Brachylaima cribbi (Digenea: Brachylaimidae): scanning electron microscopical observations of the life-cycle stages

Brachylaima cribbi is a recently described species of terrestrial trematode that infects mammals and birds with helicid land snails as its first and second intermediate hosts. The adult worm is 2.5-6.0 mm long by 0.5-0.8 mm wide being a long slender cylindrical worm with oral and ventral suckers in the anterior quarter and genital pore in the posterior quarter. Scanning electron microscopy shows that there is a dense covering of tegumental spines at the anterior end which diminishes towards the posterior extremities of the worm. Development of spines was observed in juvenile and mature adult worms. In young worms 1-3 weeks post infection (wpi) spines appear as buds with a serrated edge each having 1-4 spikes per spine. As the worm ages the spines broaden and by 5 wpi the number of spikes per spine increases to an average of 8.1. The serial development of oral sucker papillae in the cercaria, metacercaria and adult worm was observed with the finding of an elongated papilla with a bifurcated tip on the cercaria becoming a shorter and thicker elongated papilla with a large central stoma on the metacercaria. In the adult worm, this papilla becomes dome-shaped with a small central stoma. For some of these papillae a cilium could be seen extended from the central stoma. Other life-cycle stages illustrated were the hatched egg with an extruded egg membrane minus an operculum and a portion of the branched sporocyst dissected from the digestive gland of the land snail Theba pisana showing a terminal birth pore. Scanning electron microscopy morphological features of the adult worm observed for the first time in a Brachylaima were the unarmed cirrus extended from the genital pore with released sperm present and the Laurer's canal opening visible in tegumental folds on the dorsal surface approximately 300 microm posterior to the genital pore.     

Tegumental ultrastructure of the juvenile and adult Himasthla alincia (Digenea: Echinostomatidae)

The tegumental ultrastructure of juvenile and adult Himasthla alincia (Digenea: Echinostomatidae) was observed by scanning electron microscopy. One-, 5- (juveniles) and 20-day-old worms (adults) were harvested from chicks experimentally fed metacercariae from a bivalve, Mactra veneriformis. The juvenile worms were elongated and curved ventrally. The head crown bore 31 collar spines, arranged in a single row. The lip of the oral sucker had 12 paired, and 3 single type I sensory papillae, and the ventral sucker had about 25 type II sensory papillae. The anterolateral surface between the two suckers was densely packed with tegumental spines with 4-7 pointed tips. The adult worms were more elongated and filamentous, and had severe transverse folds over the whole body surface. On the head crown and two suckers, type I and II sensory papillae were more densely distributed than in the juvenile worms. Retractile brush-like spines, with 8-10 digits, were seen on the anterolateral surface, whereas claw-shaped spines, with 2-5 digits, were sparsely distributed posteriorly to the ventral sucker. The cirrus characteristically protruded out, and was armed with small spines distally. The surface ultrastructure of H. alincia was shown to be unique among echinostomes, especially in the digitation of its tegumental spines, the distribution of sensory papillae and by severe folds of the tegument.     

Surface ultrastructure of juvenile and adult Acanthoparyphium tyosenense (Digenea: Echinostomatidae)

The tegumental ultrastructure of juvenile and adult Acanthoparyphium tyosenense (Digenea: Echinostomatidae) was observed by scanning electron microscopy. One- to 3-day-old juveniles and 10-day-old adults were harvested from chicks experimentally fed metacercariae from a bivalve, Mactra veneriformis. The juvenile worms were minute, curved ventrally, and had 23 collar spines characteristically arranged in a single row. The lips of the oral sucker had 7 single aciliated sensory papillae and 4 grouped uniciliated sensory papillae. The ventral sucker had 25 aciliated round swellings on its lip. The anterolateral surface between the 2 suckers was densely packed with tongue-shaped tegumental spines, and the ventral surface just posterior to the ventral sucker was covered with peg-like spines. Retractile, peg-like spines were seen on the anterolateral surface, whereas scale-like spines with round tips and broad bases were sparsely distributed posterior to the ventral sucker. The cirrus was characteristically protruding and armed with minute spines. The surface ultrastructure of A. tyosenense was unique, especially in the number and arrangement of collar spines, shape, and distribution of tegumental spines and in distribution of sensory papillae.     

