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Thymidylate synthases from Hymenolepis diminuta and regenerating rat liver: purification, properties, and inhibition by substrate and cofactor analogues.

Comparative studies of thymidylate synthases, isolated from the tapeworm, Hymenolepis diminuta, and regenerating liver of its host, rat, aimed at a possibility of specific inhibition of the helminthic enzyme, are presented. While similar in structure (dimers with monomer molecular masses of 33.7 kDa and 34.9 kDa, respectively) and parameters describing interactions with substrates and products, the tapeworm and rat enzymes differed in the dependences of reaction velocity on temperature (Arrhenius plots biphasic and linear, respectively). The tapeworm, compared with the host, enzyme was less sensitive to the competitive slow-binding inhibition by 5-fluoro-dUMP and its 2-thio congener, but equally sensitive to inhibition by 4-thio-5-fluoro-dUMP, N4-hydroxy-dCMP and N4-hydroxy-5-fluoro-dCMP, the latter being more potent inhibitor of the parasite enzyme than 5-fluoro-dUMP. alpha-Anomer of 5-fluoro-dUMP behaved as a very weak competitive slow-binding inhibitor of both enzymes. Both enzymes differed markedly in sensitivity to inhibition by 10-propargyl-5,8-dideazafolate and its di- and triglutamates (pddPteGlu1-3), with pddPteGlu1 being stronger inhibitor of the mammalian enzyme, but pddPteGlu3 showing opposite specificity. Sulfonamidobenzoylglutamate analogue of pddPteGlu (pddPteSO2Glu) and 2-desamino-2-methyl derivative of this analogue (CH3pddPteSO2Glu) were weaker inhibitors of both enzymes than the parent compound. Substitution of the glutamyl residue in CH3pddPteSO2Glu with either norvaline or alanine increased inhibition potency, whereas similar substitutions with glycine, valine or phenylglycine were without a distinct effect with the host enzyme but weakened inhibition of the tapeworm enzyme.

Animals↗

Processes leading to a spatial aggregation of Echinococcus multilocularis in its natural intermediate host Microtus arvalis.

The small fox tapeworm (Echinococcus multilocularis) shows a heterogeneous spatial distribution in the intermediate host (Microtus arvalis). To identify the ecological processes responsible for this heterogeneity, we developed a spatially explicit simulation model. The model combines individual-based (foxes, Vulpes vulpes) and grid-based (voles) techniques to simulate the infections in both intermediate and definite host. If host populations are homogeneously mixed, the model reproduces field data for parasite prevalence only for a limited number of parameter combinations. As ecological parameters inevitably vary to a certain degree, we discarded the homogeneous mixing model as insufficient to gain insight into the ecology of the fox tapeworm cycle. We analysed five different model scenarios, each focussing on an ecological process that might be responsible for the heterogeneous spatial distribution of E. mulitlocularis in the intermediate host. Field studies revealed that the prevalence ratio between intermediate and definite host remains stable over a wide range of ecological conditions. Thus, by varying the parameters in simulation experiments, we used the robustness of the agreement between field data and model output as quality criterion for the five scenarios. Only one of the five scenarios was found to reproduce the prevalence ratio over a sufficient range of parameter combinations. In the accentuated scenario most tapeworm eggs die due to bad environmental conditions before they cause infections in the intermediate host. This scenario is supported by the known sensitivity of tapeworm eggs to high temperatures and dry conditions. The identified process is likely to lead to a heterogeneous availability of infective eggs and thus to a clumped distribution of infected intermediate hosts. In conclusion, areas with humid conditions and low temperatures must be pointed out as high risk areas for human exposure to E. multilocularis eggs as well.

Animals↗

What is Asian Taenia?

Asian Taenia is a human tapeworm which was first recognized in Taiwan aborigines and subsequently from Asian countries: Korea, Indonesia, Vietnam, and China. It was originally described as T. asiatica Eom and Rim, 1993 based on the morphology in its adult and larval stage. A taxonomic disagreement on whether it is species or subspecies level is mainly due to the morphological similarity of this tapeworm with T. saginata, but a sympatric distribution of these two tapeworms is also known in China. The life cycle is quite distinct from T. saginata in using the pig as intermediate host and parasitizing visceral organs such as liver, lung and omentum. A long unresolved question in Asian countries concerns the inconsistency between worm ratio and the food preferences was clarified with this viscerotropic tapeworm. Molecular biological differentiation is possible with DNA techniques and a complete genome of mtDNA was sequenced recently which may provide a resource for comparative mitochondrial genomics and systematic studies of parasitic cestodes. This is a young parasite discovered most recently with many research questions yet to be clarified.

