Feral frogs, native newts, and chemical cues: identifying threats from and management opportunities for invasive African Clawed Frogs in Washington state.

Invasive species threaten biodiversity globally. Amphibians are one of the most threatened vertebrate taxa and are particularly sensitive to invasive species, including other amphibians. African clawed frogs (Xenopus laevis) are native to Southern Africa but have subsequently become invasive on multiple continents-including multiple parts of North America-due to releases from the pet and biomedical trades. Despite their prevalence as a global invader, the impact of X. laevis remains understudied. This includes the Pacific Northwest of the USA, which now hosts multiple expanding X. laevis populations. For many amphibians, chemical cues communicate important information, including the presence of predators. Here, we tested the role chemical cues may play in mediating interactions between feral X. laevis and native amphibians in the Pacific Northwest. We tested whether native red-legged frog (Rana aurora) tadpoles display an antipredator response to non-native frog (X. laevis) or native newt (rough-skinned newts, Taricha granulosa) predator chemical stimuli. We found that R. aurora tadpoles exhibited pronounced anti-predator responses when exposed to chemical cues from T. granulosa but did not display anti-predator response to invasive X. laevis chemical cues. We also began experimentally testing whether T. granulosa-which produce a powerful neurotoxin tetrodotoxin (TTX)-may elicit an anti-predator response in X. laevis, that could serve to deter co-occupation. However, our short-duration experiments found that X. laevis were attracted to newt chemical stimuli rather than deterred. Our findings show that X. laevis likely poses a threat to native amphibians, and that these native species may also be particularly vulnerable to this invasive predator, compared to native predators, because toxic native newts may not limit X. laevis invasions. Our research provides some of the first indications that native Pacific Northwest species may be threatened by feral X. laevis and provides a foundation for future experiments testing potential management techniques for X. laevis.

Comparative transcriptomic analysis delineates adaptation strategies of Rana kukunoris toward cold stress on the Qinghai-Tibet Plateau.

BACKGROUND: Cold hardiness is fundamental for amphibians to survive during the extremely cold winter on the Qinghai-Tibet plateau. Exploring the gene regulation mechanism of freezing-tolerant Rana kukunoris could help us to understand how the frogs survive in winter. RESULTS: Transcriptome of liver and muscle of R. kukunoris collected in hibernation and spring were assisted by single molecule real-time (SMRT) sequencing technology. A total of 10,062 unigenes of R. kukunoris were obtained, and 9,924 coding sequences (CDS) were successfully annotated. Our examination of the mRNA response to whole body freezing and recover in the frogs revealed key genes concerning underlying antifreeze proteins and cryoprotectants (glucose and urea). Functional pathway analyses revealed differential regulated pathways of ribosome, energy supply, and protein metabolism which displayed a freeze-induced response and damage recover. Genes related to energy supply in the muscle of winter frogs were up-regulated compared with the muscle of spring frogs. The liver of hibernating frogs maintained modest levels of protein synthesis in the winter. In contrast, the liver underwent intensive high levels of protein synthesis and lipid catabolism to produce substantial quantity of fresh proteins and energy in spring. Differences between hibernation and spring were smaller than that between tissues, yet the physiological traits of hibernation were nevertheless passed down to active state in spring. CONCLUSIONS: Based on our comparative transcriptomic analyses, we revealed the likely adaptive mechanisms of R. kukunoris. Ultimately, our study expands genetic resources for the freezing-tolerant frogs.

Defenses of whirligig beetles against native and invasive frogs.

Many native insects have evolved defenses against native predators. However, their defenses may not protect them from non-native predators due to a limited shared history. The American bullfrog, Aquarana catesbeiana (Anura: Ranidae), which has been intentionally introduced to many countries, is believed to impact native aquatic animals through direct predation. Adults of whirligig beetles (Coleoptera: Gyrinidae), known for swimming and foraging on the water surface of ponds and streams, reportedly possess chemical defenses against aquatic predators, such as fish. Although whirligig beetles potentially encounter both bullfrogs and other frogs in ponds and lakes, the effectiveness of their defenses against frogs has been rarely studied. To assess whether whirligig beetles can defend against native and non-native frogs, we observed the behavioral responses of the native pond frog, Pelophylax nigromaculatus (Anura: Ranidae), and the invasive non-native bullfrog, A. catesbeiana, to native whirligig beetles, Gyrinus japonicus and Dineutus orientalis, in Japan. Adults of whirligig beetles were provided to frogs under laboratory conditions. Forty percent of G. japonicus and D.orientalis were rejected by P. nigromaculatus, while all whirligig beetles were easily consumed by A. catesbeiana. Chemical and other secondary defenses of G. japonicus and D. orientalis were effective for some individuals of P. nigromaculatus but not for any individuals of A. catesbeiana. These results suggest that native whirligig beetles suffer predation by invasive non-native bullfrogs in local ponds and lakes in Japan.

