Gosner Stage Research Articles

Older Amphibian Larvae Are More Sensitive to Ultraviolet Radiation and Experience More Sublethal Carryover Effects Post-Metamorphosis.

Elevated ultraviolet radiation (UVR) is postulated as one of multiple, interrelated environmental stressors driving amphibian population declines globally. However, key knowledge gaps remain in elucidating the link between elevated UVR and amphibian declines in a changing climate, including whether timing and irradiance of UVR exposure in early life dictates the onset of detrimental carryover effects post-metamorphosis. In this study, striped marsh frog larvae (Limnodynastes peronii) were exposed to UVR at one of two different irradiances for up to 7 days, either as hatchlings (Gosner stage 23) or as older larvae (Gosner stage 25-28). These animals were then reared to metamorphosis in the absence of UVR to examine independent and interactive carryover effects throughout development. Older larvae were more sensitive to UVR than hatchlings, with 53.1% and 15.6% mortality in larvae exposed to high and low irradiance respectively, compared with no mortality of hatchlings in either irradiance treatment. Irradiance and timing of UVR exposure had interactive effects on larval body length, causing stunted growth patterns and a lack of compensatory growth following UVR exposure, particularly in animals exposed to high irradiance UVR later in development. Timing of UVR exposure also determined the severity of carryover effects into metamorphosis, including delayed metamorphosis and the first published account (to our knowledge) of latent UVR-induced depigmentation in an amphibian. These findings highlight how acute changes to the larval UVR exposure regime can impact on amphibian health later in life, with implications for our understanding of the effects of climate change on UVR-related amphibian declines.

Lernaea cf. cyprinacea infection in agile frog (Rana dalmatina) tadpoles in Hungary.

The ectoparasitic copepod Lernaea cyprinacea (anchor worm) has more than 100 host species among teleost fishes and affects cyprinids both in fish farms and natural waters. In addition, while L. cyprinacea infection in amphibians has been recorded in Asia, North and South America, there is no data available in the literature on their presence in Europe. In this study, we first reported L. cf. cyprinacea parasitising an anuran tadpole in Europe. Specimens of L. cf. cyprinacea were observed attached to a tadpole of the agile frog (Rana dalmatina), which was caught during fishing of crucian carp fingerlings (Carassius carassius) from a small fish pond in Hungary during the summer of 2012. The infected tadpole was collected from a rearing pond, where juvenile crucian carps were kept. The tadpole was inspected in the laboratory, and digital photos were taken. The parasites were found attached to the body-tail junction and to the leg of the tadpole (at Gosner stage 41). The parasite species was identified as L. cf. cyprinacea based on morphological traits.

Effects of perfluoroalkyl sulfonic acids on developmental, physiological, and immunological measures in northern leopard frog tadpoles

The chronic toxicity of short chain perfluoroalkyl sulfonic acids (PFSAs), such as perfluorobutanesulfonic acid (PFBS) and perfluorohexanesulfonic acid (PFHxS), are relatively understudied despite the increasing detection of these compounds in the environment. We investigated the chronic toxicity and bioconcentration of PFBS and PFHxS using northern leopard frog (Rana [Lithobates] pipiens) tadpoles. We exposed Gosner stage (GS) 25 tadpoles to either PFBS or PFHxS at nominal concentrations of 0.1, 1, 10, 100, and 1000 μg/L until metamorphosis (GS42). We then assessed tadpole growth, development, stress, and immune metrics, and measured fatty acid (FA) composition and PFSA concentrations in liver and whole-body tissues. Tadpole growth and development measures were relatively unaffected by PFSA exposure. However, tadpoles exposed to 1000 μg/L PFBS or PFHxS had significantly increased hepatosomatic indexes (HSI) relative to controls. Further, tadpoles from the 1000 μg/L PFHxS treatment had altered FA profiles relative to controls, with increased total FAs, saturated FAs, monounsaturated FAs, and omega-6 polyunsaturated FAs. In addition, tadpoles from the 1000 μg/L PFHxS treatment had a higher probability of waterborne corticosterone detection. These results suggest that PFBS and PFHxS influence the hepatic health of tadpoles, and that PFHxS may alter lipid metabolism in tadpoles. We also observed a higher probability of tadpoles being phenotypically female after exposure to an environmentally relevant concentration (0.1 μg/L) of PFHxS, suggesting that PFHxS may exert endocrine disrupting effects on tadpoles during early development. The measured bioconcentration factors (BCFs) for both compounds were ≤10 L kg−1 wet weight, suggesting low bioconcentration potential for PFBS and PFHxS in tadpoles. Many of the significant effects observed in this study occurred at concentrations several orders of magnitude above those measured in the environment; however, our work shows effects of PFSAs exposure on amphibians and provides essential information for ecological risk assessments of these compounds.