Tegumental ultrastructure of juvenile and adult Echinostoma cinetorchis]

The tegumental ultrastructure of juvenile and adult Echinostoma cinetorchis (Trematoda: Echinostomatidae) was observed by scanning electron microscopy. Three-day (juvenile) and 16-day (adult) worms were harvested from rats (Sprague-Dawley) experimentally fed the metacercariae from the laboratory-infected fresh water snail, Hippeutis cantori. The worms were fixed with 2.5% glutaraldehyde, processed routinely, and observed by an ISI Korea DS-130 scanning electron microscope. The 3-day old juvenile worms were elongated and ventrally curved, with their ventral sucker near the anterior two-fifths of the body. The head crown was bearing 37-38 collar spines arranged in a zigzag pattern. The lips of the oral and ventral suckers had 8 and 5 type II sensory papillae respectively, and between the spines, a few type III papillae were observed. Tongue or spade-shape spines were distributed anteriorly to the ventral sucker, whereas peg-like spines were distributed posteriorly and became sparse toward the posterior body. The spines of the dorsal surface were similar to those of the ventral surface. The 16-day old adults were leaf-like, and their oral and ventral suckers were located very closely. Aspinous head crown, oral and ventral suckers had type II and type III sensory papillae, and numerous type I papillae were distributed on the tegument anterior to the ventral sucker. Scale-like spines, with broad base and round tip, were distributed densely on the tegument anterior to the ventral sucker but they became sparse posteriorly. At the dorsal surface, spines were observed at times only at the anterior body. The results showed that the tegument of E. cinetorchis is similar to that of other echinostomes, but differs in the number and arrangement of collar spines, shape and distribution of tegumenal spines, and type and distribution of sensory papillae.     

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.     

Anthelmintic-induced destrobilation and its influence on calculated drug efficacy in Hymenolepis diminuta infections in rats

Anthelmintic efficacy studies typically involve direct counts of worms remaining in the host shortly after drug treatment. Few such studies, however, have considered the phenomenon of tapeworm destrobilation when determining effective dosages. The present study reports on the frequency of drug-induced destrobilation and the subsequent regeneration of Hymenolepis diminuta in rats following treatment with niclosamide or praziquantel and its implications with respect to the apparent efficacy of these anthelmintics. Drug efficacies very similar to those reported in the literature were determined upon examination of infected animals 24 hr posttreatment. Small regenerating worms were, however, observed in the small intestine of rats 8 days after treatment, indicating that destrobilated worms were present, but overlooked, during the initial examination. Within several days posttreatment, destrobilated worms can regenerate to a size that is readily apparent in the gut contents, allowing the effective dosage to be determined with much greater confidence. Due to the demonstrated ability of these destrobilated worms to regenerate to the gravid state, it is imperative that a fully effective anthelmintic dosage be determined and administered.     

Surface topography of the tegument of adult Schistosoma nasale Rao, 1933 from Sri Lanka

Scanning electron microscopical studies of adult male and female Schistosoma nasale are reported. The tubercles on the dorsal and dorso-lateral surfaces of unpaired male S. nasale are devoid of spines. In paired male worms the tubercles on the dorsal surface are large and also are devoid of spines, but some tubercles on the dorso-lateral surface possess spines. Pit-like openings are visible on the surface of the smooth tubercles. The oral and ventral suckers on the male worm are well developed and are invested with spines, as are the gynaecophoric canal and flap. Ciliated sensory receptors are distributed over the surface of the male worm. The oral and ventral suckers of the female worm are much smaller than those of the male: spines occur on both suckers. The surface of the female is non-tuberculate and is thrown into transverse folds. Pit-like openings are visible at higher magnifications. The anterior end of the female is heavily invested in ciliated receptors, whereas the posterior end is heavily spined. The surface topography of S. nasale is discussed in relation to other species of Schistosoma.     

Tegumental ultrastructure of adult Gynaecotyla squatarolae (Digenea: Microphallidae)

Gynaecotyla squatarolae (Digenea: Microphallidae) adult flukes were recovered from experimental chicks at day 4-6 post-infection and their tegumental ultrastructure was observed with a scanning electron microscopy. They were pyriform in shape, and their anterior halves were concaved ventrally. The whole body surface was covered with tegumental spines, which were wide and 16-17 digitated between oral and ventral suckers. The density of spines and number of digits decreased posteriorly. The oral sucker was subterminal and the excretory pore was at the posterior end of the worm. Two ventral suckers were similar in appearance and protruded near midline of the worm. The genital atrium was dextral to the small ventral sucker. The dorsal surface was covered with tegumental spines, but the spines were sparser than on the ventral surface. On the middle portion of the dorsal surface, a small opening presumed to be the Laurer's canal was seen. From these findings, it has been confirmed that the adult G. squatarolae has unique characteristics in the surface ultrastructure.     

