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Clulow, J.

Publications and source records attributed to Clulow, J..

5 recordsLinked to original sources

Chytridiomycosis infection and heat compromises sperm quality in a threatened frog

Environmental change is reshaping wildlife reproduction through increasing temperatures and the spread of emerging infectious diseases, yet the physiological consequences of managing these stressors remain poorly understood. Amphibians are particularly vulnerable due to their ectothermy and high susceptibility to chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd). Here, we examine how Bd infection and thermal treatment interact to influence sperm quality and reproductive investment in male green and golden bell frogs (Ranoidea aurea), a species that has suffered severe population declines. Moderate Bd infection was associated with elevated sperm concentration relative to uninfected and heavily infected males, consistent with increased short-term reproductive investment under elevated mortality risk. However, severe infection led to pronounced reductions in sperm concentration and motility. Thermal treatment successfully eliminated Bd infection but imposed substantial reproductive costs: sperm concentration declined following treatment and remained significantly reduced six months later, despite partial recovery of sperm motility and membrane integrity. These results indicate persistent impairment of spermatogenic capacity rather than transient suppression. Our findings reveal that disease and thermal stress jointly shape amphibian reproductive outcomes through context-dependent trade-offs between immune defence and gamete production. While mild infection may trigger short-lived increases in reproductive output, both severe infection and pathogen clearance via thermal exposure impose lasting constraints on fertility. These results highlight an underappreciated cost of disease mitigation and suggest that increasing thermal extremes associated with climate change may further limit amphibian reproductive resilience, with important implications for conservation management and population persistence.

physiology↗

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↗

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↗