Adult Xenopus Research Articles

Effectiveness, Safety, and Pharmacokinetics of Meloxicam Formulations in African-clawed Frogs, Xenopus laevis.

Pain management in amphibians is an emerging field of veterinary medicine with only a limited number of analgesics studied for their efficacy. The African-clawed frog, Xenopus laevis, is a popular animal model in research due to its oocyte morphology and embryonic development. We investigated analgesic effects of 2 formulations of meloxicam (standard and extended release [ER]) along with their pharmacokinetics and potential toxicity in this species. Adult female African-clawed frogs (n = 6/group) received either standard (0.2, 0.4, 1, or 5 mg/kg) or ER meloxicam (0.6, 1.2, 3, or 15 mg/kg) injected into the dorsal lymph sac. The acetic acid test (AAT) was performed at -1, 1, 6, 12, 24, 48, and 72 h postadministration to evaluate pain response. In addition, a subset of frogs (n = 2/group) were euthanized 72 h postinjection and submitted for necropsy. There were no significant differences in AAT with both formulations compared with saline control. No signs of meloxicam-induced toxicity with either formulation was present in histology. A pharmacokinetic study was conducted for both the standard and ER formulation of meloxicam at 5 and 15 mg/kg, respectively. Results were consistent with the fact that both formulations of meloxicam were readily absorbed with the standard plasma concentrations peaking at 20.40 µg/mL at 2 h and ER plasma concentration at 30.4 µg/mL at 12 h. The elimination half-life was only determinable for standard formulation (7.74 h). According to the AAT, both formulations of meloxicam did not provide effective analgesia in adult female Xenopus laevis despite reaching high plasma concentrations.

Techniques for Rapidly Sampling Six Crucial Organs in Adult Xenopus.

Xenopus has been a powerful model organism for understanding vertebrate development and disease for over a hundred years. While experimental analysis and dissection techniques of the embryo have been well documented, descriptions of adult Xenopus structures and organs, together with techniques for working with adults, have not been updated to take into consideration the requirements of such modern approaches as quantitative proteomics and single-cell transcriptomics. The cell-type and gene-centric perspectives require contrasting observations in embryonic stages to those in adult tissues. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location all along the larval to adult transition, most notably during massive metamorphosis remodeling. Establishing robust standards for organ identification and dissection is crucial to ensure datasets resulting from studies performed at different laboratories can be consistent. The present protocol identifies six of the organs in the adult Xenopus, demonstrating methods for dissection and sampling of the heart ventricle, liver, fat body, pancreas, paired kidney, and skin of the adult Xenopus. Depending on the preservation methods, the dissected organs can be used for quantitative proteomics, single cell/nuclei transcriptomics, in situ hybridization, immunohistochemistry, histology, etc. This protocol aims to standardize tissue sampling and facilitate multi-lab investigations of the adult organ systems.

Distribution of XTdrd6/Xtr protein during oogenesis and early development in Xenopus laevis: Zygotic translation begins only in germ cells that have entered the genital ridge.

We previously identified Xenopus tudor domain containing 6/Xenopus tudor repeat (Xtdrd6/Xtr), which was exclusively expressed in the germ cells of adult Xenopus laevis. Western blot analysis showed that the XTdrd6/Xtr protein was translated in St. I/II oocytes and persisted as a maternal factor until the tailbud stage. XTdrd6/Xtr has been reported to be essential for the translation of maternal mRNA involved in oocyte meiosis. In the present study, we examined the distribution of the XTdrd6/Xtr protein during oogenesis and early development, to predict the time point of its action during development. First, we showed that XTdrd6/Xtr is localized to germinal granules in the germplasm by electron microscopy. XTdrd6/Xtr was found to be localized to the origin of the germplasm, the mitochondrial cloud of St. I oocytes, during oogenesis. Notably, XTdrd6/Xtr was also found to be localized around the nuclear membrane of St. I oocytes. This suggests that XTdrd6/Xtr may immediately interact with some mRNAs that emerge from the nucleus and translocate to the mitochondrial cloud. XTdrd6/Xtr was also detected in primordial germ cells and germ cells throughout development. Using transgenic Xenopus expressing XTdrd6/Xtr with a C-terminal FLAG tag produced by homology-directed repair, we found that the zygotic translation of the XTdrd6/Xtr protein began at St. 47/48. As germ cells are surrounded by gonadal somatic cells and are considered to enter a new differentiation stage at this phase, the newly synthesized XTdrd6/Xtr protein may regulate the translation of mRNAs involved in the new steps of germ cell differentiation.

