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Hormonal priming, induction of ovulation and in-vitro fertilization of the endangered Wyoming toad (Bufo baxteri).

The endangered Wyoming toad (Bufo baxteri) is the subject of an extensive captive breeding and reintroduction program. Wyoming toads in captivity rarely ovulate spontaneously and hormonal induction is used to ovulate females or to stimulate spermiation in males. With hormonal induction, ovulation is unreliable and egg numbers are low. The sequential administration of anovulatory doses of hormones (priming) has increased egg numbers and quality in both anurans and fish. Consequently, we tested the efficacy of a combination of human Chorionic Gonadotrophin (hCG) and Luteinizing Hormone Releasing Hormone analogue (LHRHa) administered as one dose, or two or three sequential doses to Bufo baxteri on egg numbers, fertilization and early embryo development. Spawning toads deposited eggs into Simplified Amphibian Ringers (SAR) solution to enable controlled in-vitro fertilization (IVF) with sperm from hormonally induced male toads. Unprimed females receiving a single mixed normally ovulatory dose of 500 IU hCG plus 4 micrograms of LHRHa produced no eggs. Whereas females primed with this dose and an anovulatory dose (100 IU hCG and 0.8 micrograms of LHRHa) of the same hormones, or primed only with an anovulatory dose, spawned after then receiving an ovulatory dose. Higher total egg numbers were produced with two primings than with one priming. Moreover, two primings produced significantly more eggs from each individual female than one priming. The cleavage rate of eggs was not found to differ between one or two primings. Nevertheless, embryo development with eggs from two primings gave a significantly greater percentage neurulation and swim-up than those from one priming. Of the male toads receiving a single dose of 300 IU hCG, 80% produced spermic urine with the greatest sperm concentration 7 hours post-administration (PA). However, peak sperm motility (95%) was achieved at 5 hours PA and remained relatively constant until declining 20 hours PA. In conclusion, Bufo baxteri egg numbers and quality benefited from sequential priming with LHRHa and hCG whereas spermic urine for IVF was produced from males with a single dose of hCG. The power of assisted reproduction technology in the conservation of endangered amphibians is shown by the release of nearly 2000 tadpoles produced by IVF during this study.

Animal Husbandry↗

Removing the threat of diclofenac to critically endangered Asian vultures.

Veterinary use of the nonsteroidal anti-inflammatory (NSAID) drug diclofenac in South Asia has resulted in the collapse of populations of three vulture species of the genus Gyps to the most severe category of global extinction risk. Vultures are exposed to diclofenac when scavenging on livestock treated with the drug shortly before death. Diclofenac causes kidney damage, increased serum uric acid concentrations, visceral gout, and death. Concern about this issue led the Indian Government to announce its intention to ban the veterinary use of diclofenac by September 2005. Implementation of a ban is still in progress late in 2005, and to facilitate this we sought potential alternative NSAIDs by obtaining information from captive bird collections worldwide. We found that the NSAID meloxicam had been administered to 35 captive Gyps vultures with no apparent ill effects. We then undertook a phased programme of safety testing of meloxicam on the African white-backed vulture Gyps africanus, which we had previously established to be as susceptible to diclofenac poisoning as the endangered Asian Gyps vultures. We estimated the likely maximum level of exposure (MLE) of wild vultures and dosed birds by gavage (oral administration) with increasing quantities of the drug until the likely MLE was exceeded in a sample of 40 G. africanus. Subsequently, six G. africanus were fed tissues from cattle which had been treated with a higher than standard veterinary course of meloxicam prior to death. In the final phase, ten Asian vultures of two of the endangered species (Gyps bengalensis, Gyps indicus) were dosed with meloxicam by gavage; five of them at more than the likely MLE dosage. All meloxicam-treated birds survived all treatments, and none suffered any obvious clinical effects. Serum uric acid concentrations remained within the normal limits throughout, and were significantly lower than those from birds treated with diclofenac in other studies. We conclude that meloxicam is of low toxicity to Gyps vultures and that its use in place of diclofenac would reduce vulture mortality substantially in the Indian subcontinent. Meloxicam is already available for veterinary use in India.

