Ecological Civilisation and Amphibian Sustainability through Reproduction Biotechnologies, Biobanking, and Conservation Breeding Programs (RBCs).

Intergenerational justice entitles the maximum retention of Earth's biodiversity. The 2022 United Nations COP 15, "Ecological Civilisation: Building a Shared Future for All Life on Earth", is committed to protecting 30% of Earth's terrestrial environments and, through COP 28, to mitigate the effects of the climate catastrophe on the biosphere. We focused this review on three core themes: the need and potential of reproduction biotechnologies, biobanks, and conservation breeding programs (RBCs) to satisfy sustainability goals; the technical state and current application of RBCs; and how to achieve the future potentials of RBCs in a rapidly evolving environmental and cultural landscape. RBCs include the hormonal stimulation of reproduction, the collection and storage of sperm and oocytes, and artificial fertilisation. Emerging technologies promise the perpetuation of species solely from biobanked biomaterials stored for perpetuity. Despite significant global declines and extinctions of amphibians, and predictions of a disastrous future for most biodiversity, practical support for amphibian RBCs remains limited mainly to a few limited projects in wealthy Western countries. We discuss the potential of amphibian RBCs to perpetuate amphibian diversity and prevent extinctions within multipolar geopolitical, cultural, and economic frameworks. We argue that a democratic, globally inclusive organisation is needed to focus RBCs on regions with the highest amphibian diversity. Prioritisation should include regional and international collaborations, community engagement, and support for RBC facilities ranging from zoos and other institutions to those of private carers. We tabulate a standard terminology for field programs associated with RBCs for publication and media consistency.

Reproduction technologies for the sustainable management of Caudata (salamander) and Gymnophiona (caecilian) biodiversity.

We review the use of reproduction technologies (RTs) to support the sustainable management of threatened Caudata (salamanders) and Gymnophiona (caecilian) biodiversity in conservation breeding programs (CBPs) or through biobanking alone. The Caudata include ∼760 species with ∼55% threatened, the Gymnophiona include ∼215 species with an undetermined but substantial number threatened, with 80% of Caudata and 65% of Gymnophiona habitat unprotected. Reproduction technologies include: (1) the exogenous hormonal induction of spermatozoa, eggs, or mating, (2) in vitro fertilisation, (3) intracytoplasmic sperm injection (ICSI), (4) the refrigerated storage of spermatozoa, (5) the cryopreservation of sperm, cell or tissues, (6) cloning, and (7) gonadal tissue or cell transplantation into living amphibians to eventually produce gametes and then individuals. Exogenous hormone regimens have been applied to 11 Caudata species to stimulate mating and to 14 species to enable the collection of spermatozoa or eggs. In vitro fertilisation has been successful in eight species, spermatozoa have been cryopreserved in seven species, and in two species in vitro fertilisation with cryopreserved spermatozoa has resulted in mature reproductive adults. However, the application of RTs to Caudata needs research and development over a broader range of species. Reproduction technologies are only now being developed for Gymnophiona, with many discoveries and pioneering achievement to be made. Species with the potential for repopulation are the focus of the few currently available amphibian CBPs. As Caudata and Gymnophiona eggs or larvae cannot be cryopreserved, and the capacity of CBPs is limited, the perpetuation of the biodiversity of an increasing number of species depends on the development of RTs to recover female individuals from cryopreserved and biobanked cells or tissues.

Semen and oocyte collection, sperm cryopreservation and IVF with the threatened North American giant salamander Cryptobranchus alleganiensis.

Semen of high to moderate quality was collected following the hormonal induction of North American giant salamanders Cryptobranchus alleganiensis . Oocytes from one female yielded the first C. alleganiensis produced while maintained in aquaria under human care and the first externally fertilising salamander produced with cryopreserved spermatozoa and IVF. Further research is needed with North American giant salamanders to establish reliable techniques to produce large numbers of viable offspring, along with the application of cryopreserved spermatozoa.

EFFICACY OF SUBCUTANEOUS IMPLANTS TO PROVIDE CONTINUOUS PLASMA TERBINAFINE IN HELLBENDERS (CRYPTOBRANCHUS ALLEGANIENSI) FOR FUTURE PROPHYLACTIC USE AGAINST CHYTRIDIOMYCOSIS.

