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Clulow, S.

Publications and source records attributed to Clulow, S..

6 recordsLinked to original sources

Imminent invasion of the chytrid fungus threatens the last naive amphibian biodiversity hotspots

While the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) is driving catastrophic biodiversity loss worldwide, some amphibian communities persist seemingly unaffected despite occurring in climates conducive to pathogen establishment. These amphibian communities may remain epidemiologically naive. As mitigation of Bd is rarely successful after establishment, identifying remaining Bd-free refuges is imperative. Presently, the only known large-scale Bd-free refuge is the island of New Guinea (NG), safeguarding Australasias amphibian phylogenetic diversity otherwise devastated by Bd. Following extensive multi-year disease surveillance, we here uncover a second large-scale Bd-free refuge in the Sierra Nevada de Santa Marta (SNSM), a Neotropical biodiversity hotspot in northern Colombia. We detected no evidence of Bd in SNSM-wide screening, while we uncovered the presence of hypervirulent Bd-GPL in adjacent areas of the tropical Andes. Population genomic analyses in an SNSM-endemic anuran found no evidence for demographic bottlenecks indicative of cryptic epizootic decline. Niche modelling highlights the high risk for Bd establishment and Bd-induced declines in both the SNSM and NG, and the important role of lowland environmental barriers in restricting Bd invasion. Infection trials using three SNSM-endemic amphibians reveal varying disease susceptibility. Together, these data identify the SNSM as an epidemiologically naive refuge likely facing imminent Bd invasion, which could result in the loss of at least 25 endemic amphibian species. We highlight the urgent need for proactive conservation action and strict implementation of biosecurity to safeguard the unique and vast amphibian diversity of the worlds last major Bd-free refuges. Significance StatementAmphibian chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) has driven unprecedented global biodiversity loss. The Neotropics and Australasia comprise epicenters of declines. Identifying remaining Bd-free refuges is crucial to curb further amphibian extinctions, but so far contemporary absence of Bd has only been demonstrated for New Guinea. Here we identify the last known major Bd-free biodiversity hotspot in the Neotropics: the Sierra Nevada de Santa Marta (SNSM) in Colombia. Our results show that amphibian communities in this hotspot are immunologically naive despite occurrence of hypervirulent Bd lineages nearby and climatic conditions within the SNSM conducive to Bd-induced declines. This creates an imminent risk for Bd-driven declines and highlights the urgent need for preventive actions to avert another wave of biodiversity loss.

ecology↗

Developing a nuclear transplantation model for Australian amphibian conservation using a common species of Australian Frog (Limnodynastes peronii)

As amphibian populations continue to decline, there is a need to establish procedures to store and recover genetic diversity. Nuclear transplantation (NT) is a potentially useful reproductive technology with applications to conservation in specific circumstances. Although sperm have been successfully cryopreserved and used in in vitro fertilization to produce sexually mature offspring, there are many endangered and even extinct amphibian species that only exist as stored frozen adult or larval tissues. The only way to recover that genetic diversity is through NT. Nuclear transplantation can introduce new genes into captive breeding programs from individuals where no viable gametes from that individual have been cryopreserved. Amphibian NT research was common during the middle to late 20th century; however, it was never conducted for amphibian conservation. Early amphibian NT experiments were conducted for the purpose of determining whether differentiation was a terminal process that involved the loss of reprogramming genes. However, following the success of mammalian NT, amphibian NT research declined, and the field has been at a standstill for decades. This study developed the first nuclear transplantation protocol for a native Australia frog using Limnodynastes peronii, as a model. Specifically, this paper aimed to test: 1) nuclear transplantation with fresh embryonic cells in Limnodynastes peronii and trial the implementation of different egg enucleation methods (pricked eggs only, UV only, and pricked + UV), 2) use of cryopreserved cells in nuclear transplantation and 3) use single nucleotide polymorphisms to determine if the nuclear genomes of nuclear transplant embryos were derived from the injected donor cell nuclei. With both fresh and cryopreserved embryonic cells as donor cell nuclei, development of the nuclear transplant embryos was low compared to the fertilized controls. Only one nuclear transplant embryo derived from a fresh embryonic cell (injected into a UV only enucleated egg) developed into a tadpole. This attained stage 36 of the Gosner Staging System for Anurans (hind limbs) before dying. As an egg activation technique, pricking the eggs resulted in higher cleavage rates and development to blastula than UV only and pricked + UV nuclear transplants (in trials with cryopreserved cells), but none of the nuclear transplants from the pricked only group developed beyond blastula. None of the nuclear transplants derived from cryopreserved embryonic cells developed to tadpoles. The most advanced stage attained from cryopreserved embryonic cells was stage 13 (neurula) from UV only eggs. Single nucleotide polymorphism (SNP) analysis testing for genotype mismatches was used to determine if the genomes of developing nuclear transplant embryos were derived from the injected donor cell nucleus. SNP analysis confirmed that two true clones were generated in this study. This is the first confirmed nuclear transplantation in any Australian native frog.