Animal Feed↗

Probabilistic risk assessment for snails, slugs, and endangered honeycreepers in diphacinone rodenticide baited areas on Hawaii, USA.

Three probabilistic models were developed for characterizing the risk of mortality and subacute coagulopathy to Poouli, an endangered nontarget avian species, in broadcast diphacinone-baited areas on Hawaii, USA. For single-day exposure, the risk of Poouli mortality approaches 0. For 5-d exposure, the mean probability of mortality increased to 3% for adult and 8% for juvenile Poouli populations. For Poouli that consume snails containing diphacinone residues for 14 d, the model predicted increased levels of coagulopathy for 0.42 and 11% of adult and juvenile Poouli populations, respectively. Worst-case deterministic risk characterizations predicted acceptable levels of risk for nonthreatened or endangered species such as northern bobwhite quail and mallards. Also, no acute toxicity was noted for snails and slugs that feed on diphacinone baits.

Animals↗

Influence of water quality and associated contaminants on survival and growth of the endangered Cape Fear shiner (Notropis mekistocholas).

The Cape Fear shiner (Notropis mekistocholas) is a recently described cyprinid species endemic to the Cape Fear River Basin of North Carolina, USA. Only five populations of the fish remain; thus, it is listed as endangered by the U.S. Government. Determining habitat requirements of the Cape Fear shiner, including water quality and physical habitat, is critical to the survival and future restoration of the species. To assess water quality in the best remaining and in the historical habitats, we conducted a 28-d in situ bioassay with captively propagated Cape Fear shiners. Fish were deployed at 10 sites in three rivers, with three cages per site and 20 fish per cage. Water and sediment samples were collected and analyzed for selected metals and organic contaminants. Passive sampling devices also were deployed at each site and analyzed for organic contaminants at test termination. Fish survival, growth (as measured by an increase in total length), and contaminant accumulation were measured on completion of the bioassay. Survival of caged fish averaged 76% (range, 53-100%) and varied significantly among sites and rivers. Caged fish accumulated quantities of cadmium, mercury, polychlorinated biphenyls, and other persistent contaminants over the test duration and grew significantly at only four sites. No apparent relations were observed between exposure to or accumulation of a specific contaminant and reduced growth or survival of fish among all the sites. However, a generalized hazard assessment showed that certain sites exhibited trends in cumulative contaminant presence with reduced fish survival and growth, thereby enabling the identification of the existing riverine habitat most suitable for reintroduction or population augmentation of this endangered fish.

Animals↗

Contaminant sensitivity of threatened and endangered fishes compared to standard surrogate species.

Standard environmental assessment procedures are designed to protect terrestrial and aquatic species. However, it is not known if endangered species are adequately protected by these procedures. At present, toxicological data obtained from studies with surrogate test fishes are assumed to be applicable to endangered fish species, but this assumption has not been validated. Static acute toxicity tests were used to compare the sensitivity of rainbow trout, fathead minnows, and sheepshead minnows to several federally listed fishes (Apache trout, Lahontan cutthroat trout, greenback cutthroat trout, bonytail chub, Colorado pikeminnow, razorback sucker, Leon Springs pupfish, and desert pupfish). Chemicals tested included carbaryl, copper, 4-nonylphenol, pentachlorophenol, and permethrin. Results indicated that the surrogates and listed species were of similar sensitivity. In two cases, a listed species had a 96-h LC50 (lethal concentration to 50% of the population) that was less than one half of its corresponding surrogate. In all other cases, differences between listed and surrogate species were less than twofold. A safety factor of two would provide a conservative estimate for listed cold-water, warm-water, and euryhaline fish species.

