Metacercarial aggregation in Digenea (Fasciola hepatica and Paramphistomum daubneyi): environmental or species determinism?

Metacercarial aggregation of Fasciola hepatica and Paramphistomum daubneyi was studied under experimental conditions to determine if the formation of these aggregates was influenced by environmental factors, or it was a characteristic of trematode species. This process was studied using the confinement of infected snails on the bottom of Petri dishes (diameter, 14 cm) for 3 days. The formation of metacercarial aggregates of F. hepatica was not significantly modified by environmental factors such as intensity and duration of lighting, quality and volume of water. Metacercariae of F. hepatica were more numerous on the Petri dish walls and 63.9% of them constituted aggregates. In contrast, most metacercariae of P. daubneyi were found on the Petri dish bottoms and 78.3% of them were isolated or in groups of two metacercariae each. The mean number of metacercariae per aggregate ranged from 6.7 to 12.2 in the case of F. hepatica, and from 2.7 to 4.5 in the case of P. daubneyi. However, these mean numbers were independent of the site of cercarial attachment. The tendency of cercariae to form metacercarial aggregations was a characteristic of F. hepatica and was species determined.     

Amiodarone — an inhibitor of phospholipase activity: a comparative study of the inhibitory effects of amiodarone,chloroquine and chlorpromazine

Amiodarone, an antiarrhythmic drug, like chloroquine and chlorpromazine, is a tertiary amine with amphiphilic properties. Chloroquine and chlorpromazine are known inhibitors of phospholipases. All three drugs produce characteristic microcorneal deposits consistent with lysosomal accumulations of phospholipid. Similar lysosomal bodies were found in leukocytes of 15 patients on chronic amiodarone treatment as well as 3 patients each on chloroquine and chlorpromazine, suggestive of widespread systemic inhibition of lysosomal phospholipases. These lysosomal inclusions were similar in morphology, irrespective of the drug given, and were of four types: multilamellar, amorphous dense, amorphous light, or a combination of 2 or more of the preceding types. There was no simple relationship between the number of inclusion bodies per cell and the cumulative dose of amiodarone (r=0.02) or amiodarone serum levels (r=0.11). An in vitro assay was used to compare the effects of the three drugs on Ca²⁺-dependent phospholipase A and C activities. Phospholipase A₂ activity was inhibited in a dose-dependent fashion (1–8 mg/assay) by all three drugs in the order: chlorpromazine > amiodarone > chloroquine. The inhibitory effect on phospholipase C was more pronounced with all three drugs, producing almost total inhibition at 8 mg/assay. In a Ca²⁺-independent lysosomal phospholipase A system, amiodarone had a greater effect, producing 85% inhibition at 1.2 mg/assay. These observations suggest that amiodarone, like other cationic amphiphiles, induces a generalized phospholipidosis by inhibiting phospholipid catabolism. Its therapeutic and toxic effects may be due to its ability to modulate both Ca²⁺-dependent membrane phospholipases and Ca²⁺-independent acid phospholipases.     

Natural infection of Lymnaea truncatula by the liver fluke Fasciola hepatica in the Porma Basin, León, NW Spain

For two years fortnightly malacological samplings were carried out to collect Lymnaea truncatula (Mollusca; Basommatophora) at five points in the upper and middle Porma river basin, León, Spain. The highest numbers of snails were collected in September, May and November. Of the 5486 molluscs examined, 11.41% harboured F. hepatica (Trematoda; Digenea) with an average intensity of 20.14. In general, the values of both infection prevalence and intensity increased with the size of the snails. It was in October when the highest figures for each parameter mentioned above were detected (18.73% and 28.48, respectively). The chi-square test showed statistically significant differences in relation to the infection prevalence among the groups of molluscs established according to: their length; the months in which they were collected; the sampling localities; monthly average ambient temperature; precipitation during the collection. Similarly, statistically significant differences were detected in the intensity of the infection among the groups of molluscs previously established, except for that based on the values of precipitation. Generally, parasites were found in the same snail at different stages of development. It seems that most mollusc infections occur in February-March and at the end of summer-beginning of autumn periods. The highest rate of rediae with mature cercariae ready to be shed were detected between September and December. Metacercariae in the grass samples were also observed at the end of autumn. For this reason, this period could be considered as the most suitable for infection of the definitive hosts to take place.     