Aquatic Soundscape Recordings Reveal Diverse Vocalizations and Nocturnal Activity of an Endangered Frog.

AbstractAutonomous sensors provide opportunities to observe organisms across spatial and temporal scales that humans cannot directly observe. By processing large data streams from autonomous sensors with deep learning methods, researchers can make novel and important natural history discoveries. In this study, we combine automated acoustic monitoring with deep learning models to observe breeding-associated activity in the endangered Sierra Nevada yellow-legged frog (Rana sierrae), a behavior that current surveys do not measure. By deploying inexpensive hydrophones and developing a deep learning model to recognize breeding-associated vocalizations, we discover three undocumented R. sierrae vocalization types and find an unexpected temporal pattern of nocturnal breeding-associated vocal activity. This study exemplifies how the combination of autonomous sensor data and deep learning can shed new light on species' natural history, especially during times or in locations where human observation is limited or impossible.

Diversity, richness, and evenness of birds in Rana Resort Forest, District Kasur, Punjab, Pakistan.

This research was carried out to determine avian diversity in the Rana Resort Forest from June to August 2023. A total of 655 birds were observed; these birds belonged to 36 different species from 11 orders; 27 families and 36 genera were found in the forest. The observed avian species that belong to the Muscicapidae family had the highest number of species, while Strigidae had the lowest number of species. The highest numbers of the order Passeriformes were recorded. Among the bird species observed, 33 were residents, 1 was a winter visitor, and 2 were summer breeders. The species diversity, richness, and evenness indices are 3.5238, 5.3973, and 0.10492, respectively. According to the index results, the Rana Resort Forest is a diverse area, but the species are not evenly distributed due to some of the threats observed during the study, such as habitat destruction, poverty, and unawareness.

An esculentin-1 homolog from a dark-spotted frog (Pelophylax nigromaculatus) possesses antibacterial and immunoregulatory properties.

BACKGROUND: Esculentin-1, initially discovered in the skin secretions of pool frogs (Pelophylax lessonae), has demonstrated broad-spectrum antimicrobial activity; however, its immunomodulatory properties have received little attention. RESULTS: In the present study, esculentin-1 cDNA was identified by analysing the skin transcriptome of the dark-spotted frog (Pelophylax nigromaculatus). Esculentin-1 from this species (esculentin-1PN) encompasses a signal peptide, an acidic spacer peptide, and a mature peptide. Sequence alignments with other amphibian esculentins-1 demonstrated conservation of the peptide, and phylogenetic tree analysis revealed its closest genetic affinity to esculentin-1P, derived from the Fukien gold-striped pond frog (Pelophylax fukienensis). Esculentin-1PN transcripts were observed in various tissues, with the skin exhibiting the highest mRNA levels. Synthetic esculentin-1PN demonstrated antibacterial activity against various pathogens, and esculentin-1PN exhibited bactericidal activity by disrupting cell membrane integrity and hydrolyzing genomic DNA. Esculentin-1PN did not stimulate chemotaxis in RAW264.7, a murine leukemic monocyte/macrophage cell line. However, it amplified the respiratory burst and augmented the pro-inflammatory cytokine gene (TNF-α and IL-1ß) expression in RAW264.7 cells. CONCLUSIONS: This novel finding highlights the immunomodulatory activity of esculentin-1PN on immune cells.

The Impact of Long-Term Hypoxia on the Antioxidant Defense System in the Siberian Frog Rana amurensis.