Animals↗

Inflammatory responses in the intestinal mucosa of gerbils and hamsters experimentally infected with the adult stage of Taenia solium.

The inflammatory response in gerbils and hamsters harbouring experimental infections with Taenia solium adult parasites as well as worm burden and duration of infections were examined. For this purpose, non-suppressed or immunosuppressed rodents were infected with eight cysticerci and necropsied at different times up to 35 days post-infection. Cells in the mucosa surrounding the implantation site of T. solium scolices (duodenum-jejunum) and in ileum were counted in stained sections. A competitive enzyme linked immunosorbent assay was used to determine histamine concentration in intestinal fluid. In non-suppressed hosts, an inflammatory reaction developed with scarce macrophages, a slight increase of plasma cells, lymphocytes and fibroblasts, a moderate increase of eosinophils and neutrophils, and high numbers of goblet and mast cells. Goblet cells began to increase at 6 days post-infection and peaked at 13 days post-infection with a four-fold increase with respect to the control group. Mast cells only increased in gerbils starting at 9 days post-infection with an eight-fold increase when cells peaked between 11 and 19 days post-infection. Histamine concentration in intestinal fluid of gerbils had a similar behaviour to mast cells. Minimal increase of mast cells was seen in hamsters. The recovery of tapeworms was inversely related to the number of both cell types, which decreased when tapeworms were eliminated. Infections lasted up to 25 days in gerbils and up to 46 days in hamsters. Worms measured only 1-2 cm in gerbils and up to 40 cm in hamsters. When gerbils were suppressed with the steroid methyl predinisolone, tapeworms could be recovered up to 35 days post-infection and tapeworms measured up to 22 cm, a minor increase of goblet and mast cells was observed and histamine concentration was similar to that in non-infected animals. Our results suggest that expulsion of T. solium in gerbils and hamsters may be related to the increase of goblet cells and mast cells, but these cells may have different roles in each rodent model of taeniosis.

Animals↗

Controlling Echinococcus multilocularis-ecological implications of field trials.

Two field trials to reduce the prevalence of Echinococcus multilocularis in foxes have been conducted in recent years. Although both trials reduced prevalence considerably, they failed to eradicate the parasite in the study region. Following the control trial in northern Germany, prevalence recovered unexpectedly and rapidly, reaching pre-control levels five quarters (15 months) after the end of control. To understand the internal dynamics of the parasite-host system's reaction to control, we developed a spatially explicit simulation model, Echi. The simulation model incorporates the information available concerning fox tapeworm population dynamics. Using epidemiological parameters to adjust pre-control prevalence, the model predicts the temporal evolution of the prevalence of E. multilocularis in controlled foxes without departing from the range of uncertainty of the field data. However, the model does not predict the rapid pre-control recovery observed in the field trial. The deviation of the model's prediction from field data indicates the involvement of processes not yet taken into account. We modified the model step by step to mimic processes with the potential to cause the rapid post-control recovery of the prevalence of E. multilocularis in foxes. Neither the longevity of tapeworm eggs nor the migratory behaviour of foxes showed any influence on the post-control reaction of the parasite-host system. However, landscape structures leading to a heterogeneous distribution of infected foxes have the potential to alter the system's reaction to control. If infected foxes are concentrated in multiple clusters in the landscape, the model prediction tallied with the range of uncertainty of the field data. Such spatial distribution of infected foxes may be caused by differential abiotic conditions influencing the survival of tapeworm eggs. The model was found to comply best with field data if the foxes acquire partial immunity by being exposed to the fox tapeworm. Both hypotheses explaining the rapid post-control recovery of the prevalence of E. multilocularis observed in the fox population were supported by field data. Both hypotheses have far-reaching consequences for future control trials. The spatial aggregation of infected foxes would enable control efforts to be concentrated on these highly infected areas. However, the acquisition of immunity acts as a buffer to control, necessitating intensified control measures.

Animals↗

Epidemiology of Anoplocephala perfoliata infection in foals on a stud farm in south-western Sweden.