In vitro evaluation of the action of the nematophagous fungi Duddingtonia flagrans, Monacrosporium sinense and Pochonia chlamydosporia on Fasciola hepatica eggs

This work evaluated the in vitro action of four isolates of the nematophagous fungi Duddingtonia flagrans (AC001), Monacrosporium sinense (SF53) and Pochonia chlamydosporia (VC1 and VC4) on eggs of Fasciola hepatica. The eggs were plated on 2% water-agar with the grown isolates and control without fungus. After 7, 14 and 21 days, the eggs were removed and classified according to the following parameters: effect type 1, lytic effect with no morphological damage to eggshells; type 2, lytic effect with morphological changes in eggshells and embryos; and type 3, lytic effect with morphological changes in embryos and eggshells, with hyphal penetration and internal egg colonization. Pochonia chlamydosporia showed ovicidal activity on F. hepatica eggs in the studied intervals of the type-3 effect, of 12.8% (VC1) and 16.5% (VC4); 14.4% (VC1) and 18.7% (VC4), 20.1% (VC1) and 21.5 % (VC4), over 7, 14 and 21 days respectively. No statistical difference was found (P > 0.01) among the isolates VC1 and VC4 for effects type 1, 2 and 3 during the studied intervals. Duddingtonia flagrans (AC001) and Monacrosporium sinense fungi only showed effect type 1, with no significant difference between them, with the following results: 60.1% (AC001) and 57.5% (SF53); 62.3% (AC001) and 62.0% (SF53); 66.5% (AC001) and 73.4% (SF53), over 7, 14 and 21 days respectively. Pochonia chlamydosporia fungi negatively influenced the in vitro F. hepatica viability. Therefore it can be considered as a potential biological control agent for this helminth.     

The effects of progressive hypoxia on the crustacean cardiovascular system: a comparison of the freshwater crayfish, (Procambarus clarkii ), and the lobster (Homarus americanus)

The heart rate of crayfish (Procambarus clarkii) and lobsters (Homarus americanus) decreased (bradycardia) as partial pressure of O₂ (P O ₂) decreased, yet cardiac output (V _b) was maintained via an increased stroke volume (S _v) to P O ₂s of 40 mmHg and 75 mmHg for crayfish and lobsters, respectively. V _b was redistributed in both animals. Flow through the anterior aorta increased while flow dropped through the posterior aorta and sternal artery to a P O ₂ of 30 mmHg; below this flow was no longer maintained in crayfish. In the lobster, flow increased to the lateral arteries and the ventral thoracic artery while flow through the anterior and posterior aortas, sternal artery and ventral abdominal artery decreased to a P O ₂ of 75 mmHg. Anterior hemolymph flow was maintained or increased in both animals presumably to supply nervous tissue and cephalic sense organs better. Crayfish showed an increase in intracardiac and mean arterial hemolymph pressures as P O ₂ declined. The increased pressures combined with the net increase in cardiac filling pressure and diastolic filling time could have accounted for the increased S _V. The cardiovascular response exhibited by both the crayfish and lobster was P O ₂ dependent; below a critical water P O ₂ active compensation was no longer observed. Accepted: 26 November 1997     

Naiveté and an aquatic-terrestrial dichotomy in the effects of introduced predators

There is much evidence that suggests that freshwater systems are more sensitive to introduced predators than are terrestrial or marine systems. We argue here that this dichotomy reflects widespread naiveté toward introduced predators among freshwater prey. Continental terrestrial animals are seldom naive toward novel predators owing to the homogenizing effects of historical biotic interchanges. Comparable biotic interchanges might have also precluded prey naiveté in most marine systems. By contrast, freshwater systems exhibit persistent large- and small-scale heterogeneity in predation regimes. This heterogeneity promotes naiveté at multiple spatial scales in freshwater prey, thereby producing a systemic vulnerability to introduced predators that is not seen in continental terrestrial or marine systems.     