First person – Kazuki Omata

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Biology Open, helping researchers promote themselves alongside their papers. Kazuki Omata is first author on ‘ Isolation and evaluation of erythroid progenitors in the livers of larval, froglet, and adult Xenopus tropicalis’, published in BiO. Kazuki is a PhD student in the lab of Takashi Kato at Waseda University, Japan, investigating the difference in the function and production mechanisms between mammalian enucleated erythrocytes and non-mammalian nucleated erythrocytes.

Isolation and evaluation of erythroid progenitors in the livers of larval, froglet, and adult Xenopus tropicalis.

Xenopus liver maintains erythropoietic activity from the larval to the adult stage. During metamorphosis, thyroid hormone mediates apoptosis of larval-type erythroid progenitors and proliferation of adult-type erythroid progenitors, and a globin switch occurs during this time. In addition, the whole-body mass and the liver also change; however, whether there is a change in the absolute number of erythroid progenitors is unclear. To isolate and evaluate erythroid progenitors in the Xenopus liver, we developed monoclonal ER9 antibodies against the erythropoietin receptor (EPOR) of Xenopus. ER9 recognized erythrocytes, but not white blood cells or thrombocytes. The specificity of ER9 for EPOR manifested as its inhibitory effect on the proliferation of a Xenopus EPOR-expressing cell line. Furthermore, ER9 recognition was consistent with epor gene expression. ER9 staining with Acridine orange (AO) allowed erythrocyte fractionation through fluorescence-activated cell sorting. The ER9+ and AO-red (AOr)high fractions were highly enriched in erythroid progenitors and primarily localized to the liver. The method developed using ER9 and AO was also applied to larvae and froglets with different progenitor populations from adult frogs. The liver to body weight and the number of ER9+ AOrhigh cells per unit body weight were significantly higher in adults than in larvae and froglets, and the number of ER9+ AOrhigh cells per unit liver weight was the highest in froglets. Collectively, our results show increased erythropoiesis in the froglet liver and demonstrate growth-dependent changes in erythropoiesis patterns in specific organs of Xenopus.

Effective Rapid Blood Perfusion in Xenopus.

Xenopus have been powerful model organisms for understanding vertebrate development and disease for over 100 years. Here, a rapid blood perfusion protocol in Xenopus, aimed at a consistent and drastic reduction of blood within all tissues, is defined. Perfusion is carried out by inserting a needle directly into the ventricle of the heart and pumping heparinized phosphate-buffered saline (PBS) through the vascular system. The procedure can be completed in approximately 10 min per animal. The blood is dominated by a few highly abundant proteins and cell types, creating numerous issues as these proteins mask most other molecules and cell types of interest. The reproducible characterization of adult Xenopus tissues with quantitative proteomics and single-cell transcriptomics will benefit from applying this protocol prior to organ sampling. The protocols for tissue sampling are defined in companion papers. These procedures are aimed at the standardization of practices across Xenopus of different sex, age, and health status, specifically X. laevis and X. tropicalis.

Establishment of the body condition score for adult female Xenopus laevis.

