The information is prepared on the basis of data from the information-analytical system RSF, informative part is represented in the author's edition. All rights belong to the authors, the use or reprinting of materials is permitted only with the prior consent of the authors.

Project titleMechanisms of teratogenesis induction caused by Strigea robusta in amphibians

KeywordsTrematodes, amphibians, teratogenesis, developmental anomalies, transcriptomics

Annotation
The study of teratogenesis in vertebrates is an important task for understanding the mechanisms of normal morphogenesis and preventing the consequences of exposure to teratogens. The increasing frequency of occurrence of morphological anomalies in amphibians around the world causes concern in the world scientific community, and the contribution to this trend of the influence of natural and anthropogenic factors is unclear. So, it turned out that many of the initially alarming cases of mass anomalies in the New World were caused by the impact of trematode invasion. The study of the P anomaly, discovered in the middle of the 20th century by the famous French biologist Jean Rostand, made it possible to reveal the trematode nature of its manifestation in tadpoles of green frogs and, thus, to discover a new case of limb deformities in vertebrates under the influence of trematodes, first discovered in the Palaearctic realm. This project is aimed at studying the characteristics of the course of infection, the species-specificity of the impact, and identifying the mechanisms of the impact of trematodes on the development of amphibian tadpoles by analyzing the transcriptomes of infected and uninfected individuals. In the course of the project, unique data will be obtained on the minimum dose of cercariae, the survival of tadpoles at different doses of cercariae, the peculiarities of the manifestation of anomalies under the influence of different doses of cercariae and their localization, and the study of the specificity of the response of amphibian species of different taxonomic groups to the effect of trematode. For the first time, sequencing, assembly, and annotation of the reference transcriptome will be carried out, followed by an experiment to identify differential gene expression in the developing buds of the extremities of tadpoles infected with the studied species of trematodes and uninfected ones. Our data will make it possible to reveal the molecular mechanisms of the response of the host organism to the introduction of the parasite and the release of morphogens by it, leading to changes in gene expression, tissue hyperplasia, and limb deformities. Thus, during the implementation of the project, not only will be obtained unique data on the P anomaly phenomenon and the molecular mechanisms of its manifestation but also new information will be obtained on the relationships in the "parasite-host" system, on the induction of teratogenesis and normal morphogenesis of the limbs of amphibians.

Expected results
During the implementation of the project, new data will be obtained on the phenomenon of anomaly P - a syndrome of morphological anomalies in amphibians, which has never been studied from the standpoint of the described trematode invasion of Strigea robusta. In the course of our experiments, it was found that the protocol for the infection of amphibians with cercariae of trematodes, described earlier in the works of P. Johnson's laboratory (Johnson et al., 1999) and taken as a basis in other studies of the effect of trematodes on amphibians (echinostomes invasion - Holland et al., 2007; Acanthostomum invasion - Rajakaruna et al., 2008) does not reflect the whole picture of infection with the trematode Strigea robusta. The protocol includes infection with doses of 16, 32 and 48 cercariae. However, our data showed the presence of anomalies at the lowest dose of 16 cercariae, which requires further development of the technique and clarification of the minimum dose of cercariae at which anomalies develop, as well as the minimum dose of cercariae, at which two forms of the syndrome develop - mild (polydactyly) and severe (a significant deformity of the hind and forelimbs). Thus, we will be the first to obtain data on the minimum dose of cercariae causing anomalies in water frogs. When staging this type of experiment, the information will be obtained on the nature of the anomalies - at what doses does the development of certain structures occur (proliferation of cartilaginous tissue, the presence of bone outgrowths, the formation of anomalies of the forelimb, the degree and severity of polydactyly, the nature of limb inversion, non-closure of the opercular chamber and the manifestation of other associated anomalies like stiff hind limbs and anophthalmia). An answer will be received to the question, what is the primary cause in the development of anomalies - the dose of encysted metacercariae or their localization? Species specificity will be the second important aspect of the issue under study. Even Jean Rostand noted in his studies that syntopically brown frogs and Lissotriton newts do not exhibit anomalies (Rostand, 1971). In our studies, it was shown that syntopic amphibian species, such as the Pallas toad (Pelobates vespertinus), the red-bellied toad (Bombina bombina) and the moor frog (Rana arvalis), do not have any of the manifestations observed in the marsh frog living in the same water bodies (Svinin et al., 2019). Thus, we plan to obtain the first information on whether the Strigea robusta trematode causes anomalies in other amphibian species and in what form they manifest themselves. To this end, we will conduct experiments on a classic laboratory facility - the clawed frog (Xenopus laevis). We also plan to conduct experiments on other species of amphibians living syntopically with a marsh frog in water bodies of "ponds with monsters" from the Ostrovtsovskaya Lesostep (Rana arvalis, Bombina bombina), as well as on species that can potentially coexist with Strigea robusta within its range (Rana temporaria, Bufo bufo, Bufotes viridis). The impact on species whose ranges are far beyond the range of Strigae robusta (including Bufotes baturae from the Himalayas and Dyscophus antongilii from Madagascar), the cultures of which are currently delivered in domestic laboratories, seems interesting. Understanding the species-specificity of the exposure may be useful in further comparing the expression spectra of different species, planned for the next projects. However, in this project, we want to show, at the molecular level, the change in gene expression spectra upon invasion by this species of trematodes. For this purpose, we will carry out sequencing, assembly, and annotation of the reference transcriptome, followed by an experiment to identify differential gene expression in the developing buds of the extremities of tadpoles infected with the studied species of trematodes and uninfected ones. Thus, during the implementation of the project, we plan to obtain information on some features of the manifestation of the trematode Strigea robusta invasion, which allows to influence on amphibian populations.

