First Record of Spirometra erinaceieuropaei spargana Infection in Amphibians of South Asia: A Causative Agent of Surficial Abnormalities.

Background: Sparganosis is a zoonotic disease caused by Plerocercoid larvae (spargana) of the genus Spirometra. We aimed to provide molecular evidence for the infection of amphibians with Spirometra sp. in the inside and outside of Horton Plains National Park (HPNP), Sri Lanka. Methods: The prevalence of sparganum infection in wild frogs (Truga eques and Minverya agricola) was investigated in the inside and outside of HPNP from June 2019 to April 2021.A total of 1,434 Amphibians samples were surveyed to examine the spargana infection from the study site. To identify the species identity of the collected spargana, a portion of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene and nuclear 18S rRNA gene were amplified, sequenced, and analyzed. Results: A total of 539 infected amphibians (T. eques and M. agricola) samples were examined to survey from the study area. Spargana were detected in all dissected specimens belonging to the species Spirometra erinaceieuropaei that were genetically confirmed using the evolutionary conserved nuclear 18S rRNA gene and then compared to the GenBank deposit, indicating that S. erinaceieuropaei is the primary causal agent of sparganosis both inside and outside the HPNP. Conclusion: Our finding is the first genetically confirmed record of S. erinaceieuropaei in amphibians in South Asia. However, further studies are needed to investigate the prevalence of sparagna infection in amphibians all over the island.

Dramatic dietary shift maintains sequestered toxins in chemically defended snakes.

Unlike other snakes, most species of Rhabdophis possess glands in their dorsal skin, sometimes limited to the neck, known as nucho-dorsal and nuchal glands, respectively. Those glands contain powerful cardiotonic steroids known as bufadienolides, which can be deployed as a defense against predators. Bufadienolides otherwise occur only in toads (Bufonidae) and some fireflies (Lampyrinae), which are known or believed to synthesize the toxins. The ancestral diet of Rhabdophis consists of anuran amphibians, and we have shown previously that the bufadienolide toxins of frog-eating species are sequestered from toads consumed as prey. However, one derived clade, the Rhabdophis nuchalis Group, has shifted its primary diet from frogs to earthworms. Here we confirm that the worm-eating snakes possess bufadienolides in their nucho-dorsal glands, although the worms themselves lack such toxins. In addition, we show that the bufadienolides of R. nuchalis Group species are obtained primarily from fireflies. Although few snakes feed on insects, we document through feeding experiments, chemosensory preference tests, and gut contents that lampyrine firefly larvae are regularly consumed by these snakes. Furthermore, members of the R. nuchalis Group contain compounds that resemble the distinctive bufadienolides of fireflies, but not those of toads, in stereochemistry, glycosylation, acetylation, and molecular weight. Thus, the evolutionary shift in primary prey among members of the R. nuchalis Group has been accompanied by a dramatic shift in the source of the species' sequestered defensive toxins.

Evolution of nuchal glands, unusual defensive organs of Asian natricine snakes (Serpentes: Colubridae), inferred from a molecular phylogeny.

A large body of evidence indicates that evolutionary innovations of novel organs have facilitated the subsequent diversification of species. Investigation of the evolutionary history of such organs should provide important clues for understanding the basis for species diversification. An Asian natricine snake, Rhabdophis tigrinus, possesses a series of unusual organs, called nuchal glands, which contain cardiotonic steroid toxins known as bufadienolides. Rhabdophis tigrinus sequesters bufadienolides from its toad prey and stores them in the nuchal glands as a defensive mechanism. Among more than 3,500 species of snakes, only 17 Asian natricine species are known to possess nuchal glands or their homologues. These 17 species belong to three nominal genera, Balanophis, Macropisthodon, and Rhabdophis. In Macropisthodon and Rhabdophis, however, species without nuchal glands also exist. To infer the evolutionary history of the nuchal glands, we investigated the molecular phylogenetic relationships among Asian natricine species with and without nuchal glands, based on variations in partial sequences of Mt-CYB, Cmos, and RAG1 (total 2,767 bp). Results show that all species with nuchal glands belong to a single clade (NGC). Therefore, we infer that the common ancestor of this clade possessed nuchal glands with no independent origins of the glands within the members. Our results also imply that some species have secondarily lost the glands. Given the estimated divergence time of related species, the ancestor of the nuchal gland clade emerged 19.18 mya. Our study shows that nuchal glands are fruitful subjects for exploring the evolution of novel organs. In addition, our analysis indicates that reevaluation of the taxonomic status of the genera Balanophis and Macropisthodon is required. We propose to assign all species belonging to the NGC to the genus Rhabdophis, pending further study.