The life history and biology of Diceratocephala boschmai (Platyhelminthes; Temnocephalida), an ectosymbiont on the redclaw crayfish Cherax quadricarinatus

Adults of the temnocephalan Diceratocephala boschmai, an ectocommensal on the crayfish Cherax quadricarinatus, deposited eggs on the host carapace. After 15 to 20 days at 28 °C, the eggs hatched into ciliated miniature adults with undeveloped reproductive organs, that remained attached to the host alongside the eggs. They grew and became gravid within 53 to 70 days if they were not dislodged. Most oviposited on the host on which they had hatched. Although larval temnocephalans have been described from in vitro work, this is the first temnocephalan life history to be described in vivo.Juvenile worms frequently changed their position on the carapace. Adult worms were sedentary just prior to, and during oviposition, and were frequently found at the base of the large chelae, the ventral surface of the antennae and antennules, and at the base of the walking legs. The ventral margins of the abdomen, the mouthparts, and the interorbital-rostral area were secondarily inhabited.It was calculated using an indirect regression that adult Diceratocephala boschmai survived on average 91 days when removed from the host but they did not produce eggs in vitro. On the host, adult worms lived an average of 48 days after the commencement of oviposition.Abbreviations C cilia - E eyespot - Es ejaculatory sac - Ex excretory canal - G gut - Gl glands - I intertentacular flange - M muscular peduncle - O ovary - P penis - Pg pharyngeal glands - Ph pharynx - R rhabdites - S seminal vesicle - T tentacles - Te testes - U uterus - Vd vas deferens - Vit vitelline glands     

体外培养日本血吸虫成虫生殖器官超微结构的观察

将日本血吸虫成虫于851培养基中培养23天后,对其生殖器官进行透射电镜观察。观察结果显示,雌虫卵巢内卵母细胞出现不同程度的变性、坏死;成熟卵黄细胞的卵黄小滴融合,脂质小滴数量增多、体积增大;培养后期卵壳形成发生障碍,最终导致无活性、无卵壳的畸形卵形成。超微结构观察首次显示,体外初产期虫卵卵壳中有条带状低电子密度区和高电子密度区相间排列。     

免疫荧光法显示贝居腹盾吸虫肌肉神经组织的实验研究

从背角无齿蚌(Anodonta woodiana)围心腔内检获自然感染的贝居腹盾吸虫(Aspidogaster conchicola),根据虫体内睾丸、卵巢、子宫及卵黄腺4种生殖器官的发育程度,将所获虫体分为幼虫组和成虫组,所有虫体用4%甲醛固定后,分别采用鬼笔环肽染色法和乙酰化微管蛋白免疫组织化学法显示贝居腹盾吸虫成虫和幼虫的肌肉纤维和神经纤维,使用激光共聚焦显微镜观察。结果显示,鬼笔环肽荧光染色的贝居腹盾吸虫成虫较幼虫肌肉组织更为发达,口周围、盾盘附着器、子宫末段及阴茎囊肌肉纤维分布较为密集。乙酰化微管蛋白荧光染色的贝居腹盾吸虫成虫较幼虫神经网络更为复杂,在部分生殖器官如子宫末段、阴茎囊和睾丸部位存在神经纤维。双重染色结果显示,该吸虫不同器官上的肌肉纤维分布与神经纤维的分布相互吻合。该结果提示,贝居腹盾吸虫可能通过神经系统对其肌肉活动进行调控。     

Growth and development of Gymnophalloides seoi in immunocompetent and immunosuppressed C3H/HeN mice

The growth and development of Gymnophalloides seoi were studied in C3H/HeN mice and effects of immunosuppression of the host on the worm development were observed. Two hundred metacercariae of G. seoi were orally administered to each mouse, and worms were recovered on days 1, 3, 5, 7, 14 and 21 post-infection (PI). The worm recovery rate was significantly higher in immunosuppressed (ImSP) mice than in immunocompetent (ImCT) mice except on days 1 and 3 PI. The worms attained sexual maturity by day 3 PI with eggs in the uterus, and worm dimensions and the number of uterine eggs continuously increased until day 14 PI in ImSP mice. Worms recovered from ImSP mice were significantly larger in size than those from ImCT mice on days 1 and 3 PI, and the number of uterine eggs was significantly larger in ImSP mice on days 5 and 7 PI. Genital organs such as the ovary, testes, and vitellaria, that were already developed in the metacercarial stage, grew a little in size until day 14 PI. The results show that the C3H/HeN mouse is, though not excellent, a suitable laboratory host for G. seoi.     

