Patterns in the morphological variation of Zonorchis and Skrjabinus (Trematoda: Dicrocoelidae) from small mammals in Malaysia.

A collection of dicroceolid flukes from rats, squirrels and Tupaia in Malaysia is found to be morphologically heterogeneous. Cluster analysis on the basis of three selected body ratios distinguishes two major types comprising highly diverse Skrjabinus from rats, and homogenous Zonorchis from callosciurid squirrels. Tupaia harbours flukes of both genera. Analysis of variance of flukes from rats, demonstrates marked uniformity among worms within individual hosts. Possible effects of allometric growth are investigated by plotting each chosen dimension against body length for the two major groups of worms. Prominent changes are noted only in the relative size of testis in Skrjabinus from rats. Patterns of morphology are discussed in relation to host ecology and distribution.     

Concurrent Capillaria and Heterakis Infections in Zoo Rock Partridges, Alectoris graeca

Two adult rock partridges raised in a city zoo were examined parasitologically and pathologically. Two distinctive eggs resembling those of Capillaria and Heterakis were detected in the feces. At necropsy, a markedly-dilated duodenum with severe catarrhal exudates, containing adult worms of Capillaria sp. and Heterakis sp. in the cecum, was observed. Male Capillaria had the cloacal aperture extended almost terminally with a small bursal lobe and an unsheathed spicule with transverse folds without spines. Female Capillaria had a vulva that was slightly prominent and slightly posterior to the union of the esophagus and intestine. The esophagus of the adult Capillaria was more than a half as long as the body in the male, but was much shorter in the female. Based on these morphological features, the capillarid nematode was identified as Capillaria obsignata. The male adult worms of Heterakis was identifiable by 2 dissimilar spicules, a unique morphological feature where the right spicule was considerably longer than the left, which is also a characteristic feature of Heterakis gallinarum. This is the first report of concurrent infections with C. obsignata and H. gallinarium in rock partridges.     

Male Courtship Pheromones Induce Cloacal Gaping in Female Newts (Salamandridae)

Pheromones are an important component of sexual communication in courting salamanders, but the number of species in which their use has been demonstrated with behavioral evidence remains limited. Here we developed a behavioral assay for demonstrating courtship pheromone use in the aquatically courting Iberian ribbed newt Pleurodeles waltl. By performing an in-depth study of the courtship behavior, we show that females invariably open their cloaca (cloacal gaping) before engaging in pinwheel behavior, the circling movement that is the prelude to spermatophore uptake. In contrast, cloacal gaping was not observed in failed courtships, where females escaped or displayed thanatosis. Since gaping mainly occurred during male amplexus and cloacal imposition, which is the obvious period of pheromone transfer, we next investigated whether male courtship water (i.e., water holding courtship pheromones) alone was able to induce this reaction in females. These tests showed that courtship water induced cloacal gaping significantly more than water, even in the absence of a male. Cloacal gaping thus provides a simple and robust test for demonstrating courtship pheromone use in the Iberian ribbed newt. Since opening the cloaca is an essential prerequisite for spermatophore pick-up in all internally fertilizing salamanders, we hypothesize that variations on this assay will also be useful in several other species.     

The normal development of the anorectum in the pig

The development of the anorectum was studied in forty-four embryos and foetuses of pig varying in length from 9 mm total length to 210 mm crownrump length and in three newborn pigs. The presence of some features during critical stages in the development of the cloaca in the pig such as an epithelial mass protruding into the dorsal cloaca near its intestinal orifice and distinct differences in the type of epithelium in different regions of the cloacal system greatly facilitated the study of the developmental process. Thus it could be established that a change in position of the dorsal cloaca and adjacent structures such as the distal part of the gut and the urorectal septum via the dorsal part of the cloacal plate towards the tailgroove is of major importance for the partition of the cloaca into a separate intestinal and urogenital division. A subsequent disintegration of the dorsal part of the cloacal plate results in two separate openings for both the systems at the same time. Disintegration of the ventral part of the cloacal plate leads only to a further widening of the external opening of the urogenital system. In the cloacal system three distinct zones were discerned, a dorsal and ventral cloacal and a cloacal plate region. From the dorsal cloacal epithelium the whole anorectal segment between the intestinal mucosa and the anal epidermis develops. The epithelium of ventral cloacal origin seems to disappear completely . Cloacal plate epithelium forms the epithelial lining of distal parts of the urogenital system. The penile urethra in the male is formed by a ventralward movement of the urogenital opening by the growing perineum and not by fusion of genital folds.     

