Allometric growth of the proglottids and strobila of the tapeworm, Hymenolepis diminuta.

HYMENOLEPIS DIMINUTA: adults were killed and fixed in fully extended positions, and the total lengths of the strobilae, numbers of proglottids, and distances of proglottids along the strobilae were determined. The relationship of proglottid number to distance along the strobila was exponential. Beginning at proglottid 100 (P100), the lengths and widths of proglottids at 100 proglottid intervals were determined, and the surfaces areas were calculated. The relationships of proglottid length and width to proglottid number were linear, but the relationship of proglottid number to surface area (SA) was exponential. The volumes of proglottids were calculated, and the relationship of volume (V) to proglottid number was exponential. The relationship of surface area to volume ratio (SAVR) to proglottid number was exponential; at the anterior end of the worm (P100), the SAVR was 14.6, while at the posterior end of the worm (P1300) the ratio was 4.2. A single exponential equation describing the relationships among proglottid number, SA, and V was derived.

Beetle-to-beetle transmission and dispersal of Hymenolepis diminuta (Cestoda) eggs via the feces of Tenebrio molitor.

When grain beetles (Tenebrio molitor) were fed eggs of Hymenolepis diminuta, many of the eggs passed intact through the beetles' intestines, and eggs were present in the beetles' feces for at least 48 hr after feeding. When uninfected T. molitor were fed beetle feces containing H. diminuta eggs, they became infected. Tenebrio molitor were fed on H. diminuta eggs and then placed in fresh bran for 48 hr. When uninfected T. molitor were placed in this bran, they became infected. Thus, feces from beetles that have ingested H. diminuta eggs serve as a source of eggs for other beetles, as well as a mechanism of egg dispersal.

Hymenolepis diminuta: glucose and glycogen gradients in the adult tapeworm.

Adult (20-day-old) Hymenolepis diminuta were cut into 12 pieces of equal length, and the individual pieces of the tapeworm's strobila were analyzed. There was a continuous gradient of decreasing concentrations of glucose (mM) and decreasing levels of glycogen (microgram/mg wt) in the strobila. The ethanol extracts of the individual pieces of strobila contained four compounds tentatively identified as disaccharides; the distributions of these compounds were different from those of glucose and glycogen. The distributions of glucose, glycogen, and the "disaccharides" changed when tapeworms were incubated for 1 h in saline or glucose. When compared, on a per-weight basis, to the most posterior sections of the strobila, the anterior sections absorbed more glucose and incorporated more glucose into glycogen. There was a continuous gradient of decreasing values along the tapeworm's strobila of the Vmax for glucose uptake, while the Kt values for glucose uptake changed only slightly. The data indicate that glucose and glycogen metabolism are most active in the anterior part of the tapeworm's strobila where new proglottids are produced and the initial stages of organogenesis occur.

Predictability of morphological gradients in the tapeworm, Hymenolepis diminuta.

The strobila of an adult tapeworm represents a continual gradient of developmental stages from immature to gravid proglottids. The purpose of this study was to determine if organogenesis (as measured by the developmental gradient) in tapeworms within a single host and among different hosts occurred at the same rates. Rats were infected with Hymenolepis diminuta and the tapeworms were recovered 20 days post-infection. The total number of proglottids in each worm was determined, and five 'benchmarks' of organogenesis were quantified. The data demonstrated that organogenesis in worms from a single host occurred at a relatively constant rate, but that rates in tapeworms from different hosts were different.

Effect of tunicamycin on the uptake and incorporation of galactose in Hymenolepis diminuta.

The effects of tunicamycin (TM) on the uptake and incorporation of tritiated galactose into the tegumental membrane and carcass from adult Hymenolepis diminuta were examined to assess the potential usefulness of this inhibitor for studying the function of the tapeworm surface glycocalyx. Hymenolepis diminuta adults (11 days old) were preincubated for 1 hr, pulsed for 30 min with [3H]galactose and [14C]leucine, and chased for 2 hr; replicate experiments were conducted in which all media contained no TM or TM at 10 micrograms/ml. Tunicamycin significantly inhibited the incorporation of tritiated galactose into the tapeworm's carcass and 30,000-g tegumental membrane fraction. Incorporation of tritiated galactose into the tapeworm's tegumental surface membrane also was inhibited significantly when expressed relative to the incorporation of [14C]leucine. Tunicamycin did not affect the amounts of free, i.e., soluble, [3H]galactose or [14C]leucine recovered from the tapeworms not did it affect the short-term (2 min) uptake of [3H]galactose by tapeworms. Thus, the inhibitory effect of TM appears to be at the level of protein glycosylation rather than carbohydrate (galactose) transport. The data indicate that TM might be useful for producing tapeworm surface membranes with diminished carbohydrate moieties.