Sequence and expression of an α-amylase gene in four related species of prickleback fishes (Teleostei: Stichaeidae): ontogenetic,dietary, and species-level effects

Partial α-amylase gene sequences were determined and α-amylase gene expression was quantified in four species of carnivorous, omnivorous, and herbivorous prickleback fishes (family Stichaeidae) to assess the effects of ontogeny, diet, and species on expression of this gene. Pairwise comparison of α-amylase nucleotide sequences revealed 96–98 % identity, and comparison of amino acid portions revealed 93–95 % similarity among the four prickleback species. Expression was determined using in situ hybridization and intensity of expression quantified using image analysis. Alpha-amylase expression level was compared in three feeding categories of the four species: (1) small, wild-caught carnivorous juveniles; (2) larger, wild-caught juveniles of the carnivorous species and the three that had shifted to herbivory or omnivory; and (3) larger, juveniles produced by feeding a low-starch artificial diet to small juveniles until they reached the size of the larger wild-caught juveniles. The results showed no dietary effect in any species but significant ontogenetic and species-level effects in Cebidichthys violaceus, as well as in the sister species Xiphister mucosus and X. atropurpureus. Based on a phylogeny for the Stichaeidae produced for this study using two mtDNA genes and one nuclear gene, the ontogenetic dietary shifts to herbivory/omnivory evolved independently in C. violaceus and in the clade containing the two species of Xiphister. All three of these species increased α-amylase gene expression with increase in size and had higher expression than Anoplarchus purpurescens, which is a member of a third, stichaeid clade comprising carnivores. These results show the importance of α-amylase in the herbivores and omnivores.     

Purification,characterization, and comparison of anα-glucosidase (endo-oligo-1, 4-glucosidase) from the mesophileBacillus subtilis and the obligate thermophileBacillus caldolyticus

Thep-nitrophenyl--d-maltoside hydrolyzing-glucosidase from the mesophileBacillus subtilis 25S and the obligate thermophileBacillus caldolyticus C2 was purified, characterized, and compared in order to determine the molecular mechanisms that may confer thermostability of starch-degrading enzymes. Both enzymes showed endo-oligo-1,4-glucosidase activity owing to their identical hydrolysis of linear malto-oligosaccharides to maltose and glucose as determined by thin-layer chromatography. Neither enzyme showed activity againstp-nitrophenyl--d-glucopyranoside, maltose, isomaltose, isomaltotriose, or panose. The enzymes may tentatively be classified as a panose-producing pullulanase owing to their hydrolysis of pullulan. The 25S and C2 enzymes were composed of two identical subunits of M_r 55,000 and 60,000 respectively. Both the 25S and C2 enzymes have a pI of 4.85, pH optimum of 7.5 and 7.0, and K_m values for the chromogenic substratep-nitrophenyl--d-maltoside of 2.96 mM and 1.31 mM respectively. The 25S enzyme exhibited optimal activity between 35 and 37°C, and complete inactivation after 10 min at 45°C, while the C2 enzyme showed optimal activity at 60°C and retained 100% of initial activity at 60°C for 2 h. The C2 enzyme required a minimum of 0.02% 2-mercaptoethanol or 0.01 mM EDTA for thermostability. A comparison of the amino acid compositions showed an increase in the number of proline, alanine, and leucine residues for the thermostable C2 enzyme. These alterations in hydrophobicity may influence enzyme thermostability; this may be a factor in the design of engineered proteins for industrial use.Florida Agricultural Experiment Station Journal Series No. 9985     

Organisation of the endosperm and endosperm–placenta syncytia in bladderworts (Utricularia, Lentibulariaceae) with emphasis on the microtubule arrangement