developmental biology↗

Goldilocks conundrum explains cryoinjury in slow-cooled amphibian embryonic cells

Cryopreservation of intact fish and amphibian eggs and embryos is challenging due to the sheer size and yolk content, which prevents proper dehydration and causes lethal intracellular ice formation during cooling. Alternatively, cryopreservation of dissociated embryonic cells allows to biobank diploid genomes. However, amphibian and fish embryos have cells of varying sizes throughout the developing embryo and cell size distribution remains understudied in cryopreservation. This study examined cell size effects during cryopreservation of dissociated embryonic cells from two amphibian species. Most work used Limnodynatses peronii blastula, gastrula, and neurula cells cryopreserved with 10% dimethyl sulfoxide (DMSO) and sucrose at 0%, 1%, or 10%. Increasing sucrose concentration improved post-thaw recovery of membrane intact cells and cell concentrations, with gastrula and neurula cells having better recovery than blastula cells. An interaction amongst cryoprotectant concentration, cell size, and embryonic stage was observed. Higher sucrose improved recovery of larger cells, but reduced recovery of smaller cells. These results supported a "Goldilocks" model of cryoinjury, in which larger cells require more time to dehydrate adequately, while smaller cells are damaged by excessive dehydration and solute effects, implying that optimal cryoprotectant conditions differ by cell size. Based on post-thaw recovery, 10% DMSO + 10% sucrose was optimal and successfully applied to the cryopreservation of neurula cells from the threatened Rawlinsonia littlejohni, marking the first report of embryonic cell cryopreservation in a threatened amphibian. Our results demonstrated the importance of cell size effects on cryoinjury and their consideration in the application of cryopreservation for amphibian conservation. In brief: Since intact amphibian eggs and embryos cannot be cryopreserved, dissociated embryonic cell cryopreservation provides an alternative avenue for biobanking embryonic diploid genomes; however, cell size effects on cryoinjury is poorly understood and the developing amphibian embryo consists of different sized cells. We demonstrated that cell size influences cryopreservation outcomes by cryopreserving embryonic cells from three developmental stages under varying cryoprotectant concentrations.

developmental biology↗

Refrigerated storage and cryopreservation of hormone induced sperm in a threatened frog