The Siberian frog Rana amurensis has a uniquely high tolerance to hypoxia among amphibians, as it is able to withstand several months underwater with almost no oxygen (0.2 mg/liter) vs. several days for other studied species. Since it was hypothesized that hypoxia actives the antioxidant defense system in hypoxia-tolerant animals, one would expect similar response in R. amurensis. Here, we studied the effect of hypoxia in the Siberian frog based on the transcriptomic data, activities of antioxidant enzyme, and content of low-molecular-weight antioxidants. Exposure to hypoxia upregulated expression of three relevant transcripts (catalase in the brain and two aldo-keto reductases in the liver). The activities of peroxidase in the blood and catalase in the liver were significantly increased, while the activity of glutathione S-transferase in the liver was reduced. The content of low-molecular-weight antioxidants (thiols and ascorbate) in the heart and liver was unaffected. In general, only a few components of the antioxidant defense system were affected by hypoxia, while most remained unchanged. Comparison to other hypoxia-tolerant species suggests species-specific adaptations to hypoxia-related ROS stress.

Multiple Transitions between Y Chromosome and Autosome in Tago's Brown Frog Species Complex.

Sex chromosome turnover is the transition between sex chromosomes and autosomes. Although many cases have been reported in poikilothermic vertebrates, their evolutionary causes and genetic mechanisms remain unclear. In this study, we report multiple transitions between the Y chromosome and autosome in the Japanese Tago's brown frog complex. Using chromosome banding and molecular analyses (sex-linked and autosomal single nucleotide polymorphisms, SNPs, from the nuclear genome), we investigated the frogs of geographic populations ranging from northern to southern Japan of two species, Rana tagoi and Rana sakuraii (2n = 26). Particularly, the Chiba populations of East Japan and Akita populations of North Japan in R. tagoi have been, for the first time, investigated here. As a result, we identified three different sex chromosomes, namely chromosomes 3, 7, and 13, in the populations of the two species. Furthermore, we found that the transition between the Y chromosome (chromosome 7) and autosome was repeated through hybridization between two or three different populations belonging to the two species, followed by restricted chromosome introgression. These dynamic sex chromosome turnovers represent the first such findings in vertebrates and imply that speciation associated with inter- or intraspecific hybridization plays an important role in sex chromosome turnover in frogs.

Female frogs communicate with males through blinking.

Blinking is a behavior unique to animal taxa with motile eyelids, such as most amphibians and reptiles as well as all birds and mammals1. Eyelid movement has physiological functions, such as lubricating the cornea and washing away dust, but its potential signaling functions are not well understood1,2. The use of eyeblinks as a social signal is currently thought to be restricted to some primates, especially humans and their companion animals, but has not been verified in other taxa1,3,4. Here, through field observation and experiments, we demonstrate that female concave-eared torrent frogs (Odorrana tormota), which inhabit noisy streams, use eyeblinks to communicate with males to urge them to initiate amplexus for mating. Our findings reveal that eyeblinks may serve as a social signal in non-primate species.

Piecing the barcoding puzzle of Palearctic water frogs (Pelophylax) sheds light on amphibian biogeography and global invasions.

Palearctic water frogs (genus Pelophylax) are an outstanding model in ecology and evolution, being widespread, speciose, either threatened or threatening to other species through biological invasions, and capable of siring hybrid offspring that escape the rules of sexual reproduction. Despite half a century of genetic research and hundreds of publications, the diversity, systematics and biogeography of Pelophylax still remain highly confusing, in no small part due to a lack of correspondence between studies. To provide a comprehensive overview, we gathered >13,000 sequences of barcoding genes from >1700 native and introduced localities and built multigene mitochondrial (~17 kb) and nuclear (~10 kb) phylogenies. We mapped all currently recognized taxa and their phylogeographic lineages (>40) to get a grasp on taxonomic issues, cyto-nuclear discordances, the genetic makeup of hybridogenetic hybrids, and the origins of introduced populations. Competing hypotheses for the molecular calibration were evaluated through plausibility tests, implementing a new approach relying on predictions from the anuran speciation continuum. Based on our timetree, we propose a new biogeographic paradigm for the Palearctic since the Paleogene, notably by attributing a prominent role to the dynamics of the Paratethys, a vast paleo-sea that extended over most of Europe. Furthermore, our results show that distinct marsh frog lineages from Eastern Europe, the Balkans, the Near East, and Central Asia (P. ridibundus ssp.) are naturally capable of inducing hybridogenesis with pool frogs (P. lessonae). We identified 14 alien lineages (mostly of P. ridibundus) over ~20 areas of invasions, especially in Western Europe, with genetic signatures disproportionally pointing to the Balkans and Anatolia as the regions of origins, in line with exporting records of the frog leg industry and the stocks of pet sellers. Pelophylax thus emerges as one of the most invasive amphibians worldwide, and deserves much higher conservation concern than currently given by the authorities fighting biological invasions.