The egg output and humoral antibody response to scolex antigens of the equine tapeworm Anoplocephala perfoliata were monitored in naturally infected foals by an egg flotation/centrifugation method and an indirect enzyme-linked immunosorbent assay (ELISA). The study was performed on a stud farm in south-western Sweden between May 1994 and April 1995. Sequential blood and faecal samples were taken from 21 foals during their first summer on pasture and until tapeworm eggs were detected. Results were expressed separately for 10 and 11 foals born before and after the end of April 1994, respectively. Increased levels of antibodies were noticed from October and onwards in both groups whereas tapeworm eggs were detected in the faeces of all foals about 4 months later. The antibody response was similar in both groups but it was more pronounced in foals born before April 1994. All foals were treated in March 1995 with an oral paste formulation of pyrantel pamoate at a dose rate of 38 mg/kg bodyweight. Most animals responded to the anthelmintic treatment and one month later, tapeworm eggs were only detected in one out of the 18 foals examined one month after treatment. Thus, the treatment reduced the number of A. perfoliata egg positive horses by 94%. A concomitant decline in antibody levels was also observed. Western immunoblot analysis of sequential individual serum samples showed that at least 10 different scolex antigens in the molecular weight range 10-200 kDa were recognised. Banding intensities, especially of the 10 kDa, 45 kDa and 66 kDa proteins were different in the sequential sera taken during the course of infection.

Administration, Oral↗

Taenia saginata (Cestoda) in western Kenya: the reliability of faecal examinations in diagnosis.

The prevalence of Taenia saginata (Cestoda) among 146 predominantly Pokot tribesmen in western Kenya was studied in an attempt to assess the reliability of detecting infections by faecal examinations. firstly, a single faecal sample was microscopically examined after being processed by a standard ether sedimentation technique. Secondly, all subjects were treated with niclosamide and a purgative and the tapeworms were recovered. In this way the efficiency of diagnosing infections by faecal examinations was estimated. T. saginata eggs were found in the faeces of 68% of the men who were eventually found to be infected. Information was collected by interview from all subjects about whether they were or ever had been infected with tapeworms and about the form of treatment previously sought or taken. Eleven out of 25 men were apparently unaware of their tapeworm infections while no evidence of infection was found among 10 of the 24 reportedly infected men. A change in the pattern of treatment for tapeworms was reported which may have resulted in a recent decline in the prevalence of infection.

Adult↗

Echinococcus granulosus coproantigens: chromatographic fractionation and characterization.

Dogs infected with adult tapeworms of Echinococcus granulosus release antigens (coproantigens) in faeces which can be detected by a capture ELISA. Supernatants prepared from E. granulosus-infected dog faecal samples were fractionated by size-exclusion fast protein liquid chromatography (FPLC) on a Superose-6 column. Coproantigen ELISA and Western blotting were used to demonstrate the immunoreactivity of eluted fractions. Two main FPLC peaks of antigenic activity were detected and designated as fraction F1 and fraction F2 with approximate relative molecular weights > 670 kDa, and in the range of 146 to 440 kDa respectively. These two antigenic fractions (F1 and F2) fractionated from infected dog faeces were heat stable and largely protease-insensitive, but were highly sensitive to sodium periodate treatment, which strongly suggested the involvement of carbohydrates. Capture IgG antibodies against E. granulosus proglottis somatic extracts, detected a molecule with an approximate molecular weight of 155 kDa in fraction F2 after immunoblotting. The 155 kDa antigen could be completely ablated by sodium periodate treatment, but not after protease or lipase treatment. A surface tegument preparation of adult E. granulosus tapeworms contained large amounts of antigen that corresponded in size range and antigenicity to that observed in the FPLC fraction F2. There was also a peak of antigenic activity at > 670 kDa corresponding to fraction F1 from a culture derived excretory-secretory (E-S) adult tapeworm preparation. The involvement of carbohydrate moieties in coproantigen activity present in the FPLC fractions F1 and F2 from faecal supernatants of E. granulosus-infected dogs was confirmed by lectin-binding assays and exoglycosidase treatment, which showed that alpha-D-mannose and/or alpha-D-glucose, beta-galactose and N-acetyl-beta-glucosamine residues were the most important carbohydrate components in putative coproantigens present in both fractions. N-acetyl-beta-glucosamine and sialic acid residues were also contained in coproantigen molecules present in fraction F2. These results suggested that coproantigens detected in faeces of E. granulosus-infected dogs are large molecular weight molecules that may be derived from the carbohydrate-rich surface glycocalyx of adult worms, and are shed, released or secreted during the life-span of the tapeworm.