Batrachochytrium dendrobatidis (Bd) is an important fungal pathogen present in wild hellbender (Cryptobranchus alleganiensis) populations that appears to cause disease during novel exposure and acute stress. Hellbender repatriation efforts are ongoing to combat declining populations, but mortality by chytridiomycosis (disease from Bd) after release has been reported. The goal was to determine whether a safe antifungal agent could be administered and provide prolonged plasma concentrations without repeated handling. A subcutaneous implant impregnated with 24.5 mg of terbinafine was tested in three juvenile eastern hellbenders (C. a. alleganiensis) raised in human care, and plasma terbinafine concentrations were recorded from weekly to biweekly for 141 days. Plasma concentrations were variable, with peak plasma concentrations of 1,610, 112, and 66 ng/ml between 28 and 56 days postimplant. Although all hellbenders achieved plasma concentrations above the published minimum inhibitory concentration for terbinafine against Bd zoospores (63 ng/ml) at several time points, only one individual remained above this threshold for more than two consecutive time intervals. Results show the potential for these implants as a prophylaxis for chytridiomycosis in captive-to-wild hellbender releases. However, further investigation will be needed to determine the plasma concentrations required to achieve prophylaxis in vivo and implant reliability.

Emerging Pathogens and a Current-Use Pesticide: Potential Impacts on Eastern Hellbenders.

Populations of the eastern hellbender Cryptobranchus alleganiensis alleganiensis have been declining for decades, and emerging pathogens and pesticides are hypothesized to be contributing factors. However, few empirical studies have attempted to test the potential effects of these factors on hellbenders. We simultaneously exposed subadult hellbenders to environmentally relevant concentrations of either Batrachochytrium dendrobatidis (Bd) or a frog virus 3-like ranavirus (RV), a combination of the pathogens, or each pathogen following exposure to a glyphosate herbicide (Roundup). Additionally, we measured the ability of the skin mucosome to inactivate Bd and RV in growth assays. We found that mucosome significantly inactivated RV by an average of 40% but had no negative effects on Bd growth. All treatments that included RV exposure experienced reduced survival compared to controls, and the combination of RV and herbicide resulted in 100% mortality. Histopathology verified RV as the cause of mortality in all RV-exposed treatments. No animals were infected with Bd or died in the Bd-only treatment. Our results suggest that RV exposure may be a significant threat to the survival of subadult hellbenders and that Roundup exposure may potentially exacerbate this threat.

GEOGRAPHIC AND INDIVIDUAL DETERMINANTS OF IMPORTANT AMPHIBIAN PATHOGENS IN HELLBENDERS (CRYPTOBRANCHUS ALLEGANIENSIS) IN TENNESSEE AND ARKANSAS, USA.

Wildlife diseases are a major threat for species conservation and there is a growing need to implement disease surveillance programs to protect species of concern. Globally, amphibian populations have suffered considerable losses from disease, particularly from chytrid fungi (Batrachochytrium dendrobatidis [Bd] and Batrachochytrium salamandrivorans [Bsal]) and ranavirus. Hellbenders (Cryptobranchus alleganiensis) are large riverine salamanders historically found throughout several watersheds of the eastern and midwestern US. Populations of both subspecies (Ozark hellbender, Cryptobranchus alleganiensis bishopi; eastern hellbender, Cryptobranchus alleganiensis alleganiensis) have experienced precipitous declines over at least the past five decades, and emerging pathogens are hypothesized to play a role. We surveyed Ozark hellbender populations in Arkansas (AR) and eastern hellbender populations in Middle Tennessee (MTN) and East Tennessee (ETN) for both chytrid fungi and ranavirus from swabs and tail tissue, respectively, from 2011 to 2017. Overall, we detected Bd on hellbenders from nine out of 15 rivers, with total prevalence of 26.7% (54/ 202) that varied regionally (AR: 33%, 28/86; MTN: 11%, 4/36; ETN: 28%, 22/80). Ranavirus prevalence (9.0%, 18/200) was comparatively lower than Bd, with less regional variation in prevalence (AR: 6%, 5/ 85; MTN: 11%, 4/36; ETN: 10%, 8/79). We did not detect Bsal in any hellbender populations. We detected a significant negative correlation between body condition score and probability of ranavirus infection (ß=-0.13, SE=0.06, 95% confidence interval: -0.24, -0.02). Evaluation of infection load of positive individuals revealed different trends than prevalence alone for both ranavirus and Bd, with MTN having a significantly greater average ranaviral load than both other regions. We documented a variety of lesions that likely have multiple etiologies on hellbenders located within all geographic regions. Our data represent a multiyear pathogen dataset across several regions of C. alleganiensis, and we emphasize the need for continued pathogen surveillance.