Histochemical studies on Raillietina (Raillietina) johri (Cestoda: Davaineidae). I. Nonspecific and specific phosphatases.

Certain phosphatases have been localized by histochemical techniques in various tissues of a pigeon cestode, Raillietina (Raillietina) johri. Acid phosphatase (AcPase), alkaline phosphatase (AlPase) and adenosine triphosphatase (ATPase) were present in almost all structures: tegument; subtegumental muscles; subtegumental cells; excretory canal; testes; sperm ductules; vas deferens; cirrus sac; cirrus; ovary; receptaculum seminis; vagina; vitelline gland cells; oocytes; uterus; embryonated eggs. AlPase was absent in parenchyma, spermatocytes, spermatids and spermatozoa. AlPase activity was more intense in the tegument of mature gravid proglottides. AcPase and ATPase were visualized in various stages of spermatogenesis of the parasite. ATPase activity was also observed in chromosomes. 5'-nucleotidase (AMPase) activity was restricted to embryonated eggs only. Functional significance of these phosphatases is discussed.     

Cardicola opisthorchis n. sp. (Trematoda: Aporocotylidae) from the Pacific bluefin tuna, Thunnus orientalis (Temminck & Schlegel, 1844), cultured in Japan

A new aporocotylid blood fluke is described, based on specimens from the ventricle of the Pacific bluefin tuna, Thunnus orientalis (Temminck et Schlegel), cultured in Wakayama and Nagasaki Prefectures, Japan. The new species is morphologically similar to the members of the genus Cardicola Short, 1953, but shows distinct differences in the body form, location of the testis and the orientation of the ootype. The body of the new species is long and slender, whereas other Cardicola species are small and generally lanceolate. The testis is mostly located posterior to the caeca and anterior to the ovary, occupying 31–45% of body length, in contrast to the known Cardicola species, whose testis is typically intercaecal. The ootype is oriented anteriorly, while in most congeners, it is directed posteriorly or horizontally. Phylogenetic analyses of this aporocotylid, together with Cardicola orientalis Ogawa, Tanaka, Sugihara et Takami, 2010 from the same host, were conducted based on DNA sequences of the ITS2 rDNA and the 28S region of ribosomal RNA. The analyses revealed that the new blood fluke belongs to the genus Cardicola despite the marked morphological differences. Thus, this aporocotylid is named Cardicola opisthorchis n. sp. and the generic diagnosis is emended in this paper. In addition, 100% identity among the ITS2 sequences from the present species, Cardicola sp. from T. orientalis in Mexico and Cardicola sp. from the northern bluefin tuna, Thunnus thynnus (Linnaeus) in Spain suggests that C. opisthorchis n. sp. has a broad geographical distribution and that it infects both the Pacific and northern bluefin tuna.     

Reproductive potential of Echinococcus multilocularis in experimentally infected foxes, dogs, raccoon dogs and cats