Transport of sperm within the cloaca of the female red-spotted newt

The transport of sperm in the cloaca and adjacent regions of the female red-spotted newt was examined. It was found that within 1 min after sperm were introduced into the vent, they progressed in a random pattern past the apertures of the spermatheca (the glandular, sperm storage organ that opens from the anterior roof of the cloaca) forward to the anterior end of the cloaca and on into the posterior regions of the hindgut and bladder. Sperm did not enter the dorsal recess of the cloaca into which the oviducts and ureters open. After 1 day, few sperm remained within the cloaca lumen. Sperm were not transported into the cloacae of artificially inseminated, anesthetized females without prior administration of norepinephrine to their cloacal mounds. Treatment of the cloacal mounds of naturally inseminated females with an antagonist of neuromuscular transmission (lidocaine) decreased the numbers of sperm in the anterior cloaca relative to those of saline-injected control specimens. Neither dead newt sperm nor live rabbit sperm entered the spermatheca. Rabbit sperm, however, entered the oviduct. It is argued that passive and active mechanisms of sperm transport work in concert. Contractions of smooth muscle, which may be initiated during courtship, probably serve to draw sperm passively into the cloaca and up to and beyond the apertures of spermathecal tubules, but sperm, once in the vicinity of those apertures, probably swim actively into them.     

Comparative anatomy and phylogeny of the cloacae of salamanders (Amphibia: Caudata). II. Cryptobranchidae,Hynobiidae, and Sirenidae

Cloacae were examined from salamanders representing the three families in which fertilization of eggs is known or inferred to occur externally. The cloacae of male and female sirenids are aglandular and lack cilia. Sexual dimorphism in sirenid cloacae occurs only in the extent of epithelial stratification in the cloacal chamber in females (entire chamber) versus males (posterior angle of the vent). Both male and female Cryptobranchus alleganiensis possess ventral glands that secrete an acid mucopolysaccharide and have ciliated cloacal linings. The ventral glands are more numerous and hypertrophied in breeding male than female C. alleganiensis, but in males, ventral glands secrete only onto the surface of the cloacal lips along the anterior three-fifths of the cloacal orifice, whereas in females, the glands secrete onto the border of the entire cloacal orifice. Except for male Onychodactylus japonicus, male and female hynobiids also possess only ventral glands and have ciliated cloacal linings. Hynobiid ventral glands secrete a glycoprotein. Much variation occurs, however, among these hynobiids in cloacal conformation, extent of epidermis into the cloaca, and anatomy of the ventral gland. Male O. japonicus possess an unciliated cloaca in which three types of cloacal glands occur, each giving unique reactions to tests for carbohydrates and proteins. The glands in male O. japonicus do not seem to be homologous to those found in spermatophore producing salamanders in the Salamandroidea, but this does not negate the possibility that O. japonicus makes spermatophores. Examination of cloacal characters in additional species of hynobiids may be useful in resolving intrafamilial phylogenetic relationships.     

The female cloaca of an oviparous caecilian amphibian (Gymnophiona): functional and seasonal aspects