Annotation of the results obtained in 2022
In the experiments on tadpoles of water frogs infection by Strigea robusta parasite, it was found that all three taxa (Pelophylax ridibundus, P. lessonae and P. esculentus) do not have specific features of infection with the trematode Strigea robusta, the tadpoles had similar deformities at doses of 16, 32 and 48 cercariae, and the occurrence of anomalies (polydactyly and severe cases of anomaly P) did not differ between species. No developmental anomalies were found in the tadpoles Bombina orientalis, Ranoidea caerulea, Triprion petasatus, Scinax ruber, Rhinella marina. Thus, a new specificity of the impact of the trematode Strigea robusta on amphibian tadpoles was shown. The presence of some other developmental anomalies was noted, such as metamorphic oedema of the abdominal cavity (Ranoidea caerulea, Triprion petasatus), hemorrhagic oedema in the area of the opercular chamber (Ranoidea caerulea), and a tail bifurcation and amelia were found in Triprion petasatus, apparently caused by other factors. The survival rate of amphibian tadpoles exposed to the studied trematode species did not differ significantly between different amphibian species, amounting to 79–98%. At the same time, in the tadpoles Triprion petasatus, Ranoidea caerulea and Bombina orientalis, the average number of cysts was significantly lower than in experiments with water frogs (4.3 cysts per individual). This is probably due to the existence of resistance in these species to infection with this species of pathogen, which makes it possible to eliminate penetrating cercariae and increase survival compared to the tadpoles of toads and water frogs. During the field studies, in the population systems of water frogs inhabited waterbodies from the north of the Middle Volga region, new hotspots with a high frequency of occurrence of the anomaly P were also found: one locality with severe forms of the anomaly and six localities with cases of symmetrical polydactyly were recorded. The occurrence of polydactyly in these localities ranged from 0.7 to 20.6%. Severe forms in the population of pond frogs inhabiting the reservoir from the Gusevo settlement accounted for 3.1%. New data on differential gene expression in the developing limbs of marsh frog tadpoles have been obtained, which provide important information for future understanding of changes in the gene expression profile of developing limbs under the influence of the Strigea robusta parasite.

Publications

1. Svinin A.O., Bashinskiy I.V., Ermakov O.A., Litvinchuk S.N. Effects of minimum Strigea robusta (Digenea: Strigeidae) cercariae doses and localization of cysts on the anomaly P manifestation in Pelophylax lessonae (Anura: Ranidae) tadpoles Parasitology Research, Vol. 122, No. 3 (year - 2023) https://doi.org/10.1007/s00436-022-07778-z

2. Svinin A.O., Ermakov O.A., Litvinchuk S.N. The incidence of the anomaly P syndrome in water frogs (Anura, Ranidae, Pelophylax) from the Middle Volga River (Russia) Herpetozoa, 35 (year - 2022) https://doi.org/10.3897/herpetozoa.35.e95928

3. - Ученые выяснили, откуда берутся аномалии конечностей у лягушек и жаб Министерство науки и высшего образования Российской Федерации, - (year - )

4. - "Пруды с монстрами". В России нашли причину загадочных аномалий у лягушек РИА Новости, - (year - )

5. - В России нашли причину загадочных аномалий у лягушек РНФ, - (year - )