Whole-genome sequencing and analysis of Chryseobacterium arthrosphaerae from Rana nigromaculata.

Chryseobacterium arthrosphaerae strain FS91703 was isolated from Rana nigromaculata in our previous study. To investigate the genomic characteristics, pathogenicity-related genes, antimicrobial resistance, and phylogenetic relationship of this strain, PacBio RS II and Illumina HiSeq 2000 platforms were used for the whole genome sequencing. The genome size of strain FS91703 was 5,435,691 bp and GC content was 37.78%. A total of 4,951 coding genes were predicted; 99 potential virulence factors homologs were identified. Analysis of antibiotic resistance genes revealed that strain FS91703 harbored 10 antibiotic resistance genes in 6 categories and 2 multidrug-resistant efflux pump genes, including adeG and farA. Strain FS91703 was sensitive to ß-lactam combination drugs, cephem, monobactam and carbapenems, intermediately resistant to phenicol, and resistant to penicillin, aminoglycosides, tetracycline, fluoroquinolones, and folate pathway inhibitors. Phylogenetic analysis revealed that strain FS91703 and C. arthrosphaerae CC-VM-7T were on the same branch of the phylogenetic tree based on 16 S rRNA; the ANI value between them was 96.99%; and the DDH values were 80.2, 72.2 and 81.6% by three default calculation formulae. These results suggested that strain FS91703 was a species of C. arthrosphaerae. Pan-genome analysis showed FS91703 had 566 unique genes compared with 13 other C. arthrosphaerae strains, and had a distant phylogenetic relationship with the other C. arthrosphaerae strains of the same branch in phylogenetic tree based on orthologous genes. The results of this study suggest that strain FS91703 is a multidrug-resistant and highly virulent bacterium, that differs from other C. arthrosphaerae strains at the genomic level. The knowledge about the genomic characteristics and antimicrobial resistance of strain FS91703 provides valuable insights into this rare species, as well as guidance for the treatment of the disease caused by FS91703 in Rana nigromaculata.

Nuclear gene introgressions in hybrid populations of water frog Pelophylax esculentus complex: geographical analysis of the phenomenon and its interpretation.

Reproduction of water frog hybrids Pelophylax esculentus (Pelophylax ridibundus x Pelophylax lessonae) is associated with hemiclonal reproduction and backcrossing. The hemiclonal mode of reproduction occurs within P. esculentus allodiploids. In this case, the unrecombined genome of one parental species is transmitted to the offspring after premeiotic elimination of the chromosome set of the second parental species. Usually, the chromosome set of P. lessonae is eliminated, and the altered genome of P. ridibundus is passed on to the progeny. The hemiclonal inheritance within diploid Pelophylax esculentus hybrids may be accompanied by certain aberrations of premeiotic elimination. As a result, the formation of P. ridibundus specimens with introgressions of the P. lessonae genetic material, or the formation of recombinant hybrids occurs, depending on which of the parental species backcrossing takes place. The aim of our study is to describe the aberration of premeiotic elimination within the water frog P. esculentus complex detected by the nuclear gene Ldh-B inheritance, with an attempt to find out the causes of this phenomenon. It has been established that aberrations of premeiotic elimination are widespread, but only within populations of water frog from the river system of Upper Dnieper within Ukraine. The highest level of introgression takes place in the water frog populations within Kiev metropolis under conditions of expressed anthropogenization, while the maximum frequency of recombinants was detected within populations from the basin of Desna River, that has preserved native ecosystems. It was demonstrated that the frequency of premeiotic aberrations does not correlate with the intensity of interspecific water frog hybridization. Populations with introgressions are more common than populations with recombinants, however, within the latter, the frequency of recombination events is higher. The primary factor of gametogenesis aberrations, most likely, is the genetic characteristics of the local populations of parental species, since unambiguous explanations of this phenomenon based on the action of environmental stress (pollution of water systems) are not obvious.