Animals↗

Long-term small effective population size, inbreeding, and a recessive lethal haplotype drive premature death in the endangered Devils Hole pupfish (Cyprinodon diabolis).

As anthropogenic habitat fragmentation and population decline accelerate globally, growing numbers of species face compounding demographic and genetic threats to long-term survival. Many populations are already forced to persist at chronically small sizes, yet the genomic and fitness consequences of this fate remain poorly understood. Here we leverage the demographic history of the Devils Hole pupfish to investigate how long-term small population size and recent bottlenecks have shaped genetic diversity, genetic load, inbreeding, and fitness through comparative population genomics, historical sequencing, and sampling embryos that died prematurely during development. We find that genetic diversity in Devils Hole pupfish is among the lowest recorded in the wild and that fixed load is high, consistent with thousands of generations of isolation at small population size. Even in the face of this low diversity and high fixed load, we show that inbreeding is still strongly associated with premature embryonic death, which affects up to 25% of offspring in the captive refuge and can be identified in advance based on a characteristic elongated heart tube and reduced heart rate. We discovered a recessive lethal haplotype segregating at ~20% frequency that accounts for 50% of embryonic deaths and contains mutations in MIB1 and MMP16, genes associated with cardiomyopathy and atrial fibrillation. Our findings link genotype, phenotype, and fitness in an iconic endangered species to provide a rare comprehensive view into the evolutionary dynamics and consequences of long-term small effective population size, demonstrating that endangered species remain vulnerable to inbreeding depression despite extremely low genetic diversity.

Journal Article↗

High population differentiation and genetic variation in the endangered Mexican pine Pinus Rzedowskii (Pinaceae).

Pinus rzedowskii is an endangered pine species from Michoacán (central México), which has been previously reported from only three localities. Classified within the subgenus Strobus, it exhibits intermediate morphological characters between subgenera Strobus and Pinus. We analyzed genetic aspects that could shed light on the evolution and conservation of this species. The genetic structure of nine populations was examined using 14 isozyme loci. Pinus rzedowskii has a relatively high level of genetic variation with 46.8% of the loci assayed being polymorphic, a total of 35 alleles, and a mean heterozygosity per population of 0.219. We calculated Wright's F(ST) statistic to estimate gene flow indirectly and to evaluate whether or not there was genetic structuring among populations. We found a marked differentiation among populations (F(ST) = 0.175) and significant inbreeding (F(IS) = 0.247). No pattern of isolation by distance was found. We also constructed a dendrogram based on a genetic distance matrix to obtain an overview of the possible historical relationships among populations. Finally, we found a convex relationship between the genetic distance among populations and the number of ancestral lineages, suggesting that demographically this species has not been at risk recently. Although endangered, with small and fragmented populations, P. rzedowskii shows higher levels of genetic variation than other conifer species with larger populations or similar conservation status.

Journal Article↗

[Fractal properties of the spatial pattern of rare and endangered plant populations in Houhe Nature Reserve in Hubei: Box-counting dimension].

The fractal properties of the spatial distribution pattern of 4 dominant plant populations and 7 rare and endangered plant populations were reflected through the box-counting dimensions. The results showed that the box-counting dimensions of 4 dominant populations ranged between 1.346 and 1.414, and occupied relatively larger ecological spaces in the community. Camellia cuspidata population occupied the largest ecological space because of its biggest box-counting dimensions. However, most of other populations occupied relatively smaller spaces due to their smaller values of the box-counting dimensions. The box-counting dimensions of 7 rare and endangered populations were smaller than 1 except Dipteronia sinensis population. The inflexion scales of 11 populations covered the range from 5 m to 12.5 m. According to the self-similarity of the fractal, the distribution pattern types under certain scales larger than the inflexion scale could be inferred as similar, and the results partly proved the ideas. The populations of Disopyros lotus, Pterostyrax psilophyllus, Davidia involucrata, Tetracentron sinense and Aesculus wilsonii were changed at the inflexion scale, and other 6 populations had the same distribution pattern types despite of the scale changed.