Multinucleate cells play an important role in higher plants, especially during reproduction; however, the configurations of their cytoskeletons, which are formed as a result of mitosis without cytokinesis, have mainly been studied in coenocytes. Previous authors have proposed that in spite of their developmental origin (cell fusion or mitosis without cytokinesis), in multinucleate plant cells, radiating microtubules determine the regular spacing of individual nuclei. However, with the exception of specific syncytia induced by parasitic nematodes, there is no information about the microtubular cytoskeleton in plant heterokaryotic syncytia, i.e. when the nuclei of fused cells come from different cell pools. In this paper, we describe the arrangement of microtubules in the endosperm and special endosperm–placenta syncytia in two Utricularia species. These syncytia arise from different progenitor cells, i.e. cells of the maternal sporophytic nutritive tissue and the micropylar endosperm haustorium (both maternal and paternal genetic material). The development of the endosperm in the two species studied was very similar. We describe microtubule configurations in the three functional endosperm domains: the micropylar syncytium, the endosperm proper and the chalazal haustorium. In contrast to plant syncytia that are induced by parasitic nematodes, the syncytia of Utricularia had an extensive microtubular network. Within each syncytium, two giant nuclei, coming from endosperm cells, were surrounded by a three-dimensional cage of microtubules, which formed a huge cytoplasmic domain. At the periphery of the syncytium, where new protoplasts of the nutritive cells join the syncytium, the microtubules formed a network which surrounded small nuclei from nutritive tissue cells and were also distributed through the cytoplasm. Thus, in the Utricularia syncytium, there were different sized cytoplasmic domains, whose architecture depended on the source and size of the nuclei. The endosperm proper was isolated from maternal (ovule) tissues by a cuticle layer, so the syncytium and chalazal haustorium were the only way for nutrients to be transported from the maternal tissue towards the developing embryo.     

Spermatogenesis in Triatoma williami Galvão,Souza and Lima (1965) (Hemiptera,Triatominae)

Cytogenetics has proven to be an important tool in the study of triatomine taxonomy. Triatoma williami is a triatomine that has been classified within the Matogrossensis subcomplex. In this study, the cytogenetic technique of lacto-aceto-orcein was used to confirm the karyotype of this species, which was found to be 2n = 22 (20A + XY). In this study, metaphases I and II were analyzed and the heteropyknotic pattern during the spermatogenesis and spermiogenesis was described, with results that corroborated the classification of T. williami in the Matogrossensis subcomplex.     

A comparison of the effects of NN′-dicyclohexylcarbodi-imide, oligomycin A and aurovertin on energy-linked reactions in mitochondria and submitochondrial particles

1. The effects of dicyclohexylcarbodi-imide, oligomycin A and aurovertin on enzyme systems related to respiratory-chain phosphorylation were compared. Dicyclohexylcarbodi-imide and oligomycin A have very similar functional effects, giving 50% inhibition of ATP-utilizing and ATP-generating systems at concentrations below 0.8nmole/mg. of submitochondrial-particle protein. Aurovertin is a more potent inhibitor of ATP synthesis, giving 50% inhibition at 0.2nmole/mg. of protein. However, aurovertin is a less potent inhibitor of ATP-utilizing systems: the ATP-driven energy-linked nicotinamide nucleotide transhydrogenase is 50% inhibited at 3.0nmoles/mg. of protein and the ATP-driven reduction of NAD(+) by succinate is 50% inhibited at 0.95nmole/mg. of protein. 2. With EDTA-particles (prepared by subjecting mitochondria to ultrasonic radiation at pH9 in the presence of 2mm-EDTA) the maximum stimulation of the ATP-driven partial reactions is effected by similar concentrations of oligomycin A and dicylcohexylcarbodi-imide, but the latter is less effective. The stimulatory effects of suboptimum concentrations of dicyclohexylcarbodi-imide and oligomycin A are additive. Aurovertin does not stimulate these reactions or interfere with the stimulation by the other inhibitors. 3. Dicyclohexylcarbodi-imide and oligomycin A stimulate the aerobic energy-linked nicotinamide nucleotide transhydrogenase of EDTA-particles, but the optimum concentration is higher than that required for the ATP-driven partial reactions. Aurovertin has no effect on this reaction. 4. The site of action of dicyclohexylcarbodi-imide is in CF(0), the mitochondrial fraction that confers oligomycin sensitivity on F(1) mitochondrial adenosine triphosphatase.     