Animals↗

In vivo and ex vivo uptake of albendazole and its sulphoxide metabolite by cestode parasites: relationship with their kinetic behaviour in sheep.

The current experiments correlate the disposition kinetics of albendazole (ABZ) following its intravenous (i.v.) and intraruminal (i.r.) administrations to Moniezia spp.-infected sheep, with the pattern of drug/metabolite uptake by tapeworms collected from treated animals. The ex vivo uptake pattern of ABZ and albendazole sulphoxide (ABZSO) by the same cestode parasite was also investigated. Naturally infected (Moniezia spp.) Corriedale lambs were treated with ABZ by either i.v. (Group A, n = 15) or i.r. (Group B, n = 15) administration at 7.5 mg/kg. Plasma and abomasal fluid samples were obtained over a 120-h period. Two animals per group were killed at 0.5, 1, 2, 4 and 6 h post-treatment; parasite material (tapeworms), bile and intestinal fluid samples were recovered. Furthermore, Moniezia spp. tapeworms obtained from sheep killed at the local abattoir were incubated with either ABZ or ABZSO for different time periods in a Kreb's Ringer Tris buffer (ex vivo experiments). Samples were analysed by high performance liquid chromatography for ABZ, ABZSO and albendazole sulphone (ABZSO2). ABZ plasma concentrations decreased rapidly and were not detectable beyond 10 h following i.v. administration. ABZSO and ABZSO2 were the metabolites recovered in plasma after both treatments. ABZ and its metabolites were extensively distributed to the digestive tract, mainly into the abomasal fluid, after the i.v. and i.r. administrations. The parent drug and its active ABZSO metabolite were recovered in tapeworms collected from both i.v. and i.r. treated lambs. However, the availability of both ABZ and ABZSO was higher in parasite material recovered from i.v. treated animals. The uptake of ABZ by the cestode parasite, both in vivo and ex vivo, was significantly greater than that of its sulphoxide metabolite, which agrees with the higher lipophilicity of the parent drug.

Abomasum↗

Pathological alterations caused by Anoplocephala perfoliata infection in the ileocaecal junction of equids.

The pathological alterations caused by Anoplocephala perfoliata in the ileocaecal junction of 28 equids slaughtered in an abattoir in Madrid (Central Spain) are described. The lesions were scored in grades based on the intensity of the damage and were related to the tapeworm number observed. The first grade (grade I) of alterations consisted of a slight enteritis associated with focal erosions observed in 43% of parasitized animals with low parasitic burden (1-26 tapeworms). The second grade (grade II) was a focal pseudomembranous enteritis, present in the ileocaecal junctions of 36% infected animals with moderate to high burden (23-188 tapeworms), and the third grade (grade III) was a regional necrotizing enteritis, present in the animals (21%) with the highest burden (72-248 tapeworms). The possible role of the lesions caused by this parasite in the aetiology of colic is discussed.

Animals↗

Validation of a centrifugation/flotation technique for the diagnosis of equine cestodiasis.

A centrifugation/flotation technique for the identification of equine tapeworm eggs is described. It was validated by using faeces samples from 80 horses of known tapeworm status, and had a sensitivity of 61 per cent and a specificity of 98 per cent. The exclusion of false negative results in animals with less than 20 tapeworms increased the sensitivity to 92 per cent. No significant correlation was found between the number of eggs observed and the number of tapeworms present in the horses.

Animals↗

Cestodes.

A review has been made of the advances in knowledge on the treatment of tapeworm infections of man and animals from the time of the introduction to dichlorophen in 1956. This opened up the era of out-patient treatment for human tapeworm infections. The drugs studied have been compared, where possible, on the basis of estimates of the single dose ED90 or the number of treatments required to reach that efficacy at safe dose levels. During the period under review, niclosamide, introduced in 1960 has been regarded as the drug of choice for the treatment of human tapeworm infections with paromomycin as a possible alternative. The bunamidine salts, introduced in 1966 permitted the treatment of Echinococcus spp in dogs. Several treatments were required to achieve acceptable efficacy. In 1975 praziquantel was introduced and based on the ED90, a single dose at no more than 10mg/kg removed all tapeworms responsible for the cestode zoonoses and for which data are available with the exception of H. nana and D. latum; these require a higher dose rate. In the mid 1970s, several benzimidazoles and praziquantel were shown to have activity against metacestodes. This has opened up a new field of research promising a practical outcome. No progress has been made during the period under review in finding effective ovicides.