The assessment of animals' health and nutritional status using a Body Condition Score (BCS) has become a common and reliable tool in lab-animal science. It enables a simple, semi-objective, and non-invasive assessment (palpation of osteal prominences and subcutaneous fat tissue) in routine examination of an animal. In mammals, the BCS classification contains 5 levels: A low score describes a poor nutritional condition (BCS 1-2). A BCS of 3 to 4 is considered optimum, whereas a high score (BCS = 5) is associated with obesity. While BCS are published for most common laboratory mammals, these assessment criteria are not directly applicable to clawed frogs (Xenopus laevis) due to their intracoelomic fat body instead of subcutaneous fat tissue. Therefore, this assessment tool is still missing for Xenopus laevis. The present study aimed to establish a species-specific BCS for clawed frogs in terms of housing refinement in lab-animal facilities. Accordingly, 62 adult female Xenopus laevis were weighed and sized. Further, the body contour was defined, classified, and assigned to BCS groups. A BCS 5 was associated with a mean body weight of 193.3 g (± 27.6 g), whereas a BCS 4 ranged at 163.1 g (±16.0 g). Animals with a BCS = 3 had an average body weight of 114.7 g (±16.7 g). A BCS = 2 was determined in 3 animals (103 g, 110 g, and 111 g). One animal had a BCS = 1 (83 g), equivalent to a humane endpoint. In conclusion, individual examination using the presented visual BCS provides a quick and easy assessment of the nutritional status and overall health of adult female Xenopus laevis. Due to their ectothermic nature and the associated special metabolic situation, it can be assumed that a BCS ≥3 is to be preferred for female Xenopus laevis. In addition, BCS assessment may indicate underlying subclinical health problems that require further diagnostic investigation.

Na+-dependent intestinal glucose absorption mechanisms and its luminal Na+ homeostasis across metamorphosis from tadpoles to frogs.

The abrupt morphological changes of the intestine during metamorphosis have been detailed in frogs. The features of intestinal metamorphosis are shortening of the intestine and remodeling of the intestinal epithelium. It is believed that the purpose of the morphological changes of the intestine is adaptation from aquatic herbivorous to carnivorous life. However, little is known about the physiological importance of these morphological changes. To elucidate the functional changes during metamorphosis, we measured luminal Na+ concentrations and Na+-dependent glucose uptake in tadpoles and adult African clawed frogs Xenopus laevis. The small intestine was isolated and divided into four segments in length, the luminal contents collected for analysis of ion concentration by ion chromatography. Phlorizin-sensitive glucose-induced short-circuit current (ΔIsc) was measured in intestinal preparations mounted in Ussing chambers. Although dietary sodium intake was extremely low in tadpoles, luminal Na+ concentration gradually increased along the proximal to the middle part of the intestine (>70 mM), and this Na+ concentration was comparable with that of carnivorous adult frogs. The increment of glucose-induced ΔIsc was observed in tadpole intestine. We also measured the ΔIsc induced by acetic acid, which is the major short-chain fatty acid produced by fermentation. The expression levels of mRNA for Na+-dependent glucose transporter 1 and tight junction protein claudin-15 in each intestinal segment was measured. These results suggest that luminal Na+ homeostasis is important and luminal Na+ is kept at a high concentration for Na+-dependent nutrient absorption mechanisms.

Adult male Xenopus laevis can tolerate months of fasting by catabolizing carbohydrates and lipids.

The African clawed frog, Xenopus laevis, has been reported to tolerate long-term fasting without dormancy. However, the strategies for energy acquisition during fasting are unclear in this species. We performed 3- and 7-month fasting experiments to investigate how the metabolism of male X. laevis changes during long-term fasting. We found that the levels of several serum biochemical parameters, such as glucose, triglycerides, and free fatty acids, as well as liver glycogen were reduced after 3months of fasting, whereas after 7months of fasting, triglyceride levels were reduced, and fat body wet weight was lower than that of fed group indicating the onset of lipid catabolism. In addition, transcript levels of gluconeogenic genes, such as pck1, pck2, g6pc1.1, and g6pc1.2, were increased in the livers of animals fasted for 3months, suggesting upregulation of gluconeogenesis. Our results raise the possibility that male X. laevis can tolerate much longer fasting than previously reported by utilizing several energy storage molecules. Further investigation of the effects of prolonged fasting on the metabolic switches from carbohydrates to lipids or amino acids in X. laevis is required.