A total of 15 red foxes, 15 raccoon dogs, 15 domestic dogs and 15 domestic cats were each infected with 20,000 protoscolices of Echinococcus multilocularis. At 35, 63, and 90 days post inoculation (dpi), five animals from each group were necropsied and the worm burdens determined. The highest worm burdens in foxes (mean of 16,792) and raccoon dogs (mean of 7930) were found at 35 dpi. These declined to a mean of just 331 worms in foxes and 3213 worms in raccoon dogs by day 63 with a further decline to 134 worms in foxes and 67 worms in raccoon dogs by day 90. In dogs, there was no significant difference between worm burdens recovered at days 35 (mean of 2466) and day 90 (mean of 1563), although reduced numbers were recovered on day 63 (mean of 899). In cats, worms were found in four animals 35 dpi (mean of 642), in three at 63 dpi (mean of 28) and in two at 90 dpi (mean of 57). Faecal egg counts were determined at 3 day intervals from 25 dpi. A mathematical model of egg excretion dynamics suggested that the mean biotic potential per infected animal was high in foxes (346,473 eggs); raccoon dogs (335,361 eggs) and dogs (279,910 eggs) but very low for cats (573 eggs). It also indicated that approximately 114, 42 and 27 eggs per worm were excreted in the faeces of dogs, raccoon dogs and foxes, respectively. The fecundity of worms in cats was low with an average of less than one egg per worm. The peak levels of coproantigen were detected earlier in foxes and raccoon dogs than in dogs. Eggs recovered from foxes, raccoon dogs and dogs resulted in massive infections in experimental mice. However, metacestodes did not develop from eggs originating from infected cats. It is concluded that foxes, raccoon dogs and dogs are good hosts of E. multilocularis. In contrast, the low worm establishment, the very few excreted eggs and the lack of infectivity of eggs strongly indicate that cats play an insignificant role in parasite transmission.     

In vitro excystation of Echinostoma paraensei (Digenea: Echinostomatidae) metacercariae assessed by light microscopy,morphometry and confocal laser scanning microscopy

Trypsin and bile salts have been identified as important triggers for excystation of Echinostoma metacercariae. Although excystation in trematodes is a well-known phenomenon, some morphological developmental changes remain to be elucidated. In order to gain further insight into the in vitro development of metacercariae, we assayed different cultivating conditions: 0.5% trypsin and 0.5% bile salts; 1% trypsin and 1% bile salts; 1% trypsin and 0.5% bile salts; 0.5% bile salts; or 0.5% trypsin. By means of light microscopy and confocal microscopy, we characterized each encysted, activated, breached and excysted stage based on the morphological features. However, breached and excysted stages were not revealed in both bile salts and trypsin-free medium. Excretory concretions (25 ± 3.9) were visualized within excretory tubules, close to the ventral sucker and genital anlage. The oral sucker armed with spines and digestive system was similar to those of adult worms. The reproductive system is composed of a genital anlage and the cirrus sac primordium. In short, trypsin and bile salts associated were fundamental for the in vitro metacercariae excystation of Echinostoma paraensei. This article presents the first detailed information of all stages of metacercariae excystation obtained through light and confocal microscopy.     

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.     

Changes in the reproductive biology of Biomphalara glabrata infected with different doses of Echinostoma paraensei miracidia

The egg-laying rate, number of egg masses, number of eggs/mass, number of eggs hatched/snail and egg viability of Biomphalaria glabrata exposed to different doses (5 and 50) of Echinostoma paraensei miracidia were analyzed as indicators of reproductive activity. Polystyrene plates were placed in aquariums containing the snails and every other day for four weeks after infection the plates were removed to count the number of egg masses and eggs laid. After this, the plates were numbered individually and placed in new aquariums free of snails and the egg masses were observed daily to determine the hatching rate. On average there was an increase in the parameters evaluated in the infected snails in relation to the controls (uninfected snails), except for egg viability, which was significantly lower in the groups infected with 50 miracidia. These findings indicate that when infected, this host snail is able to increase its reproductive activity, suggesting an ecological strategy to maintain the species.     

A Human Case of Clinostomum complanatum Infection in Korea

A 33-year-old Korean man visited a medical clinic with complaints of throat discomfort and pain for one week. The symptoms occurred one day after eating raw brackish water fish, perch. Endoscopy revealed a fluke, about 5 mm in length, attaching to and peristaltically moving on the surface of the mucosa at the arytenoid region of the larynx. Microscopically, the testes were triangular, tandem, and separated by the uterus. The ovary and cirrus pouch were placed apart from median line between testes. Numerous blood cells were observed in the ceca. The worm was identified to be Clinostomum complanatum. This is the second human case of clinostomiasis in Korea.     