Developmental sequence of the chondrocranium in the obligate carnivorous larvae of Lepidobatrachus laevis (Amphibia: Ceratophryidae).

The vertebrate head and its skull represent a significant innovation that has played a key role in the evolutionary and ecological success of vertebrates. For a global and integral understanding of the evolution of the head skeleton, it is essential to have reliable information on the development of chondrocranium in a wide range of vertebrate species. Therefore, we studied the cranial chondrogenesis of the larva of the Budgett frog, Lepidobatrachus laevis (Ceratophryidae, Neobatrachia). We studied the development using several methods, including histological preparation of transverse sections of the chondrocranium, morphological analysis of three different states of development (mesenchymal aggregation, differentiation, and chondrification), and three-dimensional digital reconstructions. As a result, we observed that the Anlage of the chondrocranium at Gosner stage 19 is laterally compressed, that is, it is initially higher than wide. It gradually flattens, enlarges, and differentiates until reaching a very wide and flat shape at Gosner stage 26. Furthermore, we show that the chondrocranial development of L. laevis takes place in a mosaic pattern, which differs to nontetrapod vertebrates in which an anterior to posterior gradient of chondrification is observed. We identified 19 developmental stages in L. laevis according to the chondrification state of its cranial structures. The first element reaching the differentiation-into-chondroblast stage is the hypobranchial plate of the branchial basket, and chondrification, that is, final differentiation, occurs simultaneously in several structures of the neurocranium and viscerocranium. We hypothesize that the rapid chondrification of L. laevis' chondrocranium is an adaptation to the semi-arid climate conditions from its type locality, El Gran Chaco in South America. Due to the only temporary availability of suitable water accumulations this apparent accelerated development would make sense to ensure the tadpoles are froglets by the time the water body disappears. Here, we provide novel information on chondrocranial development in L. laevis. We compare our results to the developmental sequences, previously known from other vertebrate taxa, particularly within the amphibian phylogeny, in an attempt to understand developmental variability and the evolutionary history of the vertebrate head skeleton.

Inter-individual variability in developmental traits in tadpoles of the Baja California chorus frog (Pseudacris hypochondriaca) in response to temperature