Impairment of the chemical defence of the beetle, Tenebrio molitor, by metacestodes (cysticeroids) of the tapeworm, Hymenolepis diminuta.

The defensive glands of beetles, Tenebrio molitor, infected with metacestodes (cysticercoids) of Hymenolepis diminuta are everted less frequently upon stimulation, and contain less toluquinone (methylbenzoquinone) and m-cresol, than glands of uninfected controls. These differences, as shown in predation trials with wild rats, increase the likelihood that both cysticercoids and beetles will be ingested by the tapeworm's definitive host. This is the first documented case of a parasite inhibiting the chemical defence of an intermediate host, and one of only a few reports of parasite-induced manipulation of host biology supported by empirical evidence implicating facilitated parasite transmission between host species.

Preliminary biochemical characterization of faeces from uninfected rats and rats infected with the tapeworm, Hymenolepis diminuta.

Faecal pellets were collected from uninfected rats and rats infected with Hymenolepis diminuta, and analysed for caloric value, lipids, carbohydrates, glucose, amino acids, and proteins. Faeces from infected rats contained significantly more lipid and less carbohydrate and glucose than faeces from control rats. The total free amino acid content of faeces from infected and control rats did not differ, but there were significant differences in the amounts of individual free amino acids. The data support the hypothesis that the nutrient composition of faeces from infected and control hosts differ. Beetles that serve as the intermediate host for H. diminuta may be able to detect these differences, and this may explain why beetles preferentially ingest faeces containing the tapeworm s eggs.

Quantification of benzoquinones in the flour beetles. Tribolium castaneum and Tribolium confusum.

Benzoquinones were extracted from flour beetles by rinsing them for 8 min in methanol followed by homogenization in fresh methanol. This procedure recovered > 96% of the total benzoquinones from both species, with the methanolic rinse and supernatant representing the "surface" and "total" benzoquinones. As determined by UV/VIS spectrometry and reversed-phase HPLC, the methanolic rinse contained methyl- and ethyl-1,4-benzoquinone (MBQ and EBQ), but no substituted hydroquinones, The methanolic supernatant contained MBQ and EBQ, and methyl- and ethyl-hydroquinone. When stored at -20 degrees C for 72 hr, the chemical composition of the methanolic rinse did not change, but the composition of the methanolic homogenate did change. Notably, the amounts of benzoquinones in the methanolic supernatant decreased during storage. The method described represents an improvement over those described previously in that it uses less hazardous solvents for extraction (methanol vs more non-polar solvents such as hexane) and allows for the differentiation and quantification of the surface and total benzoquinones of these beetles.

Increased coprophagic activity of the beetle, Tenebrio molitor, on feces containing eggs of the tapeworm, Hymenolepis diminuta.

When provided with fecal pellets from uninfected (control) rats and rats infected with the tapeworm Hymenolepis diminuta, more fed and starved (72 h) female and starved male Tenebrio molitor fed on fecal pellets from infected- than from control rats; compared to fecal pellets from controls rats, fed males avoided the infective fecal pellets. Uninfective and infective fecal pellets had similar moisture contents, so increased coprophagic activity was not due to differences in moisture content. Fed and starved males and females were fed on fecal pellets containing tapeworm eggs and examined for cysticercoids. Significantly greater numbers of starved beetles than fed beetles were infected with cysticercoids, but the numbers of infected males and females within each treatment were not significantly different. On the other hand, males contained significantly greater numbers of cysticercoids than did females, and there was no significant difference between the numbers of cysticercoids recovered from fed and starved beetles. The data support the hypothesis that the feeding behavior of T. molitor on rat feces is altered by the presence of tapeworm eggs. The data demonstrated further that transmission dynamics are affected by a complex interaction of the beetle's sex and nutritional status.