Animals↗

Effect of surgical alteration of the rat gastrointestinal tract on the growth and development of Hymenolepis diminuta.

Eight groups of rats were used to study the involvement of the enteric (ENS) and central (CNS) nervous systems in the development of Hymenolepis diminuta using surgical intestinal transection, or CNS denervation, or both procedures. The transection procedure was used to isolate the ENS of the small intestine from either orad and/or caudal portions of the alimentary system, while the CNS denervation was used to eliminate direct visceral efferent inputs from the CNS. Nine days after the surgical procedures, all rats were infected with 35 cysticercoids of H. diminuta. On 20 days postinfection, the infection intensity, tapeworm dry weight, tapeworm morphology, intestine length, and intestinal wet weight were recorded. Only the combination of the duodenal and ileal transections with a CNS denervation reduced infection intensity and prevented the increased intestinal length normally observed in infected rats. In contrast, none of the various intestinal transection procedures alone or CNS denervation alone had any effect on the survival, ability to produce oncospheres or morphology of the tapeworms. In conclusion, tapeworm survival is decreased when both CNS and ENS inputs into the small intestine are altered or absent.

Animals↗

Identification of Taenia asiatica in China: molecular, morphological, and epidemiological analysis of a Luzhai isolate.

Multiple analysis has characterized a recently described tapeworm of people, Taenia asiatica, in mainland China. Six adult tapeworms collected from people of the Zhuang minority residing in the southern part of China (Luzhai isolate) were comparatively analyzed with other tapeworms from people: T. asiatica (n = 2, South Korea), T. saginata (n = 1, Poland; n = 1, Korea), and T. solium (n = 1, People's Republic of China). Experimental infections with eggs from the Luzhai isolate in pigs and cattle produced cysticerci, each with a hookletless scolex and with wartlike formations on the external surface of the bladder wall. There were rostellar protrusions in the scolices of adult worms. Random amplified polymorphic DNA analysis using 3 arbitrary primers produced bands identical to those of the Korean T. asiatica. Conversely, T. saginata and T. solium exhibited different banding patterns. Phylogenetic relationships inferred from the complete nucleotide sequences of the internal transcribed spacer 2 placed the Chinese tapeworms consistently within the T. asiatica clade by 96% bootstrapping value in the maximum likelihood analysis, 96% in maximum parsimony, and 100% in neighbor joining. These collective data demonstrate that T. asiatica is sympatrically distributed with the other 2 species of Taenia in the human host in mainland China.

Adolescent↗

Studies on intestinal trematodes in Korea [XII. Two cases of human infection by Stellantchasmus falcatus.

Two cases of human infection by Stellantchasmus falcatus (Heterophyidae) were identified by collection of adult worms during the treatment of some tapeworm infections in Korea. The cases were 24 (Case 1) and 55-year old (Case 2) males residing in Seoul. The Case 1 had gastrointestinal troubles such as diarrhea and abdominal pain, and heart problems such as palpitation and arrhythmia, revealing the eggs of Diphyllobothrium latum in the feces. The Case 2 complained indigestion and discharge of tapeworm (Taenia saginata) segments. Praziquantel at the dose of 15-20 mg/kg body weight and 30 g magnesium salt were given to them for treatment of the tapeworm infection and the discharged strobilae were identified. However, concommitantly with the tapeworms, 188 and 5 specimens of S. falcatus were collected from Case 1 and Case 2 respectively through stereomicroscopy of the diarrheal stools. The Case 1 was infected also with 3 other kinds of heterophyid flukes. They said to have eaten raw brackish water fish such as mullets which are considered to be the source of heterophyid fluke infections.

Journal Article↗

Echinococcosis--an emerging parasite in the immigrant population.