An Apical Resection Model in the Adult Xenopus tropicalis Heart.

It is known that in adult mammals, the heart has lost its regenerative capacity, making heart failure one of the leading causes of death worldwide. Previous research has demonstrated the regenerative ability of the heart of the adult Xenopus tropicalis, an anuran amphibian with a diploid genome and a close evolutionary relationship with mammals. Additionally, studies have shown that following ventricular apex resection, the heart can regenerate without scarring in X. tropicalis. Consequently, these previous results suggest that X. tropicalis is an appropriate alternative vertebrate model for the study of adult heart regeneration. A surgical model of cardiac regeneration in the adult X. tropicalis is presented herein. Briefly, the frogs were anesthetized and fixed; then, a small incision was made with iridectomy scissors, penetrating the skin and pericardium. Gentle pressure was applied to the ventricle, and the apex of the ventricle was then cut out with scissors. Cardiac injury and regeneration were confirmed by histology at 7-30 days post resection (dpr). This protocol established an apical resection model in adult X. tropicalis, whichcan be employed to elucidate the mechanisms of adult heart regeneration.

Cellular and molecular profiles of larval and adult Xenopus corneal epithelia resolved at the single-cell level

Corneal Epithelial Stem Cells (CESCs) and their proliferative progeny, the Transit Amplifying Cells (TACs), are responsible for homeostasis and maintaining corneal transparency. Owing to our limited knowledge of cell fates and gene activity within the cornea, the search for unique markers to identify and isolate these cells remains crucial for ocular surface reconstruction. We performed single-cell RNA sequencing of corneal cells from larval and adult stages of Xenopus. Our results indicate that as the cornea develops and matures, there is an increase in cellular diversity, which is accompanied by a substantial shift in transcriptional profile, gene regulatory network and cell-cell communication dynamics. Our data also reveals several novel genes expressed in corneal cells and changes in gene expression during corneal differentiation at both developmental time-points. Importantly, we identify specific basal cell clusters in both the larval and adult cornea that comprise a relatively undifferentiated cell type and express distinct stem cell markers, which we propose are the putative larval and adult CESCs, respectively. This study offers a detailed atlas of single-cell transcriptomes in the frog cornea. In the future, this work will be useful to elucidate the function of novel genes in corneal epithelial homeostasis, wound healing and regeneration.

Cell landscape of larval and adult Xenopus laevis at single-cell resolution

The rapid development of high-throughput single-cell RNA sequencing technology offers a good opportunity to dissect cell heterogeneity of animals. A large number of organism-wide single-cell atlases have been constructed for vertebrates such as Homo sapiens, Macaca fascicularis, Mus musculus and Danio rerio. However, an intermediate taxon that links mammals to vertebrates of more ancient origin is still lacking. Here, we construct the first Xenopus cell landscape to date, including larval and adult organs. Common cell lineage-specific transcription factors have been identified in vertebrates, including fish, amphibians and mammals. The comparison of larval and adult erythrocytes identifies stage-specific hemoglobin subtypes, as well as a common type of cluster containing both larval and adult hemoglobin, mainly at NF59. In addition, cell lineages originating from all three layers exhibits both antigen processing and presentation during metamorphosis, indicating a common regulatory mechanism during metamorphosis. Overall, our study provides a large-scale resource for research on Xenopus metamorphosis and adult organs.

Synuclein Analysis in Adult Xenopus laevis.