The effect and mechanism of variable particle size microplastics and levofloxacin on the neurotoxicity of Rana nigromaculata based on the microorganism-intestine-brain axis.

Microplastics (MPs) usually appear in the aquatic environment as complex pollutants in combination with other environmental pollutants, such as levofloxacin (LVFX). After a 45-day exposure to LVFX and MPs with different particle sizes at environmental levels, LVFX was neurotoxic to Rana nigromaculata tadpoles. The order of the effects of the exposure treatment on tadpole behavior was: LVFX-MP3>LVFX-MP1>LVFX-MP2 ≥ LVFX. Results of transcriptome analysis of tadpole brain tissue showed that LVFX in combination with 0.10 and 10.00 µm MP interferes with the nervous system through the cell adhesion molecules pathway. Interestingly, the order of effects of the co-exposure on oxidative stress in the intestine was inconsistent with that of tadpole behavior. We found that Paraacteroides might be a microplastic indicator species for the gut microbiota of aquatic organisms. The results of the targeted metabolism of neurotransmitters in the intestine suggest that in the LVFX-MP2 treatment, LVFX alleviated the intestinal microbiota disorder caused by 1.00 µm MP, by regulating intestinal microbiota participating in the TCA cycle VI and gluconeogenesis and tetrapyrrole biosynthesis I, while downregulating Met and Orn, and upregulating 5HIAA, thereby easing the neurotoxicity to tadpoles exposed to LVFX-MP2. This work is of great significance for the comprehensive assessment of the aquatic ecological risks of microplastics-antibiotic compound pollutants.

Gut microbiome diversity and function during hibernation and spring emergence in an aquatic frog.

The gut microbiota maintains a deeply symbiotic relationship with host physiology, intricately engaging with both internal (endogenous) and external (exogenous) factors. Anurans, especially those in temperate regions, face the dual challenges of significant external influences like hibernation and complex internal variances tied to different life histories. In our research, we sought to determine whether different life stages (juvenile versus adult) of the Japanese wrinkled frog (Glandirana rugosa) lead to distinct shifts in gut bacterial communities during winter (hibernation) and its subsequent transition to spring. As hypothesized, we observed a more pronounced variability in the gut bacterial diversity and abundance in juvenile frogs compared to their adult counterparts. This suggests that the gut environment may be more resilient or stable in adult frogs during their hibernation period. However, this pronounced difference was confined to the winter season; by spring, the diversity and abundance of gut bacteria in both juvenile and adult frogs aligned closely. Specifically, the variance in gut bacterial diversity and composition between winter and spring appears to mirror the frogs' ecological adaptations. During the hibernation period, a dominance of Proteobacteria suggests an emphasis on supporting intracellular transport and maintaining homeostasis, as opposed to active metabolism in the frogs. Conversely, come spring, an uptick in bacterial diversity coupled with a dominance of Firmicutes and Bacteroidetes points to an upsurge in metabolic activity post-hibernation, favoring enhanced nutrient assimilation and energy metabolism. Our findings highlight that the relationship between the gut microbiome and its host is dynamic and bidirectional. However, the extent to which changes in gut bacterial diversity and composition contribute to enhancing hibernation physiology in frogs remains an open question, warranting further investigation.

Utilization of temperature-mediated activation of thyroid hormone-induced molecular memory to evaluate early signaling events in the olfactory epithelium of Rana [Lithobates] catesbeiana tadpoles.