Kuehnel, S., Herzen, J., Kleinteich, T., Beckmann, F. and Kupfer, A. 2011. The female cloaca of an oviparous caecilian amphibian (Gymnophiona): functional and seasonal aspects. —Acta Zoologica (Stockholm) 00 :1–14. Reproductive morphology is receiving increased attention in animals that have variable reproductive modes combined with internal fertilization. Exceptionally among amphibians all caecilian species practice internal fertilization via an intromittent organ: an everted part of the male cloaca (phallodeum or phallus). Because research has mostly concentrated on males, knowledge of the female cloacal morphology is scarce. Here, we present the first single‐species study of the functional morphology of the female cloaca of an oviparous, phylogenetically basal caecilian (Ichthyophis cf. kohtaoensis). We have analyzed female cloacal shape during the reproductive cycle combining conventional histology with 3D‐reconstruction. All females are similar in their overall cloacal structure with some differences in size and histology associated with the reproductive cycle. The female cloaca is divided into two distinct chambers similar to the male condition. The cranial chamber contains urogenital pockets into which oviducts and Wolffian ducts open and which may have function during oviposition. The caudal cloacal chamber bears a novel feature – dorsolateral blind sacs, which are homologous to the male condition, but are considerably smaller. The study of female cloacal morphology is essential to understanding the evolution of the caecilian reproductive system and contributes to the understanding of tetrapod genital morphology in general.     

A re-examination of the cloacal sacs and gland of the blind snake,Leptotyphlops dulcis (Reptilia: Leptotyphlopidae)

The cloacal sacs of Leptotyphlops dulcis are nonglandular, posterior evaginations of the cloaca. The median cloacal gland is tubuloalveolar. Similar unpaired cloacal glands as well as paired sacs are noted in certain colubrid snakes. Terminology applied to these cloacal derivatives is discussed, and a standardization of names is provided.     

High Prevalence of Haplorchiasis in Nan and Lampang Provinces,Thailand, Proven by Adult Worm Recovery from Suspected Opisthorchiasis Cases

Opisthorchiasis, a risk factor for cholangiocarcinoma in humans, is of public health importance in Thailand. The Annual Surveillance Reports from Nan and Lampang Provinces, Thailand, for the year 2011 showed an opisthorchiasis prevalence of over 70% by recovery of eggs in the feces. This study investigated whether most cases are actually due to minute intestinal flukes (MIF) rather than Opisthorchis viverrini, as the eggs of both can hardly be differentiated by morphology. Fifty and 100 cases from residents in Nan and Lampang, respectively, had stools positive for eggs initially assumed to be those of O. viverrini. Each patient was given praziquantel at 40 mg/kg in a single dose. After 2 hr, 30-45 ml of the purgative magnesium sulfate was given, and stools were collected up to 4 times sequentially. The stools were examined for adult worms by simple sedimentation. It was found that 39 of 50 cases (78.0%) from Nan Province had Haplorchis taichui, with intensities ranging from 5 to 1,250 with an average of 62 worms/case. Taenia saginata (7 cases) and Enterobius vermicularis (1 case) were other helminths recovered as the co-infectants. In Lampang Province, H. taichui was recovered from 69 cases (69.0%). The number of flukes recovered ranged from 1 to 4,277, with an average of 326 worms/case. Four cases had Phaneropsolus bonnei, and 10 T. saginata as the co-infectants. Adult specimens of O. viverrini were not recovered from any stool. Clearly, MIF infection, especially haplorchiasis, is more common in northern Thailand. These findings should encourage the Public Health Office to employ more specific tools than Kato''s method for surveillance of opisthorchiasis in Thailand.     

Effect of artemether,artesunate, OZ78, praziquantel,and tribendimidine alone or in combination chemotherapy on the tegument of Clonorchis sinensis

We investigated the morphological effects of half-strength treatments with praziquantel, artemether, artesunate, OZ78 and tribendimidine as well as combinations of praziquantel with artemether, artesunate, OZ78 and tribendimidine and an artesunate–tribendimidine combination in rats harboring adult Clonorchis sinensis. Rats were infected with C. sinensis, dosed orally with single agents or combination treatments and flukes recovered at 3 or 5 days post-treatment. The number of flukes was counted, the viability recorded and surface changes monitored by scanning electron microscopy. Drug effects induced by the individual drugs at sub-curative doses 3 days post-treatment were minor with the exception of flukes recovered from rats treated with artemether and tribendimidine. Treatment with the praziquantel combinations of artesunate, OZ78 and tribendimidine did not produce a greater disruption of the tegument than the individual drugs 3 days post-treatment. On the other hand, at this time point many worms treated with artemether–praziquantel had died and eruptions, roughening or blebbing were observed on all worms examined. Five days post-treatment flukes exposed to any of the praziquantel combinations in rats had died. Rats treated with an artesunate–tribendimidine combination resulted in a rapid death of flukes, 3 days post-treatment all worms had been expelled.In conclusion, we have confirmed the promising clonorchicidal properties of different drug combinations in rats. Differences in the extent and time-scale of tegumental disruption have been observed. The effect of drug combinations against C. sinensis requires further scientific inquiry, e.g. in transmission electron microscopy studies and in the C. sinensis-rabbit model.     