The α-, β- and γ-synucleins are small soluble proteins expressed in the nervous system of mammals and evolutionary conserved in vertebrates. After being discovered in the cartilaginous fish Torpedo californica, synucleins have been sequenced in all vertebrates, showing differences in the number of genes and splicing isoforms in different taxa. Although α-, β- and γ-synucleins share high homology in the N-terminal sequence, suggesting their evolution from a common ancestor, the three isoforms also differ in molecular characteristics, expression levels and tissue distribution. Moreover, their functions have yet to be fully understood. Great scientific interest on synucleins mainly derives from the involvement of α-synuclein in human neurodegenerative diseases, collectively named synucleinopathies, which involve the accumulation of amyloidogenic α-synuclein inclusions in neurons and glia cells. Studies on synucleinopathies can take advantage of the development of new vertebrate models other than mammals. Moreover, synuclein expression in non-mammalian vertebrates contribute to clarify the physiological role of these proteins in the evolutionary perspective. In this paper, gene expression levels of α-, β- and γ-synucleins have been analysed in the main organs of adult Xenopus laevis by qRT-PCR. Moreover, recombinant α-, β- and γ-synucleins were produced to test the specificity of commercial antibodies against α-synuclein used in Western blot and immunohistochemistry. Finally, the secondary structure of Xenopus synucleins was evaluated by circular dichroism analysis. Results indicate Xenopus as a good model for studying synucleinopathies, and provide a useful background for future studies on synuclein functions and their evolution in vertebrates.

Acute multidrug delivery via a wearable bioreactor facilitates long-term limb regeneration and functional recovery in adult Xenopus laevis.

Limb regeneration is a frontier in biomedical science. Identifying triggers of innate morphogenetic responses in vivo to induce the growth of healthy patterned tissue would address the needs of millions of patients, from diabetics to victims of trauma. Organisms such as Xenopus laevis—whose limited regenerative capacities in adulthood mirror those of humans—are important models with which to test interventions that can restore form and function. Here, we demonstrate long-term (18 months) regrowth, marked tissue repatterning, and functional restoration of an amputated X. laevis hindlimb following a 24-hour exposure to a multidrug, pro-regenerative treatment delivered by a wearable bioreactor. Regenerated tissues composed of skin, bone, vasculature, and nerves significantly exceeded the complexity and sensorimotor capacities of untreated and control animals’ hypomorphic spikes. RNA sequencing of early tissue buds revealed activation of developmental pathways such as Wnt/β-catenin, TGF-β, hedgehog, and Notch. These data demonstrate the successful “kickstarting” of endogenous regenerative pathways in a vertebrate model.

Sublethal toxicity of cadmium chloride (CdCl2) on the growth parameters of Xenopus laevis tadpoles (Daudin, 1802)

Intensive agricultural operations and industrial activities release enormous amount of heavy metals, such as cadmium into aquatic ecosystem. The degree of its toxicity indeed pose an environmental problem. The objective of the present work is to study the impact of sub lethal toxicity of waterborne Cadmium chloride for 8 weeks on the growth parameters of Xenopus laevis tadpoles. The research was carried out in the Fisheries Laboratory, Department of Biology, Ahmadu Bello University (ABU), Zaria, Nigeria. The tadpoles stage of development was gotten through artificial reproduction of adult Xenopus laevis collected from Ahmadu Bello University Reservoir. Some water chemistry such as temperature, pH, electrical conductivity, total dissolved solids and dissolved oxygen were monitored during the study. The tadpoles (Gosner stage 51) were exposed to Cadmium Chloride concentrations of 0.50, 1.00 and 1.50 mg/L derived from already established 96 h LC50, (8.86 mg/L CdCl2) in triplicates. The tadpoles were measured for growth parameters and the number that have completed metamorphosis were counted after an interval of two weeks for a period of eight weeks. Physicochemical parameters of water were within the normal range. The results of growth parameters of Xenopus laevis tadpoles showed that, the values of stages of development, Snout Vent Length (SVL), Hind Limb Length (HLL), Tail Length (TaL), Total Length (ToL), Live Weight Gain (LWG) and the percentage of tadpoles that completed metamorphosis of the exposed group of tadpoles decreased significantly (p

