Chytrid fungus infections in laboratory and introduced Xenopus laevis populations: assessing the risks for U.K. native amphibians.

The chytrid fungus Batrachochytrium dendrobatidis (Bd) is notorious amongst current conservation biology challenges, responsible for mass mortality and extinction of amphibian species. World trade in amphibians is implicated in global dissemination. Exports of South African Xenopus laevis have led to establishment of this invasive species on four continents. Bd naturally infects this host in Africa and now occurs in several introduced populations. However, no previous studies have investigated transfer of infection into co-occurring native amphibian faunas. A survey of 27 U.K. institutions maintaining X. laevis for research showed that most laboratories have low-level infection, a risk for native species if animals are released into the wild. RT-PCR assays showed Bd in two introduced U.K. populations of X. laevis, in Wales and Lincolnshire. Laboratory and field studies demonstrated that infection levels increase with stress, especially low temperature. In the U.K., native amphibians may be exposed to intense transmission in spring when they enter ponds to spawn alongside X. laevis that have cold-elevated Bd infections. Exposure to cross-infection has probably been recurrent since the introduction of X. laevis, >20 years in Lincolnshire and 50 years in Wales. These sites provide an important test for assessing the impact of X. laevis on Bd spread. However, RT-PCR assays on 174 native amphibians (Bufo, Rana, Lissotriton and Triturus spp.), sympatric with the Bd-infected introduced populations, showed no foci of self-sustaining Bd transmission associated with X. laevis. The abundance of these native amphibians suggested no significant negative population-level effect after the decades of co-occurrence.

The skin microbiome of Xenopus laevis and the effects of husbandry conditions.

BACKGROUND: Historically the main source of laboratory Xenopus laevis was the environment. The increase in genetically altered animals and evolving governmental constraints around using wild-caught animals for research has led to the establishment of resource centres that supply animals and reagents worldwide, such as the European Xenopus Resource Centre. In the last decade, centres were encouraged to keep animals in a "low microbial load" or "clean" state, where embryos are surface sterilized before entering the housing system; instead of the conventional, "standard" conditions where frogs and embryos are kept without prior surface treatment. Despite Xenopus laevis having been kept in captivity for almost a century, surprisingly little is known about the frogs as a holobiont and how changing the microbiome may affect resistance to disease. This study examines how the different treatment conditions, "clean" and "standard" husbandry in recirculating housing, affects the skin microbiome of tadpoles and female adults. This is particularly important when considering the potential for poor welfare caused by a change in husbandry method as animals move from resource centres to smaller research colonies. RESULTS: We found strong evidence for developmental control of the surface microbiome on Xenopus laevis; adults had extremely similar microbial communities independent of their housing, while both tadpole and environmental microbiome communities were less resilient and showed greater diversity. CONCLUSIONS: Our findings suggest that the adult Xenopus laevis microbiome is controlled and selected by the host. This indicates that the surface microbiome of adult Xenopus laevis is stable and defined independently of the environment in which it is housed, suggesting that the use of clean husbandry conditions poses little risk to the skin microbiome when transferring adult frogs to research laboratories. This will have important implications for frog health applicable to Xenopus laevis research centres throughout the world.

Assessing the Immune Response When Raising Antibodies for Use in Xenopus.

Frog-specific antibodies usually must be raised for work in Xenopus Selecting a host animal whose immune system will respond to a target antigen with an antibody response is essential to obtaining high-quality antibodies. To determine whether an immunized animal has produced antibodies against an antigen, western blotting using Xenopus embryo or egg extract as the protein source can be performed as described here. When a protein of the expected size is detected by western blotting in the immune sera but not the preimmune sera, the antibody has been successfully raised.

Purifying Antibodies Raised against Xenopus Peptides.

Antibody production for work in Xenopus involves the immunization of a host with an antigen, usually a Xenopus protein or peptide alien to the host. The antibody-containing serum, normally returned to the investigator by the company/bioresource unit where it was raised, is comprised of all proteins not used in blood clotting (coagulation) and all the electrolytes, antibodies, antigens, hormones, and any exogenous substances, such as drugs and microorganisms, that were in the blood. It is often necessary to separate the target antibody from the rest of the serum components to minimize nonspecific protein-antibody interactions in downstream applications (e.g., when performing western blotting). Most antibody production companies provide a column containing the peptide coupled to glass beads. A purification procedure for using this type of column (i.e., one that is based on controlled-pore glass beads) is described here.

Raising Antibodies for Use in Xenopus.

For work in Xenopus, frog-specific antibodies must usually be raised, although a few antibodies against mammalian proteins cross-react. To produce an immunogen for antibody production, human embryonic kidney (HEK) expression systems can be used as described here. For most laboratories, the actual method of raising the antibody is determined by local ethical regulations controlling the adjuvant and injection protocols used. Because these steps are often outsourced, they are not included in this protocol.

Confirming Antibody Specificity in Xenopus.

Verifying that a new antibody recognizes its target can be difficult. In this protocol, expression of a target protein in Xenopus embryos is either knocked down using CRISPR-Cas9 technology (for zygotic proteins) or enhanced by microinjection of a synthetic mRNA (for maternal proteins). Western blotting analysis is then performed. If the antibody recognizes the target protein, the western blot will show a relatively weak band for CRISPR-injected embryos and a relatively strong band for RNA-injected embryos. This represents a straightforward, powerful strategy for confirming antibody specificity in Xenopus.