Sperm collection and storage for the sustainable management of amphibian biodiversity.

Current rates of biodiversity loss pose an unprecedented challenge to the conservation community, particularly with amphibians and freshwater fish as the most threatened vertebrates. An increasing number of environmental challenges, including habitat loss, pathogens, and global warming, demand a global response toward the sustainable management of ecosystems and their biodiversity. Conservation Breeding Programs (CBPs) are needed for the sustainable management of amphibian species threatened with extinction. CBPs support species survival while increasing public awareness and political influence. Current CBPs only cater for 10% of the almost 500 amphibian species in need. However, the use of sperm storage to increase efficiency and reliability, along with an increased number of CBPs, offer the potential to significantly reduce species loss. The establishment and refinement of techniques over the last two decades, for the collection and storage of amphibian spermatozoa, gives confidence for their use in CBPs and other biotechnical applications. Cryopreserved spermatozoa has produced breeding pairs of frogs and salamanders and the stage is set for Lifecycle Proof of Concept Programs that use cryopreserved sperm in CBPs along with repopulation, supplementation, and translocation programs. The application of cryopreserved sperm in CBPs, is complimentary to but separate from archival gene banking and general cell and tissue storage. However, where appropriate amphibian sperm banking should be integrated into other global biobanking projects, especially those for fish, and those that include the use of cryopreserved material for genomics and other research. Research over a broader range of amphibian species, and more uniformity in experimental methodology, is needed to inform both theory and application. Genomics is revolutionising our understanding of biological processes and increasingly guiding species conservation through the identification of evolutionary significant units as the conservation focus, and through revealing the intimate relationship between evolutionary history and sperm physiology that ultimately affects the amenability of sperm to refrigerated or frozen storage. In the present review we provide a nascent phylogenetic framework for integration with other research lines to further the potential of amphibian sperm banking.

Dietary alkaloid sequestration in a poison frog: an experimental test of alkaloid uptake in Melanophryniscus stelzneri (Bufonidae).

Several lineages of brightly colored anurans independently evolved the ability to secrete alkaloid-containing defensive chemicals from granular glands in the skin. These species, collectively referred to as 'poison frogs,' form a polyphyletic assemblage that includes some species of Dendrobatidae, Mantellidae, Myobatrachidae, Bufonidae, and Eleutherodactylidae. The ability to sequester alkaloids from dietary arthropods has been demonstrated experimentally in most poison frog lineages but not in bufonid or eleutherodactylid poison frogs. As with other poison frogs, species of the genus Melanophryniscus (Bufonidae) consume large numbers of mites and ants, suggesting they might also sequester defensive alkaloids from dietary sources. To test this hypothesis, fruit flies dusted with alkaloid/nutritional supplement powder were fed to individual Melanophryniscus stelzneri in two experiments. In the first experiment, the alkaloids 5,8-disubstituted indolizidine 235B' and decahydroquinoline were administered to three individuals for 104 days. In the second experiment, the alkaloids 3,5-disubstituted indolizidine 239Q and decahydroquinoline were given to three frogs for 153 days. Control frogs were fed fruit flies dusted only with nutritional supplement. Gas chromatography/mass spectrometry analyses revealed that skin secretions of all experimental frogs contained alkaloids, whereas those of all control frogs lacked alkaloids. Uptake of decahydroquinoline was greater than uptake of 5,8-disubstituted indolizidine, and uptake of 3,5-disubstituted indolizidine was greater than uptake of decahydroquinoline, suggesting greater uptake efficiency of certain alkaloids. Frogs in the second experiment accumulated a greater amount of alkaloid, which corresponds to the longer duration and greater number of alkaloid-dusted fruit flies that were consumed. These findings provide the first experimental evidence that bufonid poison frogs sequester alkaloid-based defenses from dietary sources.