Conservation of Natural Resources↗

The endangered Illidge's ant blue butterfly (Acrodipsas illidgei) from an intertidal habitat managed for mosquito control.

Acrodipsas illidgei is an endangered butterfly inhabiting mangrove forests in southeastern Queensland, Australia. Concern over the effects of mosquito control activities prompted a broad-scale survey for the species at Coomera Island, in southeastern Queensland. The butterfly was recorded on the edge of an old-growth mangrove forest in close proximity to mosquito control runnels. Other forms of mosquito control at Coomera Island are unlikely to impact on the species because of the mode of action of larvicides used and the fact larvae occur within ant colonies formed in hollow stems and branches of mangrove trees. Further studies are required to more fully understand the relationships between mosquito control activities and the population dynamics of endangered species such as A. illidgei.

Animals↗

Cognitive improvement after extracranial reconstruction for the low flow--endangered brain.

Twelve patients with multiple occlusive neck vessel lesions were thought to possess low flow-endangered brains. These patients were studied before and after carotid reconstruction with a battery of neuropsychologic tests emphasizing memory and mental agility. A closely matched control group was selected from patients undergoing endarterectomy for hemodynamically insignificant lesions. The patients with low flow-endangered brains showed significantly greater improvements in memory and mental abilities than did the control group. Reconstruction of neck vessels may reverse such global neurologic deficits in carefully selected patients.

Analysis of Variance↗

Eliminating the risk of life-endangering complications following overstimulation with menotropin fertility agents: a report on women undergoing in vitro fertilization and embryo transfer.

OBJECTIVE: To evaluate a new method for preventing the life-endangering complications associated with inadvertent menotropin-induced severe ovarian hyperstimulation in patients undergoing in vitro fertilization and embryo transfer (IVF-ET). METHODS: Seventeen women each underwent a single cycle of controlled ovarian hyperstimulation with menotropins in preparation for IVF-ET. The indications for IVF-ET were tubal occlusion in nine, endometriosis in six, and unexplained infertility in two. The peak plasma estradiol (E2) concentration before hCG administration was greater than 6000 pg/mL and more than 30 ovarian follicles were detected by transvaginal ultrasound. Thus, life-endangering complications associated with severe ovarian hyperstimulation syndrome were highly likely to occur following hCG administration. Rather than cancel the cycle of treatment, menotropin therapy was discontinued and hCG administration was deferred for a number of days until the plasma E2 concentration fell below 3000 pg/mL ("prolonged coasting"), whereupon hCG was administered and egg retrievals and ETs were duly performed. RESULTS: None of the women developed severe ovarian hyperstimulation syndrome. There were six viable pregnancies (35.2%), which proceeded normally. CONCLUSION: This study indicates that "prolonged coasting" prevents severe ovarian hyperstimulation syndrome in severely overstimulated women undergoing IVF-ET, without necessitating cycle cancellation.

Adult↗

RAPD analysis reveals low genetic variability in the endangered light-footed clapper rail.

Numbers of light-footed clapper rails Rallus longirostris levipes, an endangered bird inhabiting southern California salt marshes, have substantially declined from historic levels. RAPD (randomly amplified polymorphic DNA) analysis was employed to assess the genetic variability within and among four of the largest remaining light-footed clapper rail populations. A single, larger population of the endangered Yuma clapper rail Rallus longirostris yumanensis was used for comparison. A total of 325 RAPD primers were tested on DNA from a subset of five clapper rails composed of a single representative for each of the four light-footed clapper rail populations and a representative for the single Yuma clapper rail population. Of the 1338 amplified bands (loci) surveyed in these five representative birds, approximately 1% were polymorphic, indicating the level of differentiation across all loci is quite low. Nine primers yielding these 16 polymorphic bands were used to analyse 48 individuals from five populations. Five of these bands were polymorphic in both subspecies, six were polymorphic only within the light-footed clapper rails, and five were polymorphic only within the Yuma clapper rail samples. Considering the few bands that were polymorphic among the light-footed clapper rail populations, a surprisingly high level of population differentiation (GST = 0.28) was found. This is in accord with the results of AMOVA analyses which show that a fairly high percentage of the limited variability among the rails is due to either differences between subspecies or differences between the light-footed rail populations. Because inbreeding depression is suspected and overall genetic distances between populations are low, movement of light-footed clapper rails from larger populations into smaller ones might be considered as a management strategy. Employing RAPDs as one of a series of assays is useful in revealing the population structure of genetically depauperate species.