Annotation of the results obtained in 2021
In the course of the project, four main tasks were completed: an experiment on the minimum dose of cercariae needs for development of morphological abnormalities in water frogs (in the case of Pelophylax lessonae) (1); experiments on infection of eight species of anurans in order to determine the selective effect of Strigea robusta metacercariae on the development of tadpoles of various amphibian species belonging to different taxonomic groups (2); a comparison of morphological deformities in amphibian species in which the development of anomalies was observed (3); transcriptom de novo annotation of developing Pelophylax ridibundus limb buds to further experiments on differential gene expression. Dose-dependent experiment to identify the minimum dose that induces anomalies in green frog tadpoles was shown that the anomaly P presents in all experimental groups of tadpoles, except for the control. Individuals (19.6%) had severe cases of the anomaly P syndrome or polydactyly (33.3%) that consists of one to four additional fingers on the hind limbs. The number of cercariae that formed cysts in tadpoles was 50.9% (221 out of 434 cercariae), i.e., almost every second cercariae encysted in tadpoles. It was found that a mild form of the anomaly P developed even after exposure to 2 cercariae. At the same time, in such individuals, the number of fingers did not exceed 6 on the hind limbs; polydactyly was not observed on the forelimbs. A severe form can develop even with minimal exposure to 2 (limb inversion) and 4 cercariae. The severe phenotype included polydactyly, limb inversion, outgrowths, and brachymelia. It was also found that cysts are localized in the same way both on the right and on the left sides of the body. Localization of cysts is observed mainly in the region of the hind limbs (25.8%), tail (22.6%), and head (20.4%), and is less common on the back (11.3%), flanks (15.4%) and forelimbs (4.5%). In order to study the influence of Strigea robusta on the development of tadpoles of different amphibian species belonging to different taxonomic groups, 702 tadpoles of 8 species of anurans (112 Rana arvalis, 70 Rana temporaria, 70 Xenopus laevis, 120 Bufotes baturae, 150 Bufo bufo, 60 Bufotes viridis , 30 Dyscophus antongilii, 90 Pelophylax lessonae) were used in experiments. All of them were divided into 41 experimental groups with a certain type of infection, or its absence (control). The anomaly P was manifested in all three studied toad species (Bufo bufo, Bufotes baturae, Bufotes viridis) and its occurrence (mainly polydactyly) varied from 19 to 78%. Individuals with severe forms of P anomaly were found in all test groups (3.7–8.0%). Despite the presence of cysts in developing tadpoles of brown frogs of the genus Rana (Rana arvalis, Rana temporaria), none of the experimental tadpoles had any deviations in morphological appearance. The survival rate of tadpoles varied from 36 to 93%; 57% on average for Rana arvalis and 77% for Rana temporaria. None of the observed experimental larvae of African clawed frogs Xenopus laevis and Madagascar tomato frog Dyscophus antongilii showed developmental anomalies. Thus, in the course of our studies, a selective effect of the impact of Strigea robusta metacercariae on developing tadpoles of anurans was found - a result that distinguishes this variant of trematode-induced deformities in amphibians from rematode-induced deformities caused by Ribeiroia ondatrae, known in North America, causing the development of additional limbs (polymelia) in all species of tailed and tailless amphibians. The trematode Strigea robusta affects the development of the limbs of water frog tadpoles of the genus Pelophylax (P. ridibundus, P. lessonae, P. esculentus) and toads of the genera Bufo (B. bufo) and Bufotes (B. viridis, B. baturae). The observed deformities were very close to those of the anomaly P in water frogs of the genus Pelophylax. They had major or minor malformations on their hind- and forelimbs. Some polydactylous individuals had 6-7 digits on the hind limbs and 5 digits on the forelimbs. In severe cases of the anomaly P in toads, inversions of the hind limbs (taumelia), tumor-like formations, polydactyly, and brachymelia were noted. At low doses of cercariae, polydactyly develops. Severe cases of the anomaly P include hindlimb inversion (thaumelia), hindlimb shortening (brachymelia), with rare exceptions accompanied by an increased number of fingers (polydactyly), as well as additional distal limb fragments in the inguinal region, tumor-like outgrowths, bony outgrowths, and hemorrhages in the hindlimbs areas. Our results on the common toad and pool frog probably support an individual immune response of tadpoles, which can explain the high level of intergroup variability observed in experiments (in addition to the influence of the dose of cercariae, established metacercariae and their localization). Assembly and annotation of the transcriptome of the developing limbs in tadpole of the marsh frog Pelophylax ridibundus yielded 1,022,972 sequences, the total length of which was 548,954,323 nt. The total number of genes found using the gVolante program was 3354, of which complete sequences includes 3024 genes (90.16% of the total number of genes), partial - 3141 (93.65%), the number of unidentified core genes was 213 (6.5%). The largest fragment included 24488 nt, while the smallest one was 176 bp, the average sequence length was 537 nt, the median was 306 nt. The total number of reads with a length of more than 1000 nt amounted to 98,981 (9.7% of the total number of reads), more than 10,000 nt – 425 sequences. The GC-content turned out to be 44.24%. The highest similarity of the studied transcriptome is observed with Lithobates catesbianus (24%), Xenopus tropicalis (17%), Leptobrachium leishanense (14%), Xenopus laevis (8%). Thirty-seven genes associated with finger morphogenesis and 91 genes associated with fore and hindimbs morphogenesis were identified. The results of transcriptome annotation will serve as an important basis for analysis of gene expression during the development of tadpole limb buds both in the normal condition and under the influence of Strigea robusta metacercariae planned for the next year of the project. Media links: 1) Tyumen State University scientists implement projects on grants from the Russian Science Foundation https://t-l.ru/306938.html 2) "Ranarium" website, "Anomalie P" page: http://www.ranaesc.ru/index/index.html

Publications

1. Svinin A.O., Matushkina K.A., Dedukh D.V., Bashinskiy I.V., Ermakov O.A., Litvinchuk S.N. Strigea robusta (Digenea: Strigeidae) infection effects on the gonadal structure and limb malformation in toad early development Journal of Experimental Zoology Part A: Ecological and Integrative Physiology, Volume 337, Issue 4 (year - 2022) https://doi.org/10.1002/jez.2599