Atrazine-Induced Hepato-Renal Toxicity in Adult Male Xenopus laevis Frogs

Atrazine (ATZ) is an herbicide commonly detected in groundwater. Several studies have focused on its immunological and endocrine effects on adult Xenopus laevis species. However, we investigated the impact of atrazine on the renal and hepatic biochemistry and histomorphology in adult male frogs. Forty adult male frogs were allocated to four treatment groups (control, one ATZ (0.01 µg/L), two ATZ (200 µg/L) and three ATZ (500 µg/L), 10 animals per group, for 90 days. Alanine aminotransferase (ALT) and creatinine levels increased significantly (p < 0.05) in the 200 and 500 μg/L groups but malondialdehyde only in the 500 μg/L group (p < 0.05). Histopathological observations of derangement, hypertrophy, vascular congestion and dilation, infiltration of inflammatory cells incursion, apoptosis and hepatocytes cell death were observed with atrazine exposure, mostly in the 500 μg/L group. Additionally, histochemical labelling of caspase-3 in the sinusoidal endothelium was observed in all the treated groups, indicating vascular compromise. Evaluation of renal histopathology revealed degradation and atrophy of the glomerulus, vacuolization, thick loop of Henle tubule epithelial cells devolution and dilation of the tubular lumen. Furthermore, expression of caspase-3 indicates glomerular and tubular apoptosis in atrazine-exposed animals. These findings infer that environmentally relevant atrazine doses (low or high) could induce hepatotoxicity and nephrotoxicity in adult male Xenopus laevis frogs and potentially related aquatic organisms.

Degenerative Osteoarthropathy in Laboratory Housed Xenopus (Silurana) tropicalis.

In this case study, 15 adult laboratory Xenopus (Silurana) tropicalis (7 adult males and 8 adult females) were examined for nodular enlargements of the clawed digits (digits 0, I, II, and III) on the hind feet. Radiographs showed smoothly margined, rounded, peripherally mineralized lesions arising from the distal phalanges of digits 0-III with osteoproductive and osteolytic components in all frogs. Micro computed tomography (microCT) scans further revealed interphalangeal (IP), metacarpophalangeal (MCP), and metatarsophalangeal (MTP) joint osteoarthritis characterized by periarticular new bone formation, rounded mineral foci both peripherally and centrally within the joints, and more rarely, linear mineralization palmar/plantar to the joints in the flexor tendons. In the nonclawed digits, the shape of the distal phalanx was variably distorted and both subluxation and malangulation of IP joints were identified. Histologically, nodules corresponded to a peripheral rim of mature cortical bone surrounding central adipose tissue, scattered hematopoietic elements, and residual bone of the distal phalanx. Occasionally, the peripheral rim of cortical bone extended proximally to encompass the distal aspect of adjacent phalanx. MCP, MTP and IP joint spaces of most digits exhibited widespread osteoarthritis characterized by periarticular cartilaginous or osseous metaplasia, bony remodeling, and less frequently, granulomatous osteomyelitis. Nutritional analyses of the feed did not indicate imbalances nor were the lesions consistent with metabolic bone disease. The exact etiopathogenesis of these lesions is unknown; however, we hypothesize that the osteoarthritic changes are due to a combination of the frogs' mature age, the unique structure of the Xenopus spp. claw, genetics and biomechanical forces on the digits and distal phalanges of the hind feet.

Cardiac and Cerebellar Histomorphology and Inositol 1,4,5-Trisphosphate (IP3R) Perturbations in Adult Xenopus laevis Following Atrazine Exposure

Despite several reports on the endocrine-disrupting ability of atrazine in amphibian models, few studies have investigated atrazine toxicity in the heart and cerebellum. This study investigated the effect of atrazine on the unique Ca2+ channel-dependent receptor (Inositol 1,4,5-trisphosphate; IP3R) in the heart and the cerebellum of adult male Xenopus laevis and documented the associated histomorphology changes implicated in cardiac and cerebellar function. Sixty adult male African clawed frogs (Xenopus laevis) were exposed to atrazine (0 µg/L (control), 0.01 µg/L, 200 µg/L, and 500 µg/L) for 90 days. Thereafter, heart and cerebellar sections were processed with routine histological stains (heart) or Cresyl violet (brain), and IP3R histochemical localization was carried out on both organs. The histomorphology measurements revealed a significant decrease in the mean percentage area fraction of atrial (0.01 µg/L and 200 µg/L) and ventricular myocytes (200 µg/L) with an increased area fraction of interstitial space, while a significant decrease in Purkinje cells was observed in all atrazine groups (p < 0.008, 0.001, and 0.0001). Cardiac IP3R was successfully localized, and its mean expression was significantly increased (atrium) or decreased (cerebellum) in all atrazine-exposed groups, suggesting that atrazine may adversely impair cerebellar plasticity and optimal functioning of the heart due to possible disturbances of calcium release, and may also induce several associated cardiac and neural pathophysiologies in all atrazine concentrations, especially at 500 µg/L.