Some influences of population density on Hymenolepis diminuta in rats.

When measured 56 days postinfection the length, wet weight and dry weight of Hymenolepis diminuta were all found to decrease with increasing number of cysticercoids given up to 20. The mean position of the worms in 10, 12 and 20 worm infections is significantly posterior to that of 1, 2 and 5 worm infections and the worms are attached over a wider area of the intestine. Egg production by the worms was followed up to day 56 postinfection; the number of eggs produced per worm and even per rat decreased with increasing population density. Thus the best way to get most eggs and to maintain the parasite in the laboratory is to have rats infected with only one tapeworm. Rats given 1-20 cysticercoids showed a mean recovery of 100-65%, while rats given 40-200 cysticercoids showed a mean recovery ranging from 13 to 2%. In addition to 'normal' worms, defined as worms greater than 10 mm, small, most probably destrobilated, worms were found. In the 50 and 100 cysticercoid infections, worm recoveries were, respectively, 8% 'normal', 16% small, and 2% 'normal', 5% small. From the significantly lower recovery from heavy infections it is concluded that a deleterious factor is operating during the 8 weeks after the infection.     

Development of Schistosoma mansoni worms in mice analyzed by bright field and confocal microscopy

The blood flukes of mammals (Digenea: Schistosomatidae) are among trematodes unique whose adult worms have separated sexes which are dissimilar in appearance. The developmental features, growth and organogenesis of Schistosoma mansoni were studied in Swiss Webster mice by a digital system for image analysis and confocal microscopy. Data so far obtained showed two phases with significative morphological changes at 3-4 weeks post-infection, and a gradual similar development onwards in the reproductive system and tegument. Our male-dependent phase demonstrated that mating occurs before sexual maturing. At week three, the majority of male worms (59%) had formed the gynaecophoric canal although testicular lobes and tegumental tubercles were absent. By this time, 33% females had an incipient ovary (without cellular differentiation). At week four, 77.2% males presented testicular lobes with few germinative cells while 26% had developing tegumental tubercles. The immature ovary was observed in 69% females. Suckers followed different pattern of growth between male and females. The size of oral and ventral suckers from six-week-old male worms grew abruptly (3.0 fold) more than that of three-week-old. In female worms, maximum growth was attained at week four, reducing in size thereafter. From sixth week onwards, all specimens showed the fully developed reproductive system. Probably, these features are morphological traits which schistosome has experienced from hermaphrodite to dioecy.     

Schistosoma mansoni: stage-dependent formation and repair of membrane pores induced by a cytotoxin from Pseudomonas aeruginosa.

Various stages of Schistosoma mansoni were treated with a cytotoxin from Pseudomonas aeruginosa and their response to the damaging effect was studied in detail. Marker release and membrane potential measurements showed that the cytotoxin formed stable pores in all developmental stages. However, in juvenile 27-day-old worms, which are refractory to the killing effect of the cytotoxin, the pores had a smaller functional diameter as compared to other stages including 31-day-old worms. Furthermore, these resistant 27-day-old worms, but not susceptible older juvenile worms were able to repair the membrane lesions as shown by restoration of the resting membrane potential. In contrast, older juvenile and adult parasites were unable to cope with the breakdown of the resting potential induced by the cytotoxin. The results demonstrate the existence in 27-day-old schistosomes of effective repair mechanisms dealing with damage to the surface membrane.     

Early patterned stimulation leads to changes in adult behavior and gene expression in C. elegans

Across phylogeny, early experience plays a critical role in nervous system development. In these experiments, we investigated the long-term effects that specific patterns of sensory experience during development had on the biology and function of the Caenorhabditis elegans nervous system. The delivery of a specific pattern of mechanosensory stimulation in the first larval stage (L1) produced significant enhancement in the tap withdrawal behavioral response, expression patterns of an ionotropic glutamate receptor (iGluR) subunit and mRNA levels for that receptor in 3-day-old adult worms and a depression of these same three measures in 5-day-old adult worms. A critical period for the 3-day enhanced behavior and GLR distribution was observed in L1, whereas there was no critical period for the depressed effects observed in 5-day-old worms. The spaced pattern of stimulation was essential for expression of this effect: Various forms of massed training produced neither the enhancement at 3 days nor the depression at 5 days. The 5-day depressed behavioral response had many features in common with long-term memory, including sensitivity to disruption following retrieval. The different behavioral and molecular effects that early patterned mechanosensory stimulation produced in 3 and 5-day-old worms led us to hypothesize that separate cellular phenomena produced the enhanced 3-day and depressed 5-day behaviors and molecular effects.     