Benzoquinones of the beetles, Tribolium castaneum and Tribolium confusum.

Tribolium castaneum and T. confusum were washed in HPLC-grade methanol, and the methanolic washes were analyzed by UV spectroscopy, reversed phase HPLC, and GC/MS. The methanolic washes from both species contained methyl-1,4-benzoquinone (MBQ) and ethyl-1,4-benzoquinone (EBQ). The amounts of MBQ recovered from the two species were not significantly different, but the amounts of EBQ and total benzoquinones (MBQ+EBQ) recovered from T. castaneum were significantly greater than for those recovered from T. confusum. The methods described are superior to previous methods for isolating, identifying, and quantifying the benzoquinones in these beetles, since they are relatively simple, fast, do not require handling of the beetles, and are sensitive enough to quantify the benzoquinones of a single beetle.

Hymenolepis diminuta metacestodes (Cysticeroids) are unable to utilize exogenous trehalose.

Cysticercoids (metacestodes) of Hymenolepis diminuta were incubated in Tyrode's salt solution (pH 7.2) containing 1.25 mM or 40 mM trehalose for 5 or 18 hr, and the amounts of trehalose remaining in the media were determined using high-pressure liquid chromatography. No differences were detected in the amounts of trehalose remaining in control (no cysticercoids) or experimental incubations under any of the experimental conditions. Thus, cysticercoids are apparently unable to utilize trehalose present in the external medium.

Partial characterization of the carbohydrates of the eggs (oncospheres) of the tapeworm, Hymenolepis diminuta.

The carbohydrate constituents of whole eggs and fractions derived from the eggs of Hymenolepis diminuta were partly characterized. Whole eggs contained 9.6 ng of carbohydrate (phenol-sulphuric acid-positive material) per egg, and 3.6 ng of glucose. Analysis of hydrolysed eggs by HPLC demonstrated the presence of glucose, galactose, glycerol, N-acetylgalactosamine, and 2-deoxyribose. Isolated egg shells and the KOH-stable, ethanol-precipitable fraction (putative glycogen) of eggs contained 26 and 76%, respectively, of the total carbohydrate associated with eggs. Glucose and galactose were present in both of these samples, but only glucose was present in the KOH-stable, ethanol-precipitable fraction isolated from chemically deshelled eggs; thus, the source of the galactose in the KOH-stable, ethanol-precipitable fraction isolated from whole eggs was the egg-shells. These data confirm earlier reports that the shells contain carbohydrate; the data demonstrate further that only two monosaccharides (glucose and galactose) are present in the shells, and that they are present as high molecular weight polymers. The ethanol-soluble fraction of eggs contained 2.5% of the total carbohydrate in eggs, and glucose accounted for < 1% of the ethanol-soluble carbohydrate. Analysis of the ethanol-soluble fraction by HPLC demonstrated that the predominant monosaccharides were glycerol and mannose, with smaller quantities of 2-deoxyribose and 2-deoxyglucose. The absence of a 'free pool' of glucose in eggs, and the presence of large amounts of glycerol and mannose, suggest that the pathways of intermediary carbohydrate metabolism in eggs might be very different from those in adult tapeworms.

Partial characterization of the protein and carbohydrate constituents of the egg shell of Hymenolepis diminuta (Cestoda).

The egg shell of Hymenolepis diminuta is composed of 31.7% protein and 2.9% carbohydrate (by weight), as determined using the Lowry and phenol-sulfuric acid assays and bovine serum albumin and glucose, respectively, as standards. Amino acid analyses of egg shells demonstrated the presence of 16 naturally occurring amino acids and six unidentified residues. Histidine accounted for > 22% of the amino acid residues. Hypochlorite-solubilized egg shells were fractionated using a combination of gel, hydroxyapatite and anion-exchange chromatography, and the fractions were analyzed by isoelectric focusing and gel chromatography. The results demonstrate that the shell consists of a complex mixture of proteins (almost all of which have acidic isoelectric points), glycoproteins and possibly free complex carbohydrates.