Analysis of Variance↗

Population history rather than tree age contributes to the evolutionary importance of ancient trees in an endangered conifer.

Ancient trees are in global decline and face increasing conservation challenges. Their exceptional longevity has fostered the view that they are genetic reservoirs, yet whether old age is synonymous with unique genetic variation remains unclear. Here we assembled a ~8-Gb chromosome-level reference genome for the critically endangered conifer Glyptostrobus pensilis, now largely restricted to southern China with scattered populations in Vietnam and Laos, and resequenced 147 individuals, including 64 ancient (>100 years old and persisting in human-dominated landscapes), 33 wild and 50 recently cultivated individuals. Ancient individuals comprised both likely natural relics and historically introduced individuals and formed two deeply divergent lineages and one ancestral-admixed group, each with distinct demographic histories of prolonged contraction and genomic erosion. Lineage identity explained more variation in genome-wide diversity, inbreeding and genetic load than the three conservation types, despite broad differences in age structure. Rare-allele analyses revealed pronounced heterogeneity among ancient trees: only relic and ancestral-origin individuals from high-diversity lineages contributed substantial unique variation, much of which is poorly represented in wild and cultivated populations. Together, our findings suggest that ancient trees are not uniformly genetically irreplaceable and that, at least in this conifer, evolutionary importance is shaped more strongly by population history than by age alone.

Endangered Species↗

A telomere-to-telomere gap-free genome assembly of the endangered humphead wrasse (Cheilinus undulatus).

Humphead wrasse, Cheilinus undulatus, is an endangered fish species with high economic and ecological value as well as natural sex change from female to male, while sexual selection occurs in breeding aggregations. In our present study, we constructed the first gap-free telomere-to-telomere (T2T) genome assembly for humphead wrasse, by integration of PacBio HiFi, ONT Ultra-long and Hi-C sequencing techniques. With 99% of the entire sequences anchored into 24 chromosomes, this haplotypic genome assembly spans approximately 1.25 Gb and presents a complete set of 48 telomeres and 24 centromeres. In terms of correctness (quality value QV: 53.447) and completeness (BUSCO score: 99.3%), this chromosome-scale assembly is indeed of high quality. We predicted 658.03 Mb of repetitive sequences and annotated 26,609 protein-coding genes in the assembled genome. This high-quality T2T genome assembly not only facilitates the genetic conservation of humphead wrasse, but also offers fundamental genomic data for supporting in-depth investigations on functional genomics, genetic diversity, and selective breeding for this economically important teleost.

Animals↗

High connectivity and genetic diversity in the Endangered endemic white-spotted sand bass 'camotillo' (Paralabrax albomaculatus) within the Galápagos Marine Reserve.