A new species of Petasiger (Digenea: Echinostomiformes: Echinostomatidae) in the brown pelican, Pelecanus occidentalis, (Aves: Pelecaniformes: Pelecanidae), from the Area de Conservación Guanacaste, Costa Rica

A new species of Petasiger inhabits Pelecanus occidentalis, from the Area de Conservación Guanacaste, Costa Rica. The new species most closely resembles Petasiger novemdecim Lutz 1929 and Petasiger caribbensis Nassi, 1980 by having 19 circumoral spines and vitelline follicles confluent between the ventral sucker and gonads. The new species differs from both of these species by having a cirrus sac that is anteroposteriorly elongate and that reaches posteriorly to the midventral sucker, an ovary and Mehlis' gland that overlap the anterior testis dorsally, a uterus that lies dorsal to the ventral sucker, and a dextromedial genital pore. In P. novemdecim and P. caribbensis the cirrus sac is round and does not extend posteriorly to the anterior margin of the ventral sucker, both the ovary and Mehlis' gland are anterior to the anterior testis, the uterus runs lateral to and not dorsal to the ventral sucker, and the genital pore opens medially and sinistromedially, respectively.     

Reproduction of the blacktip sawtail catshark,Galeus sauteri,in the waters off northeastern Taiwan

The reproductive biology of blacktip sawtail catsharks,Galeus sauteri, in northeastern Taiwan waters was investigated. Male catsharks possessed paired testes producing spermatozoa, which were then stored in the epididymides all year round. No spermatophores were observed in the lower ductus deferens. Only the right ovary was functional in females, oogonia being formed in the ovarian cortex and then developing into mature ova. Ova exceeding 17 mm in diameter were ovulated. Pregnant individuals contained one eggcase only, in each side of the uterus. No hatched embryos were observed in such eggcases. The size at which 50% of blacktip sawtail catshark specimens were mature was 410–420 mm and 350–360 mm for females and males, respectively. The species does not have a well-defined reproductive season.     

Structure of Reproductive System of the Amphicotyllide Cestode Eubothrium rugosum (Cestoda, Pseudophyllidae)

A tendency for increase both of numerical and of ecological factors of infestation in pseudophyllid cestodes is realized due to morphological peculiarities of the structure of their reproductive system. In the species E. rugosum with the closed bursiform uterus and intrauterine development of eggs, there are revealed the same degree of maturity of oocytes in ovary, the lack of sphincter or filtering cell at the base of the ovicapt infundibulum, and the presence of a large vitelline reservoir. By analogy with trematodes, an interconnection is traced between the degree of egg maturation in the uterus and morphology of uterus and vittelicles. The thickening of the egg membrane has been shown to occur not only by deposits of individual vitelline globules at the internal superficial egg layer, but also by binding of lipid droplets excreted from the surrounding parenchyma to the uterine tube along its entire length to the external egg surface. The male reproductive system of Eubothrium rugosum is characterized by the presence of a small cirrus pouch, by the cirrus supplied with microtrichia, which corresponds to the similar vagina structure, and by localization of prostate glands outside the cirrus pouch. Comparison is presented with other ultrastructural peculiarities of organization of the reproductive system in the earlier studied species of pseudophyllid cestodes, Diphyllobothrium latum.     

Neohaematotrephus arayae n. sp. (Digenea: Echinostomiformes: Cyclocoelidae) in Jacana spinosa (Aves: Charadriiformes: Jacanidae) from the Area de Conservación Guanacaste, Costa Rica

Specimens of a species of cyclocoelid digenean inhabiting Jacana spinosa from the Area de Conservación Guanacaste, Costa Rica, most closely resemble Haematotrephus facioi (Brenes and Arroyo, 1962) Yamaguti, 1971, in the same host from Aranjuez, Puntarenas Province, Costa Rica, in having confluent vitelline follicles posteriorly, diagnostic of Neohaematotrophus, and in pharynx length, ovary width, and cirrus sac on the sinistral side. The new species is also highly similar in appearance to H. gendrei Dubois, 1959, also inhabiting a jacanid (from West Africa), which has vitelline follicles confluent posteriorly, and extending anteriorly to the intestinal bifurcation and genital pore opening immediately posterior to the anterior margin of the pharynx. Like H. facioi, H. gendrei has a relatively much shorter and broader cirrus sac than does the new species. Examination of the holotype and paratype of H. facioi confirmed that the specimens from Guanacaste differ in having a longer body, a larger ovary and eggs, and smaller testes. They also have the ovary on the sinistral rather than the dextral side of the body, genital pore anterior to the pharynx rather than at or posterior to the level of the posterior margin of the pharynx, longer and thinner cirrus sac, and eggs without eyespotted miracidia. Half the eggs in both specimens of H. facioi have well-developed eyespotted miracidia, whereas the typical condition for cyclocoelids is for virtually all eggs to exhibit eyespotted miracidia. Both H. facioi and H. gendrei are transferred to Neohaematotrophus, along with the new species.     