Copepod consumption by amphibians and fish with implications for transmission of Dracunculus species.

Parasitic nematodes in the genus Dracunculus have a complex life cycle that requires more than one host species in both aquatic and terrestrial habitats. The most well-studied species, Dracunculus medinensis, is the causative agent of human Guinea worm disease (dracunculiasis). There are several other Dracunculus species that infect non-human animals, primarily wildlife (reptiles and mammals). The classic route of D. medinensis transmission to humans is through the ingestion of water containing the intermediate host, a cyclopoid copepod, infected with third-stage larvae (L3s). However, many animal hosts (e.g., terrestrial snakes, dogs) of other Dracunculus sp. appear unlikely to ingest a large number of copepods while drinking. Therefore, alternative routes of infection (e.g., paratenic or transport hosts) may facilitate Dracunculus transmission to these species. To better understand the role of paratenic and transport hosts in Dracunculus transmission to animal definitive hosts, we compared copepod ingestion rates for aquatic species (fish, frogs [tadpoles and adults], and newts) which may serve as paratenic or transport hosts. We hypothesized that fish would consume more copepods than amphibians. Our findings confirm that African clawed frogs (Xenopus laevis) and fish consume copepods, but that fish ingest, on average, significantly higher numbers (68% [34/50]) than adult African clawed frogs (36% [18/50]) during a 24-h time period. Our results suggest that amphibians and fish may play a role in the transmission of Dracunculus to definitive hosts. Still, additional research is required to determine whether, in the wild, fish or frogs are serving as paratenic or transport hosts. If so, they may facilitate Dracunculus transmission. However, if these animals simply act as dead-end hosts or as means of copepod population control, they may decrease Dracunculus transmission.

Microvascular anatomy of the non-lobulated liver of adult Xenopus laevis: A scanning electron microscopic study of vascular casts.

The microvascular anatomy of the non‐lobulated liver of adult Xenopus laevis was studied by scanning electron microscopy of vascular corrosion casts. Hepatic portal veins and hepatic arteries entered hepatic lobes at the hiluses, hepatic veins left at these sites. Intraparenchymal, hepatic portal veins branched up to 10 times before terminal portal venules supplied liver sinusoids. Hepatic arteries closely followed portal vessels. Arteriolar side branches formed anastomoses with close by portal venules (arteriolar‐portal anastomoses; APAs), liver sinusoids (arteriolar‐sinusoidal anastomoses; ASAs), and peribiliary plexus vessels. Distally, hepatic arteries anastomosed with terminal portal venules having >100 μm in diameter. Liver sinusoids formed a dense three‐dimensional network displaying signs of non‐sprouting and sprouting angiogenesis evidenced by “holes” and blind ending tapering cast vascular structures (sprouts), respectively. Sinusoids drained via efferent hepatic veins. Right and left hepatic veins drained into the posterior caval vein. Locally, a dense honeycomb‐like 3D‐meshwork of resin structures was found around terminal portal venules and hepatic arteries. These networks were fed by hepatic arterioles and drained into adjacent terminal portal venules. As their morphologies differed significantly from sinusoids and they were found at sites where diffuse lymphoid tissue is described, we are convinced that they represent the vasculature of diffuse lymphoid tissue areas. Frequencies and diameter ratios of hepatic portal venules versus hepatic arterioles anastomosing with the former (APAs) implicate that the arterial supply contributes to the oxygenation of parenchymal and stromal cells rather than to a significant increase in blood flow towards hepatic sinusoids.