Phenotypic plasticity is the intraspecific expression of different phenotypes in response to environmental conditions. Inter-individual variability refers to the range of phenotypes produced between conspecifics within a population, and such variability is ubiquitously observed among animals. Investigating inter-individual variability can help develop a more comprehensive understanding of how phenotypes respond to environmental change. The Baja California chorus frog ( Pseudacris hypochondriaca) is found in a variety of ecosystems that differ in temperature and elevation throughout its latitudinal range (∼1400 km) from Santa Barbara County, California to Baja California, Mexico. The chorus frog experiences a variable thermal environment across its life history and males initiate mating calls at a wide range of water temperatures, between 4 and 20°C. Embryos develop successfully between 8 and 29°C, and tadpoles between 0 and 33°C. We have previously observed inter-individual variability in developmental traits, such as development rate and metabolic rate, and therefore aimed to investigate this variability within and between temperatures more closely. Chorus frog eggs were collected from a wetland on the CSUSM campus the day after being laid, and incubated at 15°C or 22°C. Once tadpoles reached the target stage (Gosner Stage 33; hind limb toe differentiation), oxygen consumption rate (Vȯ2) was measured using intermittent respirometry, and body length, wet mass and dry gut-free masses were measured. We also investigated the effect of temperature on swimming speed and time to exhaustion. Average time to stage, wet mass, dry gut-free mass and body length all decreased at 22°C, while mass-specific Vȯ2 increased. We observed decreased variability at 22°C compared to 15°C in time to reach stage (days from first to last tadpole to reach stage: 30.2 ± 2.7 days at 15°C, 15.4 ± 1.1 at 22°C), wet mass (wet mass range: 148.8 ± 14.2 mg at 15°C, 105.1 ± 11.3 mg at 22°C), and dry gut-free mass (dry gut-free mass range: 14.2 ± 1.2 mg at 15°C, 7.3 ± 0.7 mg at 22°C). Variability in body length did not differ between temperatures (total length range: 7.7 ± 0.4 mm at 15°C, 6.4 ± 0.5 mm at 22°C). In contrast to other traits, variability in mass-specific Vȯ2 increased at 22°C compared to 15°C (mass-specific Vȯ2 range: 0.61 ± 0.07 μl h−1 mg−1 at 15°C, 1.63 ± 0.21 μl h−1 mg−1 at 22°C). Overall, there is a correlation between the average effect of temperature and the inter-individual variability of phenotypic traits, with higher temperature resulting in less inter-individual variability in development rate and morphological characteristics, which are faster and smaller at higher temperature, while variability in mass-specific Vȯ2 increased with temperature, reflecting the increase in average Vȯ2 that occurs at higher temperature. CS supported by a Beckman Scholarship. CAM supported by a CSUSM Research, Scholarship and Creative Activities Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Rearing American bullfrog tadpoles in different tank colors: Effect on metamorphosis, performance, and biochemistry responses

This study examined the influence of tank color on the performance, time to metamorphosis, skin pigmentation, and biochemistry parameters of American bullfrog tadpoles and froglets. Six hundred tadpoles (0.94 ± 0.11 g) were selected (Gosner stage – 25) and distributed in a completely randomized design in 15 tanks of 40 L (1 tadpole L−1) in a recirculating aquaculture system, five repetitions of each color (blue, black, and white). Biometrics were performed every 14 days in all experimental units, and when the tadpoles at metamorphic climax and following complete transformation. Animals were weighed, measured, and photographed to evaluate the growth performance, uniformity, and skin pigmentation analyses. At the conclusion of metamorphosis, twenty-four animals randomly selected per treatment were euthanized. Twelve of them were macerated to performed of the whole-body biochemistry analysis, and the other twelve were dissected to obtain the biological indexes. The tadpoles raised in blue, and black tanks reached the metamorphosis climax and transitioned into froglets more rapidly (P < 0.05) than animals held in white tanks. However, tadpoles at metamorphic climax reared in black tanks had prolonged time to complete the metamorphosis (P < 0.05). The tadpoles raised in blue background had the higher values for final body weight throughout the experiment (P < 0.05). The tadpoles raised at blue and black tanks had the higher values for body weight gain, body length, body length gain, protein efficiency ratio, specific growth rate and lower feed conversion ratio throughout the experiment (P < 0.05). The pre- and pro-metamorphic uniformity of tadpoles was affected by the color of the tank, with the white treatment having lower uniformity at the 14, 42, 56, 70 and 98 days of the experiment (P < 0.05). In addition, tadpoles on metamorphic climax and froglets raised on black, and blue tanks had darker color of skin compared with the animals raised in white tanks, with animals raised in black tanks showing darker skin tones (P < 0.05). However, neither the growth, and uniformity of tadpoles on metamorphic climax and froglets, nor the biological indexes and whole-body biochemistry of froglets were affected by tank color (P > 0.05). Therefore, considering the homogeneity in growth, and time to metamorphosis, blue colored tanks represent best choice for reared of American bullfrog tadpoles.