The amphibian olfactory system is highly distinct between aquatic tadpole and terrestrial frog life stages and therefore must remodel extensively during thyroid hormone (TH)-dependent metamorphosis. Developmentally appropriate functioning of the olfactory epithelium is critical for survival. Previous studies in other Rana [Lithobates] catesbeiana premetamorphic tadpole tissues showed that initiation of TH-induced metamorphosis can be uncoupled from execution of TH-dependent programs by holding tadpoles in the cold rather than at warmer permissive temperatures. TH-exposed tadpoles at the nonpermissive (5 °C) temperature do not undergo metamorphosis but retain a "molecular memory" of TH exposure that is activated upon shift to a permissive warm temperature. Herein, premetamorphic tadpoles were held at permissive (24 °C) or nonpermissive (5 °C) temperatures and injected with 10 pmoles/g body weight 3,5,3'-triiodothyronine (T3) or solvent control. Olfactory epithelium was collected at 48 h post-injection. RNA-sequencing (RNA-Seq) and reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) analyses generated differentially expressed transcript profiles of 4328 and 54 contigs for permissive and nonpermissive temperatures, respectively. Translation, rRNA, spliceosome, and proteolytic processes gene ontologies were enriched by T3 treatment at 24 °C while negative regulation of cell proliferation was enriched by T3 at 5 °C. Of note, as found in other tissues, TH-induced basic leucine zipper-containing protein-encoding transcript, thibz, was significantly induced by T3 at both temperatures, suggesting a role in the establishment of molecular memory in the olfactory epithelium. The current study provides critical insights by deconstructing early TH-induced induction of postembryonic processes that may be targets for disruption by environmental contaminants.

Investigation of heavy metals in tissues and habitats of three edible frogs from Türkiye.

The soil, water, and organisms have been contaminated by heavy metals due to human activities and industrialization, which has produced a major environmental problem that has a deleterious effect on human health and food quality. Frogs, one of the good bioindicators for environmental pollution, are also among the alternative essential protein sources for humans. In Türkiye, three of these frogs are edible: Pelophylax ridibundus, Pelophylax bedriagae, and Pelophylax caralitanus, also known as Anatolian water frogs. Hence, to assess the possible health risks that might result from consuming frog legs in addition aquatic habitat of Anatolian water frogs, the water, sediment, and frog tissue samples (muscle and liver) were obtained from 11 different provinces covering all regions of Türkiye and analyzed to determine Cd, Cu, Cr, Zn, Pb, and As concentrations. The results revealed considerable variations in heavy metal concentrations among frog tissues, influenced by the sampling sites and species (ANOVA: p < 0.05). The Estimated Daily Intake (EDI) values, calculated based on the average serving size, were also lower than the Provisional Tolerable Daily Intake (PTDI) levels for adult consumers. Furthermore, the study computed the Target Hazard Quotient (THQ) values for heavy metals, all of which were below the critical value of 1, indicating that consuming the hind leg muscles from these frog species would not pose an adverse health risk for humans.

Cross-species gut microbiota transplantation predictably affects host heat tolerance.

The gut microbiome is known to influence and have regulatory effects in diverse physiological functions of host animals, but only recently has the relationship between host thermal biology and gut microbiota been explored. Here, we examined how early-life manipulations of the gut microbiota in larval amphibians influenced their critical thermal maximum (CTmax) at different acclimation temperatures. We stripped the resident microbiome from egg masses of wild-caught wood frogs (Lithobates sylvaticus) via an antibiotic wash, and then inoculated the eggs with pond water (control), no inoculation, or the intestinal microbiota of another species that has a wider thermal tolerance - green frogs (Lithobates clamitans). We predicted that this cross-species transplant would increase the CTmax of the recipient wood frog larvae relative to the other treatments. In line with this prediction, green frog microbiome-recipient larvae had the highest CTmax while those with no inoculum had the lowest CTmax. Both the microbiome treatment and acclimation temperature significantly influenced the larval gut microbiota communities and α-diversity indices. Green frog microbiome-inoculated larvae were enriched in Rikenellaceae relative to the other treatments, which produce short-chain fatty acids and could contribute to greater energy availability and enhanced heat tolerance. Larvae that received no inoculation had a higher relative abundance of potentially pathogenic Aeromonas spp., which negatively affects host health and performance. Our results are the first to show that cross-species gut microbiota transplants alter heat tolerance in a predictable manner. This finding has repercussions for the conservation of species that are threatened by climate change and demonstrates a need to further explore the mechanisms by which the gut microbiota modulate host thermal tolerance.

Blood parameters of adult marsh frogs Pelophylax ridibundus (Amphibia: Ranidae) in rice paddies subjected to intense agrochemical use.