On the Early Development of the Protonephridial Systems in Some Species Belonging to the Genera Diphyllobothrium, Triaenophorus and Schistocephalus (Cestoda, Pseudophyllidea)

Malmberg, G. {Department of Zoology, University of Stockholm, Stockholm, Sweden.) On the early development of the protonephridial systems in some species belonging to the genera Diphyllobothrium, Triaenophorus and Schistocephalus {Cestoda, Pseudophyllidea). Zool. Scripta 1 (5): 227–228, 1972.–The protonephridial systems of coracidia (oncospheres) and procercoids of four Diphyllobothrium species, of Triaenophorus nodulosus (Pallas) and of Schistocephalus solidus (Muller) were studied. In the oncospheres of Diphyllobothrium and Schistocephalus the two flame bulbs of the primary protonephridial system were present, but not in the oncospheres of Triaenophorus. In Schistocephalus the two flame bulbs were found to be inactive in the oncosphere (studied inside the coracidium), but very active in the youngest procercoids, which may imply that the primary protonephridial system does not start its function until the oncosphere has entered the copepod body cavity. The primary protonephridial system of the Triaenophorus procercoids was totally (most specimens) or partly reduced. The secondary protonephridial system, however, began developing more or less simultaneously with the integumental hooklets, the cercomer and the first calcareous bodies, which is in accordance with what is described concerning Diphyllobothrium. The ciliated, excretory bladder described by Rosen in 1919 was found to be a posteriorly open invagination, surrounding the “cercomer shaft”. Reverse bends of the posterior main canals of the secondary system are located closely around the wall of, though very likely not emptying into this invagination.     

日本血吸虫在小鼠体内发育形态的扫描电镜观察

日本血吸虫童虫在移行和发育过程中体形发生了明显的演变,并形成了口吸盘和排泄孔,同时也伴随着许多细微结构的改变。这些改变包括:体褶和体嵴数量及高度的增加、体棘数目和位置的改变、感觉器数量和分布的变化。     

Surface ultrastructure of the developmental stages of Heterophyopsis continua (Trematoda: Heterophyidae).

Ultrastructural observations were made on the tegument of juvenile and adult stages of Heterophyopsis continua using scanning electron microscopy. On the surface posterior to the ventral sucker, the tegumental processes were bandlike in the metacercariae, cobblestonelike in the flukes 2 days postinfection (PI), and velvety at 3 days PI. The anterior surface between the oral and ventral suckers of the metacercariae was packed densely with tegumental spines having a 10- to 14-pointed tip. In flukes 6 days PI, the number of points increased to 15-17. The tegumental spines immediately behind the ventral sucker on the metacercariae surface possessed 5-7 points; posteriorly the points were reduced in size and in number. Ciliate sensory papillae (type I), as single or clumped forms of 2 or 3, were abundant around the oral and ventral suckers of metacercaria and adult worms. The clumped papillae appeared bilaterally symmetrical on the dorsal and ventral surfaces. The ciliate papillae may function in tango-, rheo-, and/or chemoreception. On the lip of the ventral sucker, 6-7 aciliate domed papillae (type II) were arranged in an equidistant manner. At 2 days PI each type II papilla became a clumped form having 2 or 3 papillae. Type II papillae may function as tango- and/or pressure-receptors. The structure and distribution of papillae suggest that the ventral sucker likely functions as a holdfast organ and the oral sucker as a probing organ involved in feeding.     

A review of protonephridial excretory systems in Acanthocephala

Our present understanding of the excretory system of Acanthocephala is largely the result of work done by 5 German scholars: Kaiser, Schepotieff, Meyer, Kilian, and von Haffner. Present studies indicate that a protonephridial system is restricted to the family Oligacanthorhynchidae. However, many members of this family have not had a protonephridial system described. Three nephridial designs have been described: 1) dendritic type, organized as branches of a tree where each final branch terminates in a ciliated bulb; 2) capsular type, in which all ciliated bulbs empty directly into a common chamber; and 3) rudimentary type, consisting of a single cell with a patent ciliary pouch but no ducts to the outside. The first 2 types are a syncytia with 3 nuclei located in the capsule or stem wall and none in the flame bulbs. These excretory systems consist of 2 clusters of flame bulbs that empty separately into an expandable excretory bladder which in turn empties into ducts of the reproductive system. This urogenital system empties to the outside through a gonopore located at the tip of the penis in males and the posterior terminus of the vagina in females. Cilia occur in certain excretory tubes, depending on sex and species, but are unknown in the excretory bladder or ducts leading into it. The rudimentary type consists of a cell whose posterior extension terminates near the bursal lumen, but it is not known if this is significant for the discharge of material. There is no information on the physiology or biochemistry of the excretory system or its products.     