The effects of two short-chain perfluoroalkyl carboxylic acids (PFCAs) on northern leopard frog (Rana pipiens) tadpole development

Short-chain perfluoroalkyl carboxylic acids (PFCAs) have been detected in the environment globally. The presence and persistence of these compounds in the environment may lead to chronic wildlife exposure. We used northern leopard frog (Rana pipiens) tadpoles to investigate the chronic toxicity and the bioconcentration of two short-chain PFCAs, perfluorobutanoic acid (PFBA) and perfluorohexanoic acid (PFHxA). We exposed Gosner stage 25 tadpoles to PFBA and PFHxA (as individual chemicals) at nominal concentrations of 0.1, 1, 10, 100, and 1000 µg/L for 43–46 days. Tadpoles exposed to 0.1 to 100 µg/L of PFBA and PFHxA had significantly higher mean snout-to-vent lengths, mean masses, and scaled mass indexes than control tadpoles. These results indicate that exposure to short-chain PFCAs influences tadpole growth. Further investigation into the mechanism(s) causing the observed changes in tadpole growth is warranted. We observed a significantly higher proportion of males in the PFBA 1 µg/L treatment group, however further histological analyses are required to confirm visual sex identification before making concrete conclusions on the effects of PFCAs on amphibian sex ratios. PFBA concentrations in tissues were higher than PFHxA concentrations; a pattern that contrasts with previously published studies using fish, suggesting potential differences between taxa in PFBA and PFHxA bioconcentration. Bioconcentration factors were <10 L/kg wet weight, indicating low bioconcentration potential in tadpoles. Our results suggest that PFBA and PFHxA may have effects at environmentally-relevant concentrations (0.1–10 µg/L) and further investigation is required before these compounds can be deemed a “safe” alternative to their long-chain counterparts.

Rearing density and food variety impact growth, development, and survival of larvae in the declining amphibian, Pseudacris maculata.

Boreal chorus frogs (Pseudacris maculata Agassiz 1850) are a widespread amphibian in North America, but several populations are in decline. Specifically, we are developing captive breeding and reintroduction methods for the Great Lakes/St. Lawrence-Canadian Shield population. Here we present the effects of tadpole density, food variety, and addition of supplemental minerals to rearing water on the growth, development, and survival during the larval and metamorph/juvenile stages. We conducted two experiments using a factorial design. We found that low tadpole density (1vs. 2 tadpoles/L) and high food variety (fivefood items vs. three food items) significantly increased tadpole body length and Gosner stage after 2 weeks (p < .001), increased survival to metamorphosis (p < .001), decreased time to metamorphosis (p < .001), and increased weight after metamorphosis (p < .001). On average, tadpoles in the high density/low food treatment, compared to the low density/high food, were 25% smaller after 2 weeks, had 3.9× lower survival to metamorphosis, took 1.25× longer to reach metamorphosis, and weighed 1.5× less after metamorphosis. In contrast, neither density (0.5vs. 1 tadpole/L) nor mineral supplemented water affected growth and development, but tadpole survival was higher at 1 tadpole/L. Our findings demonstrate the feasibility of rearing boreal chorus frogs in captivity and provide guidelines for rearing this and similar species in a laboratory environment.

Assessment of toxicity, genotoxicity and oxidative stress in Fejervarya limnocharis exposed to tributyltin.

Tributyltin (TBT) is widely used in various commercial applications due to its biocidal properties. Toxicological and genotoxicological data on TBT exposure to amphibians is insufficient. Our study aimed to determine the acute toxicity and genotoxic potential of TBT in Fejervarya limnocharis tadpoles. Furthermore, oxidative stress was also investigated in TBT-treated tadpoles. Tadpoles of Gosner stage (26-30) were screened and subjected to increasing concentrations of TBT (0, 3, 7, 11, 15, 19, 23µg/L) for determining the LC50 values for 24h, 48h, 72h, and 96h. LC50 values of TBT for 24h, 48h, 72h, and 96h were found to be 19.45, 15.07, 13.12, and 11.84μg/L respectively. Based on the 96h LC50 value (11.84µg/L), tadpoles were exposed to different sub-lethal concentrations of TBT for the evaluation of its genotoxic potential and effects on oxidative balance. The role of TBT on survivability, growth, and time to metamorphosis was also assessed. TBT exposure significantly altered the life history traits measured, increased mortality, and delayed the time taken to metamorphosis. Results indicated significant induction of micronucleus (MN, p < 0.001) and other erythrocytic nuclear aberrations (ENA, p < 0.01) in the TBT-treated groups. Significant alterations in comet parameters and oxidative balance were also observed in the treated groups. The present study findings might add to the cause of the gradual population decline seen in the amphibians. This study also demonstrates the alteration of the life-history traits, oxidative balance, and DNA damage upon TBT exposure which can have long-term consequences for the anuran amphibian F. limnocharis.