Studies on the in vitro effects of bithionol and menichlopholan on flukes of Clonorchis sinensis, Metagonimus takahashii and Paragonimus miyazakii.

The present study was carried out in order to obtain information on the action of bithionol and menichlopholan on Clonorchis sinensis, Metagonimus takahashii and Paragonimus miyazakii. The drugs inhibited the motility of the newly excysted juveniles and adult worms in vitro. Moreover, the in vitro treatment slightly decreased lactate dehydrogenase activity in homogenates of adult worms. The drug treatments of adult worms in vitro also inhibited the activity of fumarate reductase, malic enzyme, phosphogluconate dehydrogenase and succinate dehydrogenase in the homogenate. In addition to this, these drugs caused morphological alterations in the ultrastructure of the tegument of the flukes.     

The cloaca of Myxine glutinosa (Cyclostomata): a scanning electron microscopical and histochemical investigation

The cloaca of Myxine glutinosa was examined by histochemical and scanning electron microscopical methods. No copulation organ could be found in Myxine and no detectable differences in the anatomy of the cloaca between male and female Myxine glutinosa. The anal gland which is the only gland in the cloacal region is situated between rectum and ductus coelomaticus. Like the lateral mucous glands in the epidermis it consists of large mucous gland cells, thread cells and undifferentiated cells. The cloacal epithelium neither develops a spatial separation by folds nor a ciliation is present in the caudal and dorsal part of the cloacal chamber. Therefore female and male myxinoides do not show any structures which would allow transportation of sperm into the abdominal cavity or out of it.     

Comparative anatomy and phylogeny of the cloacae of salamanders (Amphibia: Caudata). VI. Ambystomatidae and Dicamptodontidae.

Histology of the cloacae of Rhyacotriton olympicus and representative species from the genera Ambystoma and Dicamptodon was examined by light microscopy. Females of Ambystoma possess sperm storage glands, the spermathecae, as well as ventral glands and dorsal glands, both of uncertain function. Females of Ambystoma examined from the subgenus Linguaelapsus differ from those in the subgenus Ambystoma by possessing more extensive ventral gland clusters and a shorter cloacal tube. Females of Dicamptodon possess spermathecae and ventral glands, but differ in cloacal conformation from females of Ambystoma and lack the dorsal glands. Females of R. olympicus possess more extensive epidermal lining in the cloaca than that found in females of Ambystoma and Dicamptodon, and the only glands present are spermathecae, which cluster around a tube in the dorsal roof. Males of Ambystoma, Dicamptodon, and R. olympicus possess five types of cloacal glands (dorsal pelvic glands, lateral pelvic glands, anterior ventral glands, posterior ventral glands, and Kingsbury's glands) that function in spermatophore formation, and vent glands that may produce a courtship pheromone. In Ambystoma and Dicamptodon, vent glands secrete along the medial borders of the cloacal orifice. Males of A. opacum and A. talpoideum differ from males of other species examined from the two genera by possessing more extensive vent glands. Males of R. olympicus possess unique vent glands in which tubules secrete onto the surface of vent lobes lateral to the posterior end of the cloacal orifice, and distal ends of the glands pass anteriorly, superficial to the fascia enclosing the other cloacal glands. The results from analysis of cloacal anatomy support other data indicating that Ambystoma and Dicamptodon are sister groups, and that Rhyacotriton olympicus is not closely related to either of the other two genera and merits placement in a separate family.     