A scanning electron microscope study on the tegument of Proalarioides kobayashii Park, 1940 (Trematoda)

A SEM study was performed on the surface of adult P. kobayashii Park, 1940, recovered from the snake, Elaphe rufodorsata. The anterior part of the worms was cup-shape and equipped with oral, ventral suckers, pseudosuckers, and tribocytic organ, and the posterior one was finger-like and round-ended. The tegument of the anterior body was covered with 3-4 pointed small spines on the mid-ventral surface and 1-2 pointed ones on the lateral surface. Sensory papillae such as type II, dome-shape ones, and papillae with an opening were distributed over the ventral surface of the anterior portion. The round tribocytic organ was bearing small stout spines laterally, whereas the surface which comes in contact with the host tissues consisted of numerous long fibrillar fibers. The lip of the oral sucker contained type II papillae. Lateral margin of the anterior body revealed type III papillae.     

Possible immunological damage to the tegument of Hymenolepis diminuta in mice and rats.

Opaque or darkened areas (DA) of variable size and position occur on Hymenolepis diminuta in mice and rats. In mice DA normally first appear in the neck region of the worm but subsequently they appear elsewhere and increase in number until destrobilation or worm expulsion. The posterior of destrobilated worms is often darkened. In the more immunogenic infections with six cysticercoids there are more DA per worm than in infections with one cysticercoid. DA are areas of the tegument with a homogeneous increase in electron density; abnormal mitochondria; reduced granular endoplasmic reticulum, Golgi complexes and discoidal secretory bodies; and accumulation of lipid droplets. DA disappear from worms maintained for up to 4 h in Hanks' balanced salt solution and can be induced by mechanical damage to the worms. As the numbers of DA increase with the duration and intensity of infection and have similarities with types of cell injury, they are probably sites of worm pathology induced by host immunity.     

Transplantation of Hymenolepis diminuta into naive, immune and irradiated mice.

Almost 100% of 7- to 10-day-old Hymenolepis diminuta became established when surgically transplanted from donor mice into the duodenum of naive recipient mice. Transplanted tapeworms survived 8-12 days, by which time they had survived much longer in total than they would have done in the donor. Mice previously immunized by a primary infection rejected transplants within 4 days. Sub-lethal irradiation (550 rad) delayed rejection by immune mice but such mice still rejected worms much more quickly than did naive mice. Surgery was shown to delay by 2-3 days the rejection of worms by naive mice, and the importance of circumventing surgery by administering the worms per os is emphasized. Prospects for reconstituting irradiated immune mice are considered vis-à-vis work with nematodes, and the differences which, on present knowledge, appear to exist between nematode and cestode rejection are briefly discussed.     

Echinostoma revolutum: resistance to secondary and superimposed infections in mice

A complete or almost complete resistance (94-100%) to a superimposed Echinostoma revolutum infection existed in mice harboring 20-, 30-, and 40-day-old infections in the range of 2-4 to 30-35 worms, but no resistance was found at challenge Day 10. A similar high level of resistance (85-100%) also existed in mice for at least 6 weeks after natural expulsion of a primary 6 metacercarial infection and for at least 5 weeks after anthelmintic termination of a 30-day-old 20 metacercarial infection. Thymus-deficient nude mice failed to develop resistance to a superimposed infection, and the resistance in normal mice was inhibited by corticosteroid treatment. These findings are all in favor of a host immune response being responsible for the resistance against both a secondary and a superimposed infection. Nearly all the worms of a superimposed infection were, in resistant mice, expelled prior to 24 hr following infection (rapid expulsion), and the few worms circumventing this early expulsion persisted for at least 8 days. Newly excysted juvenile worms implanted intraduodenally into resistant mice were rejected to the same degree as juvenile worms from an oral metacercarial infection indicating that the newly excysted juvenile worms are the target of the host immune response. However, 7-day-old worms implanted intraduodenally into resistant mice survived indicating that adaptation to the host immune response had occurred. In conclusion, this host-parasite model is an example of concomitant immunity because the immunological mechanism responsible for the expulsion of the superimposed infection had no effect on the number of primary worms present.