Green toads – sex determination and other interesting facts

In the vast majority of vertebrates, two biological sexes (sexes) are observed, although within the same sex there can be several alternative reproductive strategies each, as we mentioned earlier. True hermaphroditism or sex changes are encountered in certain fish, such as the clownfish known from the cartoon Where’s Nemo? The biological sex of vertebrates almost always depends on the presence of sex chromosomes, although in reptiles and certain fish it is determined by temperature. From high school, we remember that male mammals have distinct XY chromosomes, while female birds have distinct ZW chromosomes. In amphibians, the situation remains rather unclear.

They are only invisible

It has been widely accepted that amphibians also have separate heterochromosomes. However, they cannot be distinguished by shape or staining under a microscope. And it might seem that sex determination in these animals should be perfectly worked out. After all, the clawed platypus Xenopus laevis is one of the most widely studied model species! At least until now, since it has now been banned from breeding and trade without special permits.

Also, the genomes of other amphibian species are being studied quite closely due to the harmfulness of these animals to native wildlife (aga Rhinella marina) or active conservation (tree frog Hyla arborea). The aquatic frog Pelophylax cl. esculentus is analyzed because of its intriguing gamete-stealing system. Our lowland toads Bombina bombina and mountain toads B. variegata, on the other hand, are good models for studying hybrid zones and their role in evolution [2].

Big can do more

Although humans are considered the crown of creation, amphibian genomes tend to be much larger than human genomes, and thus more difficult to analyze. This is not so surprising when one recalls that frogs and newts undergo complete transformation. Their genomes must therefore handle the functioning of two strongly dissimilar organisms: a gill-breathing tadpole feeding on rotting plant remains and an adult amphibian breathing with lungs and hunting.

Nevertheless, to date we know virtually nothing for sure regarding sex differentiation in the more than 8,600 species of this cluster other than the platypus. All we have discovered is that this determination is disrupted every now and then by polluting the water with pesticide residues, contraceptives or microplastics. Distinguishing males from females by genetic methods would be extremely useful both in amphibian aquaculture for human food and in basic biomedical research or gene banks. Besides, slaughter use and active conservation are sometimes carried out by the same entities, as we see in China in the case of giant salamanders Andrias davidianus [1, 2].

You praise what you don’t know

Scientists decided to grab the bull by the horns, and more specifically to work out the genetic and epigenetic mechanisms determining sex in green toads Bufotes viridis. This is a species or group of poorly distinguishable – both by morphological and genetic methods – species, native to Europe (including Poland), Kazakhstan and Asia Minor. It is a true rarity among Amphibia, as it tolerates drought well, and, according to some herpetologists, even avoids wetlands – although at night it visits bodies of water to replenish the body’s fluid supply. In some places it remains numerous, as it adapts quite well (for a toad…) to human pressure.

For example, it readily uses artificial, shallow reservoirs and watercourses as breeding sites. On the basis of previous studies of sex changes forced either surgically or hormonally, it was supposed that sex determination in green toads takes place similarly to that in mammals – that is, the heterogametic sex would be XY males, while platypuses develop like birds with heterogametic ZW females. The presence of two different mechanisms of sex determination within one cluster is quite surprising, but possible – after all, it happens similarly in fish and butterflies [1, 2].

Toads green like people

A paper presenting the preliminary results of this work has just been published in the prestigious journal Nature Communications. Only thorough sequencing and whole-genome analysis of the female and male have identified structural differences between the otherwise nearly identical X and Y chromosomes in Bufotes viridis. The sex-determining site (locus) is located in the 5′ regulatory region of the bod1l gene. After transcription of the aforementioned gene, a long non-coding RNA protein (IncRNA) is produced. Previously, such ribonucleic acid transcripts, lacking open reading frames, were considered junk or observational artifacts.

Today we know that many of them are true gray eminences or golden hands, responsible for translation of RNA into proteins, maturation and stability of transcripts, DNA transport and replication, cell differentiation or tissue specificity. This mysterious IncRNA is expressed only in male green toads. This is a strong clue, though still not proof, that this very locus of the aforementioned bod1l gene is responsible for male differentiation. It has also been identified in five, related species(Bufotesbalearicus, B. shaartusiensis, B. siculus, B. turanensis and B. variabilis). The next step will be to control gene expression using the simple and inexpensive, yet so precise CRISPR/Cas9 genome editing technique [1].

Problems with sex determination in amphibians are just one of the challenges – along with the impracticability of cryopreserving oocytes and tadpoles – delaying the creation of gene banks for these animals. Meanwhile, amphibians are, along with dioecious fish and many cartilaginous animals, the closest group of vertebrates to extinction. Fortunately, breeding in terraria is making great strides.

In the article, I used, among other things. z:

1. Kuhl, H., Tan, W. H., Klopp, C., Kleiner, W., Koyun, B., Ciorpac, M., … & Stöck, M. (2024). A candidate sex determination locus in amphibians which evolved by structural variation between X-and Y-chromosomes. Nature Communications, 15(1), 4781.
2. Yoshimoto, S., & Ito, M. (2011). A ZZ/ZW-type sex determination in Xenopus laevis. The FEBS journal, 278(7), 1020-1026.