Partial biochemical characterization of venom from the ant, Pseudomyrmex triplarinus.

Venom from the ant, Pseudomyrmex triplarinus, contains 12 proteins with mol. wts of > 100,000-4200, and they constitute 41.5% of the dry weight. In comparison with published data on ant, wasp, and bee venoms, whole venom has intense phospholipase activity and intermediate hemolytic activity. Four major proteins were isolated and purified by low pressure chromatography. The most abundant protein had a mol. wt of 4200 and weak hemolytic activity. The second most common protein was 20,400 and had phospholipase A2 activity. The other two major proteins had mol. wts of 24,500 and 14,100 and both exhibited phospholipase and direct hemolytic activities. There are eight minor proteins (> 100,000-40,000), each present at about 1% or less of the total protein. Assayed as a mixture, they had hyaluronidase activity. Seventeen free amino acids were detected with aspartic acid, glutamic acid, and proline together making up 72% of the total mass of amino acids. Glycerol was present at a concentration of 3.1% of the dry weight and the venom was devoid of lipids.

Temporal changes in glucose and carbohydrate metabolism in the oncospheres of the cyclophyllidean tapeworm, Hymenolepis diminuta.

When incubated in vitro for 24 h, oncospheres of Hymenolepis diminuta absorb and metabolize radioactive glucose. Between 0 and 12 h post-activation, oncospheres absorb glucose, but glucose is neither metabolized into other carbohydrates nor incorporated into the ethanol-precipitable fraction (which would contain glycogen). Between 12 and 24 h post-activation glucose is incorporated into a number of higher molecular weight carbohydrates that are demonstrable in ethanol extracts of the larvae, as well as the incubation media. Furthermore, measurable amounts of radioactivity are incorporated into the ethanol-precipitable carbohydrate fraction of oncospheres. To determine if these temporal changes in carbohydrate metabolism occurred spontaneously following activation, oncospheres were pre-incubated for 12 h (0-12 h post-activation) in the absence or presence of glucose, and then transferred to media containing radioactive glucose for an additional 12 h (12-24 h post-activation). In these latter experiments, glucose absorption and metabolism between 12 and 24 h post-activation were virtually identical to glucose metabolism in oncospheres that were incubated in radioactive glucose for 0-12 h immediately following activation. Thus, these data do not support the hypothesis that the temporal shift in carbohydrate metabolism occurs spontaneously.

A study of carbohydrate metabolism in the eggs (oncospheres) of the tapeworm, Hymenolepis diminuta, with special reference to glucose.

When incubated in vitro for 24 h, intact eggs, chemically shelled eggs (obtained by treating intact eggs with NaOCl), activated larvae (eggs in which the outer shell and inner envelope were removed), and oncospheres (activated larvae treated with papain to remove the embryophore) absorb and metabolize radioactive glucose. Intact eggs, which are covered by the impermeable shell, absorb only small amounts of exogenous radioactive glucose, while chemically shelled eggs, activated larvae, and oncospheres absorb much larger amounts. Only very small amounts of the exogenous glucose are incorporated into the ethanol-precipitable carbohydrate fraction (which would include glycogen) by any of the preparations of eggs/larvae. However, the glucose is incorporated into higher molecular weight end-products that are liberated into the incubation medium. There is a temporal shift in the ability of activated larvae and oncospheres to metabolize exogenous glucose. Activated larvae and oncospheres absorb but do not metabolize glucose during the first 8 h post-activation. Between 8 and 16 h post-activation, however, virtually all of the absorbed glucose is metabolized into higher molecular weight end-products that are liberated into the incubation media. This temporal shift suggests that activation of oncospheres and cysticercoid morphogenesis are accompanied by distinct changes in carbohydrate metabolism.

Purification, quantification and mechanical hatching of eggs of the tapeworm, Hymenolepis diminuta.

Eggs of Hymenolepis diminuta were separated from host's feces using a combination of NaCl flotation, filtration through nylon monofilament screen cloths, and centrifugation. The resulting purified egg preparations (PEPs) were verified microscopically to be virtually free of contaminating debris. The relationship of absorbance (500 nm) of PEPs and 'number of eggs per unit volume' was linear, and such measurements provided a rapid, reproducible method for quantifying PEPs. The average wet and dry weights of individual eggs were 2.86 x 10(-7) and 0.458 x 10(-7) g, respectively. Eggs were mechanically hatched using a Dounce homogenizer with pestle 'A'. This technique caused no detectable damage to larvae and resulted in a high percentage of 'activated' (i.e. motile) larvae.