There are strong potential benefits of incorporating assisted reproductive technologies (ARTs) into conservation programs for the management of threatened amphibians as the global amphibian decline continues. As sperm cryopreservation and other ARTs advance in common species, focus on non-lethal sperm collection methods for threatened amphibians is imperative. We aimed to realise this goal by testing various doses of exogenous hormones for non-lethal induction of spermiation in a threatened frog (Litoria aurea) and develop cold storage and cryopreservation protocols following the recovery of urinic sperm. Our major findings include: (1) that sperm release could be induced in high concentrations with 20 IU/g bodyweight of human chorionic gonadotrophin (hCG); (2) high levels (>50%) of live, motile sperm could be recovered post-cryopreservation by treating the sperm with 15% v/v DMSO and 1% w/v sucrose pre-freeze; and (3) urinic sperm stored at 5{degrees}C retained motility over a 14-day period. Our findings demonstrate that it is possible to obtain and store large quantities of quality sperm from a threatened amphibian via non-lethal means, representing an important step forward for the use of ARTs in conservation programs for rare and threatened species. Lay SummaryAmphibians are undergoing an extinction crisis unparalleled in any other taxa. The development of assisted reproductive technologies, such as sperm cryopreservation and IVF have an important role to play in the conservation management of amphibians globally. Here we report non-lethal methods of sperm collection and storage in a highly threatened amphibian. Major findings include successfully induced sperm release in high concentrations, retention of [~]50% live, motile sperm after 7 days storing it at 5{degrees}C, and successful recovery of of more than 50% live, motile sperm following cryopreservation. Our results demonstrate the viability of obtaining and storing sperm from rare and threatened species via non-lethal means, providing an important step forward for the use of ARTs in conservation programs around the globe.

cell biology↗

Sex-linked markers in an Australian frog Platyplectrum ornatum with a small genome and homomorphic sex chromosomes

Amphibians have highly diverse sex-determining modes leading to a notable interest in vertebrate sex determination and sex chromosome evolution. The identification of sex-determining systems in amphibians, however, is often difficult as a vast majority consist of homomorphic sex chromosomes making them hard to distinguish. In this study, we used Diversity Array Technology sequencing (DArTseq) to identify the sex-determining system in the ornate burrowing frog from Australia, Platyplectrum ornatum. We applied DArTseq to 44 individuals, 19 males and 25 females, collected from two locations to develop sex-linked markers. Unexpectedly, these 44 individuals were classified into two distinct population clusters based on our SNP analyses, 36 individuals in cluster-1, and 8 individuals in cluster-2. We then performed sex-linkage analyses separately in each cluster. We identified 35 sex-linked markers from cluster-1, which were all associated with maleness. Therefore, P. ornatum cluster-1 is utilising a male heterogametic (XX/XY) sex-determining system. On the other hand, we identified 210 sex-linked markers from cluster-2, of which 89 were male specific, i.e., identifying XX/XY sex determining system and 111 were female specific, i.e., identifying ZZ/ZW sex determining system, suggesting existence of either male or female heterogametic sex determining system in cluster-2. We also performed cytogenetic analyses in 1 male and 1 female from cluster-1; however, we did not detect any visible differentiation between the X and Y sex chromosomes. We also mapped sex-linked markers from the two clusters against the P. ornatum genome and our comparative analysis indicated that the sex chromosomes in both clusters shared homologies to chromosome 10 (autosome) of Rana temporaria and ZWY sex chromosome of Xenopus tropicalis. It is plausible that the cluster-2 has a potential to be either male or female heterogamety in sex determination, requiring further investigation.

genomics↗

Diving beetle offspring oviposited in amphibian spawn prey on the tadpoles upon hatching

In highly ephemeral freshwater habitats, predatory vertebrates are typically unable to become established, leaving an open niche often filled by macroinvertebrate predators. However, these predators are faced with the challenge of finding sufficient food sources as the rapid rate of desiccation prevents the establishment of extended food chains and limits the number of prey species present. It could therefore be advantageous for adults to oviposit their offspring in the presence of future prey within sites of extreme ephemerality. We report the first case of adult diving beetles ovipositing their eggs within spawn of the sandpaper frog, Lechriodus fletcheri. This behaviour was found among several pools used by L. fletcheri for reproduction. Beetle eggs oviposited in frog spawn were found to hatch within 24 hours of the surrounding L. fletcheri eggs, with the larvae becoming voracious consumers of the hatched tadpoles. Although it has yet to be established experimentally whether this is an adaptive behaviour, the laying of eggs among potential future tadpole prey in this instance should confer significant fitness benefits for the offspring upon hatching, ensuring that they are provided an immediate source of food at the start of their development and potentially throughout. This oviposition behaviour may be common among diving beetles and could form a significant predatory threat for amphibians with a free-swimming larval stage in ephemeral freshwater habitats.

zoology↗