Chytridiomycosis causes high amphibian mortality prior to the completion of metamorphosis

Amphibian populations are undergoing extensive declines globally. The fungal disease chytridiomycosis, caused by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), is a primary contributor to these declines. The amphibian metamorphic stages (Gosner stages 42–46) are particularly vulnerable to a range of stressors, including Bd. Despite this, studies that explicitly examine host response to chytridiomycosis throughout the metamorphic stages are lacking. We aimed to determine how Bd exposure during the larval stages impacts metamorphic development and infection progression in the endangered Fleay's barred frog (Mixophyes fleayi). We exposed M. fleayi to Bd during pro-metamorphosis (Gosner stages 35–38) and monitored infection dynamics throughout metamorphosis. We took weekly morphological measurements (weight, total body length, snout-vent-length and Gosner stage) and quantified Bd load using qPCR. While we observed minimal impact of Bd infection on animal growth and development, Bd load varied throughout ontogeny, with an infection load plateau during the tadpole stages (Gosner stages 35–41) and temporary infection clearance at Gosner stage 42. Bd load increased exponentially between Gosner stages 42 and 45, with most exposed animals becoming moribund at Gosner stage 45, prior to the completion of metamorphosis. There was variability in infection outcome of exposed individuals, with a subgroup of animals (n = 5/29) apparently clearing their infection while the majority (n = 21/29) became moribund with high infection burdens. This study demonstrates the role that metamorphic restructuring plays in shaping Bd infection dynamics and raises the concern that substantial Bd-associated mortality could be overlooked in the field due to the often cryptic nature of these latter metamorphic stages. We recommend future studies that directly examine the host immune response to Bd infection throughout metamorphosis, incorporating histological and molecular methods to elucidate the mechanisms responsible for the observed trends.

Toxic, genotoxic, mutagenic, and bioaccumulative effects of metal mixture from settleable particulate matter on American bullfrog tadpoles (Lithobates catesbeianus)

Amphibians are more susceptible to environmental stressors than other vertebrates due to their semipermeable skin and physiological adaptations to living in very specific microhabitats. Therefore, the aim of the present study was to investigate the effects of a metal mixture from settleable particulate matter (SePM) released from metallurgical industries on Lithobates catesbeianus tadpoles. Endpoints analyzed included metal bioconcentration, morphological (biometrical indices), hematological parameters (hemoglobin and blood cell count), and erythrocyte DNA damage (genotoxicity and mutagenicity). American bullfrog tadpoles (Gosner's stage 25) were kept under control condition (no contaminant addition) or exposed to a sub-lethal and environmentally relevant concentration (1 g.L−1) of SePM for 96 h. Tadpoles exposed to SePM exhibited elevated whole blood levels of Fe56, AL, Sn, Pb, Zn, Cr, Cu, Ti, Rb, V, Ce, La, Ag, As. SePM-exposed tadpoles showed a significant decrease in condition factor (12%) and increases in hepatosomatic index (25%), hemoglobin concentration (17%), and total leukocytes (82%), thrombocytes (90%), and monocytes (78%) abundance. In addition, exposed tadpoles showed higher MN and ENAs (340 and 140%, respectively) frequencies, and erythrocyte DNA damage with approximately 1.2- to 1.8-fold increases in comet parameters. Taken together, these results suggest that the multimetal mixture found in SePM is potentially genotoxic and mutagenic to L. catesbeianus tadpoles, induces stress associated with hematological changes, and negatively affects growth. Although such contamination occurs at sublethal levels, regulatory standards are needed to control the emission of SePM and protect amphibian populations.