Developmental and age-related changes in proteins in the female reproductive tract of Heligmosomoides polygyrus (Nematoda)

Proteins in the female reproductive tract of Heligmosomoides polygyrus at days 8, 16, 35, 90, and 140 postinfection (PI) were examined using polyacrylamide gel electrophoresis. Sixteen-day-old and 140-day-old worms also were examined histochemically. Egg production of these worms was assessed for each age group. In analyzing proteins using electrophoresis, the reproductive tracts were separated into 3 sections: the tip, or anteriormost part of tract, containing oogonia; the middle region, containing developing oocytes; and the posterior region, containing the uterus with fertilized eggs. Three major reproductive tract proteins were identified as having molecular weights of greater than 140 kDa, 115 kDa, and 82 kDa. These were found in all parts of the reproductive tract from worms of all ages except those at 8 days PI (which are too young to produce eggs) and are believed to be yolk proteins. Four low molecular weight proteins (L1-4) are believed to be nucleoproteins. L4 was absent from the posterior section of the reproductive tracts and L3 was limited to the posterior sections and may be associated with sperm stored in the uterus. Of 5 high molecular weight proteins the second heaviest, designated H2, appeared to be relatively more concentrated in the posterior sections of the reproductive tract. An 85-kDa protein was limited to the tip and middle sections of reproductive tracts. Histochemical tests on sectioned H. polygyrus showed strong positive reactions for protein in cytoplasmic granules in developing oocytes and in eggs of younger worms (16 days) but a reduced reaction in older worms (140 days). Strains for collagen showed a slight positive reaction in and between developing oocytes and a strong reaction in the egg shells. Stains for nucleoproteins particularly reacted with sperm stored in the uterus, and slightly reacted with fertilized eggs and the nucleoli of the intestinal and ovarian epithelium. Egg production by H. polygyrus increased to 123 eggs/female/day by 16 days PI but declined from 121 eggs/female/day at 35 days PI to 64 eggs/female/day in worms 140 days old. Electrophoresis indicated no loss in the different types of proteins in the reproductive tract of older worms, but histochemistry and protein content assays suggest that older worms that produce fewer eggs contain a relatively smaller amount of protein in the female reproductive tract.     

濒危植物马蹄参的大孢子发生和雌配子体发育

为探讨马蹄参(Diplopanax stachyanthus Hand.-Mazz.)濒危机制与雌性生殖发育的关系,采用石蜡切片法观察马蹄参大孢子形成和雌配子体的发育过程。结果表明,马蹄参雌蕊单心皮,子房下位,1室,1枚胚珠。胚珠横生于短片状胎座上,具单珠被,厚珠心。单孢原细胞自珠心1~2层表皮细胞处分化;大孢子四分体为直线形。成熟胚囊中,2个极核在受精前融合为次生核,3个反足细胞不发达,较早退化;二核胚囊时期出现二核分裂不均且较小核退化消失的异常发育现象。因此,马蹄参雌配子体发育过程中出现异常现象是造成其结实率低的主要原因。     

Galaxitaenia toloi n. gen., n. sp. (Eucestoda: Pseudophyllidea) from Galaxias platei (Pisces: Osmeriformes, Galaxiidae), in the Patagonian region of Argentina