Mechanism of 3-0-methylglucose uptake by Hymenolepis diminuta.

The mechanism by which Hymenolepis diminuta (Cestoda) absorbs 3-0-methylglucose (30MG) in vitro was analyzed. Influxes of 0.1 and 0.01 mM-3H-30MG during incubations ranging from 5 s to 60 min were not affected significantly when 10 mM-unlabeled 30MG was present as an inhibitor. After 60 min in 0.1 mM-3H-30MG, the concentration of labeled substrate within tapeworms (0.04 mumol ml-1 worm water = 0.04 mM) was less than that of the bathing medium. Tapeworms incubated for 1 h with either 5 mM-glucose or 5 mM-beta-methylglucose (beta MG) gained 15-20% more water than did tapeworms in saline alone, but addition of 5 mM-30MG to the saline had no significant effect on weight change. When the 3H-30MG concentration was varied from 0.01 to 10 mM, influxes were a linear function of substrate concentrations. These analyses show that H. diminuta absorbs 30MG by simple diffusion alone. Thus, use of this monosaccharide to estimate the internal concentration of actively transported sugars (e.g. glucose or beta MG) in H. diminuta is invalid.

Partial purification and characterization of a soluble acid phosphatase from the tapeworm, Hymenolepis diminuta.

An acid phosphatase activity (APA; EC 3.1.3.2) was demonstrated in homogenates of adult Hymenolepis diminuta. The APA was soluble based on the observation that it did not sediment at 130,000 g. APA was partially purified using a combination of differential centrifugation, ammonium sulphate precipitation, chloroform extraction, and gel and fast-protein-liquid-chromatography. This combination of techniques resulted in a preparation with a specific activity approximately 500 times greater than the crude enzyme preparation. The temperature and pH optima of the partially purified APA were 44 degrees C and pH 5.0. The enzyme appeared to be a monomer with a molecular weight of approximately 62,000. APA had a higher affinity for a greater activity towards aromatic than aliphatic phosphoesters, and phosphoryl transferase activity was demonstrable using 1-butanol and ethylene glycol as acceptors. APA was inhibited significantly by sodium dodecyl sulphate, fluoride, molybdate and tartrate, but CuSO4 and Fast Garnet GBC were poor inhibitors. The precise cellular localization and function of this enzyme remains unknown since it possesses characteristics of both cytoplasmic and lysosomal APA's of other organisms.

Hymenolepis diminuta: further characterization of the membrane-bound acid phosphatase activity associated with the brush border membrane of the tapeworm's tegument.

The acid phosphate activity (APA) associated with the isolated brush border membrane of the tapeworm, Hymenolepis diminuta, hydrolyzed p-nitrophenyl phosphate (PNPP), pyrophosphate (PPi), and beta-glycerophosphate (beta GP). Inhibition of PNPP hydrolysis at pH 4.0 was inhibited in a competitive manner by the following compounds (listed in order of decreasing affinity with their apparent inhibitor constants (Ki')): molybdate (0.031 mM); PPi (0.147 mM); NaF (0.150 mM); o-carboxyphenyl phosphate (0.261 mM); inorganic phosphate (0.770)); arsenate (3.45 mM); tartrate (22.1 mM); and beta GP (29.8 mM). Cu2+, formaldehyde, and arsenite at 10:1, 80:1, and 200:1 inhibitor to substrate ratios did not inhibit APA. The maximal rate of hydrolysis (Vmax) of each substrate was greater at pH 4.0 than 5.0. The apparent Michaelis constant (Km') for PNPP increased from 0.233 to 0.351 mM when the pH was raised from 4.0 to 5.0. The Km' for PPi decreased from 0.101 to 0.046 mM, while the Km' for beta GP changed from 2.04 to 2.22 mM under similar circumstances. APA and alkaline phosphatase activity increased as a function of temperature up to 45 degrees C.