Impacts of isolated or mixed Roundup® Original DI and Boral® 500 SC herbicides on the survival and metamorphosis of Melanophryniscus admirabilis tadpoles

The bufonid species Melanophryniscus admirabilis is restricted to a single location in the southern Atlantic Forest, Brazil. Although the site of occurrence of M. admirabilis is covered with native forest and it is not directly exposed to pesticides application, the area is surrounded by agricultural activity. Our objectives were to evaluate possible alterations in morphological parameters (body mass, snout-vent length, and body index), metamorphosis (time to reach Gosner stages 42, 46 and to complete metamorphosis), and survival of M. admirabilis exposed to isolated Roundup® Original DI (R1: 234 and R2: 2340 µg.L−1 of glyphosate) and Boral® 500 SC, (B1: 130 and B2: 980 µg.L−1 of sulfentrazone) or mixed (R1+B1, R2+B1, R1+B2, R2+B2). Spawns of M. admirabilis were collected in natural lakes in the municipality of Arvorezinha and taken to laboratory cultivation. After the tadpoles acquired free swimming, the animals were acclimated for five days and fed ad libitum. The aquariums were contaminated with herbicides on the sixth day of cultivation, and the animals stayed in these aquariums for four days. Afterwards, the tadpoles were transferred to aquariums with clean water and monitored until metamorphosis (Gosner stage 46), when they were weighed, measured (snout-cloacal length) and cryoeuthanized. We observed no alterations in morphological parameters; however, survival was reduced in exposed groups (mortality index: 71 % in R2 and 29–64 % in mixed groups), suggesting energy allocation for metamorphosis at the expense of survival. Boral did not alter metamorphosis time. Roundup isolated and mixed with Boral altered the timing of Gosner stages 42 and 46 and reduced metamorphosis time, suggesting endocrine disruption. Thus, monitoring the presence and limiting the use of these pesticides in the area where M. admirabilis occurs can be crucial for conservation strategies.

Husbandry and first record of captive breeding of the Asian giant river toad Phrynoidis asper

The Asian giant river toad Phrynoidis asper is a large species of bufonid from south-east Asia that is apparently obtained by the international exotic-pet trade from wild populations. Captive breeding of this species seems not to have been documented. The donation to Chester Zoo in October 2021 of an adult group of five males and one female of this toad species provided an opportunity to study captive breeding. The specimens were maintained separated by sex until signs of reproductive condition were apparent and then they were placed together in a large breeding enclosure. This had three distinct environmental zones simulating a riverbank. Six days after mixing the sexes a large spawn mass was laid. On hatching, the tadpoles were transferred to a rearing aquarium and then, after reaching Gosner stage 42, the developing toads were transferred to terrestrial vivaria to complete development. Captive breeding of this species could replace collection from the wild.

Tadpoles of Papurana milleti (Amphibia: Anura): molecular identification and morphological description

Based on molecular identification, we described the tadpole morphology of Dalat forest frog, Papurana milleti. Twenty tadpole specimens of P. milleti (Gosner stage 25–42) were collected from Di Linh plateau, southern Vietnam. Detailed descriptions of external morphological features, morphometric measurements, color patterns in life and in preservation and ecological notes are provided. Tadpoles of P. milleti are distinguished from those of its congeners by having yellowish brown body, LTRF 2(2)/3(1) and single submarginal papillae from the lateral of row P-1 to under row P-3.

Adaptive plasticity in anuran metamorphosis: response of tadpoles of Polypedates maculatus (Anura: Rhacophoridae) to pond drying

&#x0D; &#x0D; &#x0D; &#x0D; The influence of desiccation on metamorphic traits (larval duration and size at emergence) was studied in Polypedates maculatus under laboratory conditions. Gosner Stage 23 tadpoles were exposed to decreasing water levels (gradual or rapid) until the beginning of metamorphic climax (Stage 42). A control group was reared in constant water levels. Tadpoles reared in decreasing water levels reached the metamorphic climax earlier and metamorphosed at a smaller size than those reared in constant water levels. Further, tadpoles experiencing rapid depletion of water reached the metamorphic climax earlier and metamorphosed at a smaller size than those experiencing gradual depletion of water levels. Tadpoles of P. maculatus showed adaptive plasticity in metamorphosis to pond drying. Survival of tadpoles in treatments and the control was 100%. The study revealed that tadpoles of P. maculatus have plastic development in response to water levels; the trade-off between growth and development favors development, which results in early metamorphosis at a small size.&#x0D; &#x0D; &#x0D; &#x0D;