Echinococcus tapeworms are parasites that infect dog species worldwide and occasionally are transmitted to humans. Infection occurs most commonly in persons who raise sheep or cattle, and who have contact with dogs. The tapeworm eggs are typically ingested during play with dogs or through consumption of garden vegetables or water contaminated by dog feces. The most common sites of tapeworm cyst formation within the body are the liver and lungs, although any internal organ or bone can be infected. The tapeworm infection is generally asymptomatic for 10 to 20 years, until the cyst grows large enough to cause problems. Untreated infection can be fatal. The possibility of this uncommon infection is most often considered when cysts are found on imaging studies of affected organs. Serologic testing has variable sensitivity, but it can be helpful. Surgical removal of cysts is the usual treatment, often with perioperative use of anthelmintic medication to prevent recurrence of disease caused by later growth of undetected daughter cysts.

Bosnia and Herzegovina↗

[The history of the exploration of the Diphyllobothrium latum life cycle].

Diphyllobothrium latum typically occur in the subarctic and temperature zones of the Eurasian Continent, however it is also observed in the Arctic Region and Australia. Raw fish meat and raw shell-fish containing plerocercoids is the main source of human infections. Humans are the principial final host of D. latum. Under primitive conditions human excrements with immature tapeworm eggs are deposited anywhere. After embrionic development which occurs in the water, the eggs are transformed into coracidia. Two intermediate hosts (cyclop and fish) are needed for further development. The free-swimming coracidium must enter the stomach of different species of Copepoda. After contact with the intestinal juice of the cyclop, the coracidium loses its ciliated envelope and the hooks become mobile. The metamorphosis of the oncosphere to the procercoid occurs. The discovery of the procercoid done by Janicki and Rosen in 1917 implied that the missing link in the cycle of the broad tapeworm had been found. The copepod including the procercoid is consumed by the second intermediate host. The larvae escape from the digestive tract of the fish and are transformed into a plerocercoid. The plerocercoids develop slowly in the fish, and they must reach a certain degree of maturity to be capable of infecting the final host. Today the life cycle of the tapeworm is well recognized and well illustrated in parasitological textbooks and websites. In this paper the history of the exploration of the D. latum life cycle is described. Additionally the main scientific researches carried out on life stages of the broad tapeworm have been reviewed.

Animals↗

Water balance and its relation to fermentation acid production in the intestinal parasites Hymenolepis diminuta (Cestoda) and Moniliformis moniliformis (Acanthocephala).

Water balance and its relation to carbohydrate metabolism was examined in Hymenolepis diminuta in parallel with the putative osmoconformer Moniliformis moniliformis. Worms were removed from rat intestines, weighed, and incubated (37 C) 1 hr in rat serum and various salines, some with mannitol to vary osmotic concentration from 150 to 400 mOsm/L. Worms were removed at 15-min intervals, weighed, and returned to the test solution. Rat serum and a Ringer's saline (pH 7.4 and 300 mOsm/L) with or without 5 mM glucose were isotonic to M. moniliformis, which behaved like an osmometer, shrinking, or swelling in proportion to external osmotic changes. Hymenolepis diminuta rapidly lost 20-25% wet weight in these solutions and regained lost water when 5 mM glucose was added to the saline. Tapeworms maintained constant body weight between 210 and 335 mOsm/L, but they rapidly gained or lost water outside of this range. Glucose metabolism and uptake of [3H]glucose from the medium increased progressively between 210 and 310 mOsm/L, whereas uptake rates of [3H]leucine, 22Na+, and 36Cl- were not affected. Unbuffered saline (initial pH 6.5 and 300 mOsm/L) had a lower pH (5.0) and higher osmolality (307 mOsm/L) after a 1-hr incubation with tapeworms. Such saline was less hypertonic than unconditioned saline to freshly obtained worms. A Ringer's saline (300 mOsm/L) containing 50 mM acetate- was also hypertonic (greater than 20% weight loss) to tapeworms at pH 7.4, but it was hypotonic (greater than 20% weight gain) at pH 5.0. Isotonicity at 300 mOsm/L was achieved with pH 5.0 and 20 mM acetate-, the approximate pH and fermentation acid concentration in an infected rat intestine. Rats infected with tapeworms (12 days old) were fasted for 2 days. Starved worms were smaller but had the same percentage of body water and internal osmolality as controls. These results show that H. diminuta can regulate its body water content and that water balance is closely related to the fermentation acid concentration and pH of the bathing medium.

Animals↗