Galaxitaenia n. gen. (Eucestoda: Pseudophyllidea) is proposed to accommodate Galaxitaenia toloi n. gen., n. sp. from Galaxias platei, a freshwater fish inhabiting Andean lakes in Argentine Patagonia. Galaxitaenia belongs to the Triaenophoridae because it possesses a marginal genital pore, a ventral uterine pore, and a follicular vitelline gland. The new genus can be distinguished from other triaenophorids by the following combination of characters: a scolex with a prominent rectangular apical disc without hooks, grooves, or indentations; neck present; segments wider than long to quadrangular; testes in 2 lateral fields, often connected anteriorly; cirrus unarmed; an ovary situated posteriorly; a vagina posterior to the cirrus sac; vitelline follicles medullary, in 2 ventral fields forming 2 wings interrupted medially; a uterus saccate to branched; and eggs without operculum. The types, distribution, and density of microtriches were analyzed on the surface of the tegument of scolex, neck, and immature segments. The presence of tumuli were observed on the apical disc of the scolex. Galaxitaenia toloi n. sp. is the first triaenophorid species recorded in a South American freshwater fish and the fourth helminth parasitizing the intestine of G. platei.     

Development in vitro of the neuromusculature of two strigeid trematodes,Apatemon cobitidis proterorhini and Cotylurus erraticus

Confocal microscopy interfaced with cytochemical procedures has been used to monitor development of the major muscle systems and associated serotoninergic (5-HT, 5-hydroxytryptamine) and peptidergic (FaRP, FMRFamide-related peptide) innervation of the strigeid trematodes, Apatemon cobitidis proterorhini and Cotylurus erraticus during cultivation in vitro. Sexually undifferentiated metacercariae were successfully grown to ovigerous adults using tissue culture medium NCTC 135, chicken serum and egg albumen. Eggs were produced after 5 days in culture but had abnormal shells and failed to embryonate. 5-HT and FaRP (the flatworm FaRP, GYIRFamide) were localised immunocytochemically in both central and peripheral nervous systems of developing worms. During cultivation, the central serotoninergic and FaRPergic neuronal pathways of the forebody became more extensive, but retained the same basic orthogonal arrangement as found in the excysted metacercaria. Longitudinal extensor and flexor muscles of the hindbody provide support for the developing reproductive complex. The male reproductive tracts were established in advance (day 3) of those of the female system (day 4); completion of the latter was marked by the appearance of the ootype/egg chamber. The inner longitudinal muscle fibres of the female tract appeared prior to the outer and more densely arranged circular muscles. Circular fibres dominate the muscle complement of both alimentary and reproductive tracts. 5-HT- and GYIRFamide-immunoreactivities were demonstrable in the central nervous system (CNS) and subtegumental parasympathetic nervous system (PNS) throughout the culture period, but innervation of the developing reproductive structures was reactive just for 5-HT. Only at the onset of egg production was FaRP-IR observed in the reproductive system and was expressed only in the innervation of the ootype, a finding consistent with the view that FaRPs may regulate egg assembly in platyhelminths.     

Cytopathological process by multiple nucleopolyhedrovirus in the testis of Bombyx mori L., 1758 (Lepidoptera: Bombycidae)

A cytopathological methodology was used to analyze infection by Bombyx mori multiple nucleopolyhedrovirus (BmMNPV), a geographic isolate of the family Baculoviridae, in the caterpillar testes of the B. mori. Japanese B. mori strain caterpillar, fifth instar, were inoculated with BmMNPV and their testes were collected and processed for light and transmission electronic microscopy. Epithelial coating cells and interfollicular septa in testes were susceptible to BmMNPV. The first evidence of infection was detected on the 6th day post-inoculation (p.i.) in the external epithelium, and on the 7th day p.i. in the internal epithelium and interfollicular septa. Cytopathological characteristics consisted of hypertrophied nuclei, the formation of virogenic stroma, and the occlusion of virions in polyhedron protein crystals in several stages of development. At the end of the infectious process, cell lysis and release of polyhedra into the extracellular medium occurred. Histopathology revealed early infection foci in the surrounding regions of tracheal insertions, thus underlining the role of the trachea as an infection-spreading organ in insects. This spreading occurs through penetration of the basal lamina, which facilitates entry of the budded virus into the testis. Additionally, an alignment of a partial sequence of the ORF 14 of the BmMNPV geographic isolate with other NPV certified the virus genera.