Gonadal morphogenesis in the South American toad Rhinella Arenarum (Anura, Bufonidae) unveils an extremely delayed rate of sex differentiation.

Among anurans, Bufonids are recognized for their retarded sex differentiation. However, few studies have addressed gonadal morphogenesis in this family. Here, we analyzed the early gonadogenesis in laboratory-reared Rhinella arenarum. Few germ cells were identified in the genital ridge at Gosner stage 26. At metamorphosis, somatic cells and germ cells were observed in the outer region of the undifferentiated gonad, whereas the central region was occupied by stromal tissue. A cortico-medullary organization was first recognized on Day 7 postmetamorphosis. The cortex was composed of germ cells and encompassing epithelial cells, whereas the medulla contained cells presumptively derived from the coelomic epithelium. Medullary somatic cells formed metameric knots along the length of the undifferentiated gonad. Consequently, a series of 12-14 gonomeres became recognizable externally. The first sign of ovarian differentiation was observed on Day 15 postmetamorphosis, when a cavity was formed within each gonomere. In contrast, testes were recognized by a uniform distribution of germ cells and intermingled somatic cells, as the division into cortex and medulla was lost. By Day 50 postmetamorphosis, the gonadal metameric organization was still apparent both in the ovaries and testes. Follicles containing diplotene oocytes were observed within the ovary. In the testis, an incipient lobular architecture was recognized without initiation of meiosis within the seminiferous cords. These observations reveal an extremely delayed gonadal development in R. arenarum, not reported previously for other anuran species. In addition, the late differentiation of the gonads contrasted with the early appearance of follicles in the Bidder's organ. Lastly, we observed that delayed metamorphs exhibited an undifferentiated gonad, demonstrating that gonadogenesis in this species is more dependent on somatic development than on age.

The succession of gut microbiota in the concave-eared torrent frog (Odorrana tormota) throughout developmental history.

The gut microbiota of amphibians plays a crucial role in maintaining health and adapting to various developmental stages. The composition of gut microbial community is influenced by the phylogeny, habitat, diet, and developmental stage of the host. The present study analyzed the microbiota in the intestine of O. tormota at 11 developmental stages (from the tadpole at Gosner stage 24 to the 3-year-old adult) using high-throughput 16S rRNA sequencing. Alpha diversity index analysis of the microbiota revealed that the index decreased from tadpole at Gosner stage 24 to adult frog stage, remained stable during the adult frog stages, but increased significantly at the early metamorphosis and hibernation preparation stages. The gut microbiota structure is similar in adult frogs but differs significantly in other developmental stages. Furthermore, the dominant phyla of gut microbiota in tadpoles were Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes, whereas those in adult frogs were Proteobacteria, Firmicutes, Bacteroidetes, and Verrucomicrobia. Host and environmental factors jointly affected the gut microbial diversity and community composition of O. tormota, but developmental stage, feeding habit, and habitat type had a more significant influence. The microbial community in the gut varies with the developmental stage of the host and constantly adapts to the survival requirements of the host. These findings advance our understanding of the evolutionary mechanism of amphibian gut microbiota in maintaining health homeostasis and adaptation.