We present the results of an in situ study of a set of blood parameters in adult marsh frogs (Pelophylax ridibundus (Pallas 1771) from populations inhabiting the largest system of rice fields in Bulgaria, the Tsalapitsa rice fields (TRF), under chronic stress conditions. This study was conducted in spring 2022 to assess the health status of TRF frogs compared to that of frogs occupying a reference site (RS). Furthermore, this study also compared the results obtained for the TRF population with those obtained in a study conducted at the exact same location with P. ridibundus individuals in 2013 (Zhelev et al. 2018). This comparison highlights the potential effects of persistent use of agrochemicals (pesticides and fertilizers) on the marsh frogs of later generations. Our results suggest that the general health of marsh frogs in the polluted site (PS) in southern Bulgaria has severely deteriorated. Frogs of both sexes were anemic with weakened immune systems compared to those living in the RS. The long-term use of agrochemicals in the PS affected males to a greater extent than it did females. Statistically significant hypochromia was observed in males, combined with general leukopenia, neutrophilia, lymphopenia, monocytosis, eosinophilia, and higher neutrophil/lymphocyte (N/L) ratios.

Transcriptomic analysis of skin immunity genes in the Chinese spiny frog (Quasipaa spinosa) after Proteus mirabilis infection.

Recently, populations of Chinese spiny frogs (Quasipaa spinosa), an important amphibian species in China, have decreased, mainly due to a disease caused by the gram-negative bacteria Proteus mirabilis. To elucidate the immune response of the frogs, this study aimed to identify novel candidate genes functionally associated with P. mirabilis infection-induced "rotting skin" disease. Chinese spiny frogs were infected with P. mirabilis, and the skin transcriptome was sequenced using the MGISEQ-2000 platform. A total of 233,965 unigenes were obtained by sequencing, of which 27.23 % were known genes. Screening of differentially expressed genes (DEGs) indicated 210 unigenes differentially expressed after P. mirabilis infection, of which 132 unigenes were up-regulated, and 78 unigenes were down-regulated. Using Kyoto Encyclopedia of Genes and Genomes enrichment analysis, DEGs were identified as enriched in signal pathways, such as oxidative phosphorylation, apoptosis, and the Janus kinase-signal transducer and activator of transcription pathway. Of the DEGs, there was a significant upregulation of the colony stimulating factor 2 receptor beta common subunit, interleukin 2 receptor subunit gamma, cathelicidin antimicrobial peptide, interleukin-17 receptor E, receptor-interacting serine/threonine-protein kinase 3, and pulmonary surfactant-associated protein D immune genes following P. mirabilis infection. Conversely, scavenger receptor cysteine-rich domain-containing group B protein, tumor protein p53 inducible nuclear protein 2, suppressor of cytokine signaling 2, and metalloreductase STEAP3 were significantly downregulated. In conclusion, the first skin transcriptome database of Chinese spiny frogs was established, and several immune genes were identified to elucidate the pathogenic mechanism of "skin rot" in Chinese spiny frogs and other cultured frogs.

Unleashing diversity through flexibility: The evolutionary journey of sex chromosomes in amphibians and reptiles.

Sex determination systems have greatly diversified between amphibians and reptiles, with such as the different sex chromosome compositions within a single species and transition between temperature-dependent sex determination (TSD) and genetic sex determination (GSD). In most sex chromosome studies on amphibians and reptiles, the whole-genome sequence of Xenopous tropicalis and chicken have been used as references to compare the chromosome homology of sex chromosomes among each of these taxonomic groups, respectively. In the present study, we reviewed existing reports on sex chromosomes, including karyotypes, in amphibians and reptiles. Furthermore, we compared the identified genetic linkages of sex chromosomes in amphibians and reptiles with the chicken genome as a reference, which is believed to resemble the ancestral tetrapod karyotype. Our findings revealed that sex chromosomes in amphibians are derived from genetic linkages homologous to various chicken chromosomes, even among several frogs within single families, such as Ranidae and Pipidae. In contrast, sex chromosomes in reptiles exhibit conserved genetic linkages with chicken chromosomes, not only across most species within a single family, but also within closely related families. The diversity of sex chromosomes in amphibians and reptiles may be attributed to the flexibility of their sex determination systems, including the ease of sex reversal in these animals.