BACKGROUND: Paralabrax albomaculatus, known as the camotillo or white-spotted sand bass, is a finfish endemic to the Galápagos Islands. An important species in the artisanal fishery, P. albomaculatus has undergone heavy population declines in recent decades, and is categorised as Endangered by the IUCN. Despite its socio-economic importance and endemic status, nothing is currently known about the population structure of the species, impeding evidence-based fisheries management. In this study, we use microsatellite markers to investigate its population structure and genetic diversity over the east, south and west of the Galápagos archipelago. RESULTS: We found evidence of high connectivity across the archipelago, with the species constituting a single population. We also found that genetic diversity was high, despite fishing pressure and recorded ongoing population declines. CONCLUSIONS: Our results suggest that P. albomaculatus should be managed as a single fishery stock across the Galápagos. As a single-population species and fishery, managers should also be aware that P. albomaculatus is inherently vulnerable to perturbation as recruitment from other populations is not possible in the event of population decline. However, high genetic diversity gives cause for cautious optimism regarding the genetic capacity for resilience of the population to novel environmental stressors. These results should aid in the implementation of a species-specific management plan for this Galápagos endemic.

Animals↗

Genomic early growth mechanisms of two endangered Mexican spruces.

This study elucidated the genomic basis of family-level growth variance in the critically endangered endemic Mexican spruces Picea martinezii and P. mexicana by: (i) analyzing family- and population-level variations in seedling basal diameter and height after 12 months of growth under common garden conditions and seed weight as maternal provisioning trait; and (ii) identifying genomic loci (SNPs) associated with these traits. Despite limited sample sizes (77 and 74 families representing all known populations of both species), 32 and 10 outlier SNPs were identified yielding 17 and six annotated candidate genes in P. martinezii and P. mexicana, respectively. These genes showed contrasting multivariate associations suggesting species-specific hypothesized growth strategies at the family level: defense-oriented framework in P. martinezii and plasticity-driven response in P. mexicana. Notably, several candidate genes encode key components of growth hormone pathways, including a gibberellin-regulated protein, a cytokinin hydroxylase and the AP2-like transcription factor ANT, providing valuable insights into how maternal genetic variation corresponds to the hormonal pathways that govern cell proliferation and organ size in the progeny. Integration of these findings with the contrasting demographic histories of both species revealed that population bottlenecks enhance the detectability of growth-associated variants by reducing background genetic variation. These genomic resources provide actionable information for prioritizing conservation measures, implementing assisted gene flow to maintain adaptive potential under climate change and designing future breeding programs. With 80.9-99.6% sequence identity to conserved Picea abies homologs, these findings may extend across the genus.

Picea↗

Two-dimensional electrophoresis for the study of blood/serum proteins of the otter, an endangered species.

Studies on serum/blood samples from European otters (Lutra lutra), a species under protection in most countries, were undertaken. Building of a two-dimensional electrophoresis (2-DE) protein identification map was started, identifying serum proteins by pattern comparison with cat or human serum protein maps and by immunoblotting using cross-reacting antibodies against the respective human serum proteins. The capture of endangered species is very restricted, thus making fresh samples almost unavailable; most investigations therefore had to be performed on samples collected from animals found dead. Patterns obtained from specimens of this source were compared to the established pattern of serum proteins and of blood or liver cells. Some of the major differences observed can be explained by contamination with cellular proteins and/or the onset of lytic processes generating breakdown products, as shown by preliminary in vitro experiments. Better knowledge of "normal" serum protein patterns and interfering products will facilitate interpretation of data collected from samples with a less defined history.

Animals↗

DNA polymorphism analysis in five endangered species of Meconopsis (Himalayan poppy) using multi-copy sequence-based probes.

An attempted to understand the genetic basis of extinction of Meconopsis (Himalayan poppy), an endangered genus of ornamental value and confined to alpine Himalayas, is described. Multicopy DNA sequences were isolated from a Sau3A partial genomic plasmid library of M. simplicifolia and M. paniculata. Genetic polymorphism between five species of Meconopsis belonging to geographically isolated populations was evident, albeit at a low level, in genomic Southerns when probed with repetitive DNA clones isolated from this library. Intra-population monomorphic band patterns for different populations of a species was evident with respect to a number of enzymes suggesting very low or no genetic variability and/or low mutation rate within a given population of Meconopsis.

Base Sequence↗