Exposure to nitrate induced growth, intestinal histology and microbiota alterations of Bufo raddei Strauch tadpoles

Nitrate (NO3−) is one of the ubiquitous environmental chemicals which multiplies negative impacts on aquatic life such as amphibian larvae. However, the data involving the dynamics of amphibians in response to NO3-N are scarce. This study investigated the effects of NO3-N on locomotor ability, growth performance, oxidative stress parameters, intestinal histology, and intestinal microbiota of Bufo raddei Strauch tadpoles. The tadpoles were chronically exposed to different concentrations of NO3-N (10, 50, 100, and 200 mg/L) from Gosner stage 26 to 38. Our results revealed that NO3-N exposure caused significantly reduced body weight and length, impaired locomotor activity, and severe oxidative damage to liver tissue. Moreover, the high NO3-N (50, 100, and 200 mg/L) exposure caused irregular arrangement and indistinct cell borders of mucosal epithelial cells in the tadpoles intestine. The NO3-N exposure significantly changed the structure of the intestinal microbiota. The phylum Cyanobacteria occupy the main niche of intestinal microbes and have a certain negative correlation with the growth and motility of tadpoles. In addition, the functional prediction revealed that NO3-N exposure obviously downregulated the metabolism of enzyme families in tadpoles. Our comprehensive research shows the toxicity of NO3-N exposure in B. raddei Strauch, explores the potential links between development and intestinal microbiota of tadpole, and provides a new framework for the potential health risk of nitrate in amphibians.

Chemical communication in wood frog (Rana sylvatica) tadpoles is influenced by early-life exposure to naphthenic acid fraction compounds

Environmental pollutants can disrupt chemical communication between aquatic organisms by interfering with the production, transmission, and/or detection of, as well as responses to, chemical cues. Here, we test the hypothesis that early-life exposure to naphthenic acid fraction compounds (NAFCs) from oil sands tailings disrupts antipredator-associated chemical communication in larval amphibians. Wild adult wood frogs (Rana sylvatica) captured during their natural breeding period were combined (1 female:2 males) in six replicate mesocosms filled with either uncontaminated lakewater or with NAFCs isolated from an active tailings pond in Alberta, Canada, at nominal 5 mg/L concentrations. Egg clutches were incubated and tadpoles maintained in their respective mesocosms for ∼40 days post-hatch. Tadpoles (Gosner stage 25-31) were then transferred individually to trial arenas filled with uncontaminated water and exposed to one of six chemical alarm cue (AC) stimuli solutions following a 3 × 2 × 2 design (3 AC types × 2 stimulus carriers × 2 rearing exposure groups). Relative to control tadpoles, NAFC-exposed tadpoles demonstrated higher baseline activity levels (line crosses and direction changes) when introduced to uncontaminated water. Antipredator responses differed in graded fashion with AC type, with control ACs eliciting the greatest latency to resume activity, water the least, and NAFC-exposed ACs intermediate. Pre- to post-stimulus difference scores were non-significant in control tadpoles, while NAFC-exposed tadpoles demonstrated significantly greater variation. While this suggests that exposure to NAFCs from fertilization through hatching may have interfered with AC production, it is unclear whether the quality or quantity of cues was affected. There was also no clear evidence that NAFC carrier water interfered with ACs or the alarm response in unexposed control tadpoles. These results emphasize the importance of understanding how behavioral and physiological effects of early-life NAFC exposure on critical antipredator responses may persist across life history stages.

Copper-mediated shifts in transcriptomic responses of intestines in Bufo gargarizans tadpoles to lead stress.

The differential transcriptomic responses of intestines in Bufo gargarizans tadpoles to Pb alone or in the presence of Cu were evaluated. Tadpoles were exposed to 30μg/L Pb individually and in combination with Cu at 16 or 64μg/L from Gosner stage (Gs) 26 to Gs 38. After de novo assembly, 105,107 unigenes were generated. Compared to the control group, 7387, 6937, and 11139 differentially expressed genes (DEGs) were identified in the treatment of Pb + Cu0, Pb + Cu16, and Pb + Cu64, respectively. In addition, functional annotation and enrichment analysis of DEGs revealed substantial transcriptional reprogramming of diverse molecular and biological pathways were induced in all heavy metal treatments. The relative expression levels of genes associated with intestinal epithelial barrier and bile acids (BAs) metabolism, such as mucin2, claudin5, ZO-1, Asbt, and Ost-β, were validated by qPCR. This study demonstrated that Pb exposure induced transcriptional responses in tadpoles, and the responses could be modulated by Cu.