12 dinucleotide repeats and four of 12 trinucleotide repeats were polymorphic. The polymorphic loci showed a mean 7.5 +/- 4.8 alleles per locus and their mean heterozygosity was 55.8 +/- 7.7%.     

Levels and patterns of genetic variation in the endangered species Abronia macrocarpa

Genetic variation was evaluated in the federally endangered species Abronia macrocarpa (large-fruited sand-verbena), an herbaceous perennial restricted to deep sandy soils and endemic to three counties of east-central Texas. Seven of the ten known populations were sampled and analyzed using starch gel electrophoresis of eight enzymes coded by 18 interpretable loci. Duplicate gene expression was observed for four loci, suggesting polyploid ancestry for the lineage that includes A. macrocarpa. Values for estimators of genetic polymorphism within populations (ranges: P = 38.9%-61.1%, A = 1.7-2.1, H = 0.122-0.279) exceeded average values for seed plants (P = 34.2%, A = 1.53, H = 0.113). Genotype proportions at most loci in most populations were in Hardy-Weinberg equilibrium, consistent with obligate outcrossing previously documented for this species; exceptions could be attributed to population substructure. Values of F(ST) tended to be high, ranging from 0.021 to 0.481 for individual loci (mean F(ST) = 0.272), indicating substantial divergence and limited gene flow among populations, despite their close geographic proximity. Pairwise values of Nei's genetic identity between populations ranged from 0.799 to 0.975 and tended to be influenced by geographic proximity of population pairs. Collectively, these data suggest a long history of isolation among populations that have not been subjected to bottlenecks. Isolation of A. macrocarpa populations apparently results from the disjunct occurrence of suitable habitat and perhaps has been accentuated by human disturbance.     

Genetic variation in Sri Lankan leopards

Electrophoretic variation of 50 gene-enzyme systems was typed in a population of 33 captive leopards (Panthera pardus) from the island of Sri Lanka. The captive leopard population was composed of several lineages: (1) wild-caught leopards of the island subspecies (P. p. kotiya), (2) captive-born animals of the same subspecies, (3) a melanistic lineage whose founders were obtained from Malaysia (P. p. delacouri), and (4) leopards of known mixed lineage and unknown status. Two loci, APRT and PGD, were polymorphic in all samples, whereas 48 loci were invariant. Percent polymorphism (P) and percent average heterozygosity (H) were calculated as 4% and 1.4%, respectively, for the wild-caught leopards; 4% and 1.2% for the captive-born kotiya leopards; and 4% and 2.0% for the melanistic lineage. The overall results revealed a detectable decrease in genetic variability compared with a previous study of captive leopards from mainland origins. The mainland leopards had three additional polymorphic loci, ADA, ESI, and HBB. Reexamination of the TF locus using a revised protocol resolved a new allele in the sample of mainland leopards but not in the Sri Lankan sample. With this new polymorphism, recalculated P and H values for the mainland sample are 10% and 3.1%, respectively. No significant differences in polymorphic loci were observed between the leopard subspecies examined.     

4种形态类型诸葛菜的等位酶变异及遗传多样性分析

以采自江苏省南京市4个样点的4种不同形态类型(紫花毛果、紫花光果、白花光果和白花毛果)诸葛菜〔Orychophragmus violaceus(L.)O.E.Schulz〕63个单株为研究对象,采用不连续聚丙烯酰胺凝胶电泳技术(SDS-PAGE)对过氧化物酶同工酶(POD)、乙醇脱氢酶(ADH)、谷氨酸脱氢酶(GDH)、超氧化物歧化酶(SOD)和多酚氧化酶(PPO)酶谱进行了分析,并基于5个等位酶的酶谱分析结果对4种形态类型诸葛菜的遗传多样性进行了研究。结果表明:5个等位酶系统共包含12个位点32个等位基因,其中POD-1、POD-4、POD-5、POD-6、ADH-1、PPO-1、SOD-1和SOD-2为多态性位点;除具有共有谱带外,不同类型诸葛菜还具有各自特有的酶谱特征,其中,GDH-2的b带和c带分别是紫花类型和白花类型的特有谱带。紫花毛果、紫花光果和白花毛果类型的多态位点百分率(PPL)均为58.33%,而白花光果的PPL为66.67%;紫花毛果、紫花光果、白花光果和白花毛果每个位点的平均等位基因数分别为2.42、2.25、2.42和1.83,平均期望杂合度分别为0.35、0.30、0.38和0.29。4种形态类型诸葛菜的总基因多样度平均值为0.61,类型内基因多样度平均值为0.49,明显大于类型间基因多样度平均值(0.12);类型间的基因分化系数平均值为0.195,表明总基因多样性的80.5%来源于类型内。紫花光果和白花毛果类型间的遗传一致度最低(0.724 4)且遗传距离最大(0.322 5),而2个白花类型间的遗传一致度最高(0.954 1)且遗传距离最小(0.047 1)。研究结果显示:诸葛菜的种内变异程度很大、遗传分化程度较高,但在各类型内具有较高的遗传相似性。