Histological study of the cloacal region and associated structures in the hedgehog tenrec Echinops telfairi

The present investigation is based on several careful dissections and on extensive series of histological sections. It has led us to the conclusion that adult male and female Echinops telfairi are in the possession of a cloaca which represents a primitive feature among mammals. This cloaca is a small, bowl-shaped pouch at the ventro-posterior end of the body. Intestinal, genital and urinary tract open into this cloaca. The opening of the intestinal tract into the cloaca is regulated by a sphincter muscle. In the female the genital and the urinary tract open into the urogenital sinus, a subcompartment of the cloaca. The cloaca of the lesser hedgehog tenrec is lined by a multilayered, non-keratinized squamous epithelium without skin glands. In a small transitory zone between the cloaca and the outer skin the epithelium changes into the keratinized, multilayered squamous epithelium of the epidermis with eccrine and holocrine glands as well as hairs. In addition, there is a distinct circular cloacal sphincter muscle, built up by cross-striated skeletal muscle tissue.

In the terminal parts of intestinal, urinary and genital tracts of male animals the following glandular structures were observed: prostate gland, Cowper's glands and strongly pigmented seminal vesicles; in female animals: the urethral and the Bartholin glands. Both males and females, in addition, possess (a) a cloacal gland, the excretory ducts of which open into the cloaca and (b) a pericloacal gland which is located in the adipose tissue on both sides of the cloaca; it presumably also opens into the cloaca.     

Acanthoparyphium shinanense n. sp. (Digenea: Echinostomatidae) from Experimental Chicks Infected with Metacercariae Encysted in Brackish Water Clams in the Republic of Korea

Acanthoparyphium shinanense n. sp. (Digenea: Echinostomatidae) is described from chicks experimentally infected with the metacercariae encysted in 2 brackish water clam species, Ruditapes philippinarum and Coecella chinensis, in the Republic of Korea. The metacercariae were round to oval, armed with 23 collar spines, and 0.216 (0.203–0.226) mm in diameter. From 5 chicks experimentally infected each with 200 metacercariae, 34 juvenile (5-day-old worms) and 104 adult flukes (7-day-old worms) were harvested from their small intestines, with the average worm recovery rate of 13.8%. The adult flukes were 3.18 (2.89–3.55) mm long and 0.68 (0.61–0.85) mm wide, with an elongated, posteriorly tapering body, and a prominent head collar armed with 23 collar spines arranged in a single uninterrupted row. The posterior testis of A. shinanense was longitudinally elongated, which is similar to Acanthoparyphium spinulosum Johnston, 1917 but unique from the other closely related species, including Acanthoparyphium tyosenense Yamaguti, 1939, Acanthoparyphium kurogamo Yamaguti, 1939, and Acanthoparyphium marilae Yamaguti, 1934. The eggs of A. shinanense were larger than those of A. spinulosum, and the anterior extent of 2 lateral groups of vitellaria was slightly more limited in A. shinanense than in A. spinulosum. Molecular analysis of nuclear and mitochondrial genes revealed low homology with A. spinulosum from USA (96.1% in 5.8S rRNA) and Ukraine (97.9% in 28S rRNA), Acanthoparyphium n. sp. from USA (98.0% in 28S rRNA), and Acanthoparyphium sp. from Australia, Kuwait, and New Zealand. Biological characteristics, including its first intermediate host and natural definitive hosts, as well as its zoonotic capability, should be elucidated.     

Bmp7 expression and null phenotype in the urogenital system suggest a role in re-organization of the urethral epithelium

Signaling by Bone morphogenetic proteins (Bmps) has multiple and diverse roles in patterning and morphogenesis of the kidney, eye, limbs and the neural tube. Here, we employed the Bmp7^lacZ strain to perform a detailed analysis of Bmp7 expression and the null phenotype during development of the mouse urogenital system. The urethral compartment originates in mid-embryogenesis from the ventral part of the cloaca, a transient cavity at the caudal end of the hindgut. At mid-gestation, Bmp7 expression was detected within several specific domains in the cloacal epithelium and mesenchyme. In late embryogenesis, Bmp7 expression was present in the urethra, rectum, the urethral glands, corpus cavernosum, and in the male and female genital ducts. Importantly, loss of Bmp7 resulted in arrest in cloacal septation, and severe defects in morphogenesis of the genital urethra and mesenchyme. Together, our analysis of Bmp7 expression and the null phenotype, indicates that Bmp7 may play an important role in re-organization of the epithelium during cloacal septation and morphogenesis of the genital tubercle.     

Defining the molecular pathologies in cloaca malformation: similarities between mouse and human

Anorectal malformations are congenital anomalies that form a spectrum of disorders, from the most benign type with excellent functional prognosis, to very complex, such as cloaca malformation in females in which the rectum, vagina and urethra fail to develop separately and instead drain via a single common channel into the perineum. The severity of this phenotype suggests that the defect occurs in the early stages of embryonic development of the organs derived from the cloaca. Owing to the inability to directly investigate human embryonic cloaca development, current research has relied on the use of mouse models of anorectal malformations. However, even studies of mouse embryos lack analysis of the earliest stages of cloaca patterning and morphogenesis. Here we compared human and mouse cloaca development and retrospectively identified that early mis-patterning of the embryonic cloaca might underlie the most severe forms of anorectal malformation in humans. In mouse, we identified that defective sonic hedgehog (Shh) signaling results in early dorsal-ventral epithelial abnormalities prior to the reported defects in septation. This is manifested by the absence of Sox2 and aberrant expression of keratins in the embryonic cloaca of Shh knockout mice. Shh knockout embryos additionally develop a hypervascular stroma, which is defective in BMP signaling. These epithelial and stromal defects persist later, creating an indeterminate epithelium with molecular alterations in the common channel. We then used these animals to perform a broad comparison with patients with mild-to-severe forms of anorectal malformations including cloaca malformation. We found striking parallels with the Shh mouse model, including nearly identical defective molecular identity of the epithelium and surrounding stroma. Our work strongly suggests that early embryonic cloacal epithelial differentiation defects might be the underlying cause of severe forms of anorectal malformations in humans. Moreover, deranged Shh and BMP signaling is correlated with severe anorectal malformations in both mouse and humans.KEY WORDS: Anorectal malformation, Cloaca, Patterning, Epithelial differentiation, Sonic hedgehog     

Development of Schistosoma mansoni in the laboratory rat analyzed by light and confocal laser scanning microscopy

The kinetic of maturation (schistogram) of Schistosoma mansoni worms grown in laboratory rats was studied by light and confocal laser scanning microscopy. Infected rats with the BH strain were weekly euthanized 3-9 weeks pi. Recovered flukes stained with hydrochloric carmine were preserved as whole-mounts and analyzed by confocal and brightfield microscopy. Worms displayed varying degrees of maturation of the reproductive system at weeks 3-6. Male worms showed complete maturation of the reproductive system at week 6, while female worms completed their maturation at week 7. Males presented few tubercles in tegument in all weeks. Despite the presence of a developing embryo within the ootype, no uterine egg was found. The schistogram in rats follows a pattern similar to that observed in mice hosts.     

Geographical distribution of Metagonimus yokogawai and M. miyatai in Shizuoka Prefecture, Japan, and their site preferences in the sweetfish, Plecoglossus altivelis, and hamsters

Sweetfish, Plecoglossus altivelis, obtained from 18 rivers in Shizuoka Prefecture were examined for metacercarial infection of 2 flukes, Metagonimus yokogawai and Metagonimus miyatai. The infection rate and density of metacercariae in the fish were higher in eastern and western regions than in central region of the prefecture. After infection of hamsters with metacercariae derived from the scale, 98.7% of the adult worms obtained from the intestine was found to be M. miyatai. Conversely, from infection with metacercariae from the flesh, 90.0% of the worms was M. yokogawai. Since the worms had no exclusivity in the tissues, we conclude that the flukes have location preference with the former primarily preferring the scale, and the latter the flesh. Fish from two rivers located in adjacent areas in the western region had relatively a higher ratio of M. yokogawai in the scale relative to other rivers, suggesting an intraspecific genetic variation due to geographical isolation. On examination of adult worms in the hamster's intestine, M. yokogawai was mainly located towards the anterior part of the intestine, unlike M. miyatai, suggesting that in mammalian host too, the parasites have site preference.