Cardiovascular effects of bPTH-(1–34) and isoproterenol in the frog,Rana tigrina

1. The cardiovascular effects of bovine parathyroid hormone fragment [bPTH-(1–34)] and isoproterenol (ISO) on frog (Rana tigrina) isolated atria and helical strips of blood vessels were examined since PTH produces a β-adrenergic-like effect in the mammal.2. Data showed that both bPTH-(1–34) and ISO were vasorelaxant in KC1 and arginine vasoctocin (AVT) preconstricted dorsal aorta, iliac and femoral arteries.3. They both relaxed extracellular calcium-dependent contrations.4. There was no additive nor synergistic effect between them in AVT preconstricted strips.5. Both bPTH-(1–34) and ISO were positively inotroplc but differed in their chronotroplc effects, being negative and positive.6. In the tiger frog, bPTH-(1–34) shows β-adrenergic like contractile responses in both the cardiac and vascular smooth muscle as in the mammal, but not in the heart rate.     

Identification of new polymorphic positions in rDNA sequences of the “intermediate” Fasciola forms

Fascioliasis is a foodborne zoonotic disease generally caused by the parasitic flukes Fasciola gigantica and Fasciola hepatica in class Trematoda. An “intermediate” Fasciola forms between F. gigantica and F. hepatica has been shown to exist. However, the relationships among F. gigantica, F. hepatica, and “intermediate” Fasciola forms remain unclear. In this study, we found five new polymorphic positions in 18S and 28S rDNAs sequences of “intermediate” Fasciola forms. According to the high-throughput sequencing results, all known 16 polymorphic positions of “intermediate” Fasciola forms show a clear and consistent tendency for F. gigantica or F. hepatica, and the percentages of the most frequently occurring bases were different in specimens. In the three ITS sequence fragments, hybrid-type base combinations of the polymorphic positions were detected, and the percentages of the most frequent base combinations were different in specimens too. In addition, interestingly, the newly detected ITS-802 position was not a traditional polymorphic position in “intermediate” Fasciola forms, and the bases in ITS-802 position are not same as the allele bases of F. gigantica or F. hepatica. Our results will be helpful to investigations into the molecular taxonomy, population genetics, and ecology of F. gigantica, F. hepatica, and “intermediate” Fasciola forms.     

The role of estradiol 17β in the activation of the uterus during premature labor and the effect of naproxen,an inhibitor of prostaglandin synthesis

Fifty-two pregnant rats were ovariectomized on day 16 of gestation to induce estrogen and progesterone deficiencies and the animals were divided into four Groups. Ovariectomy alone (Group A) resulted in the premature delivery of 21% of the fetuses. When ovariectomy was followed by estrogen treatment restoring normal estrogen levels (Group B), premature delivery of the fetuses increased to 96%. Daily injections of 25mg/kg b.w. Naproxen (Group C), given from the day of ovariectomy to reduce prostaglandin synthesis, completely prevented premature delivery if the animals received no estradiol treatment and reduced prematurity to 50% if estradiol had been administered (Group D).It is concluded that the estrogen and progesterone deficiency, induced by ovariectomy, provokes a regulatory imbalance which promotes premature delivery. This imbalance is enhanced when the estradiol levels are restored to normal values, probably because estradiol increases the synthesis of prostaglandin, the intrinsic myometrial stimulant. Naproxen, an inhibitor of prostaglandin synthesis, restores the regulatory balance, partially or completely, depending on the estrogen levels.     

The effects of precocene treatment on egg wax production in Gené's organ and egg viability in the cattle tickBoophilus microplus (Acarina ixodidae): An ultrastructural study

Was synthesis by Gené's organ, the egg waxing organ of ticks, is disrupted by precocene treatment of engorged femaleBoophilus microplus, resulting in desiccated nonviable eggs due to the absence of a waterproofing wax layer. Electron microscopy shows that precocene has a destructive effect on the glandular cells of Gené's organ. The precocene also inhibited in vitro was synthesis by the gland cells, indicating a selective cytotoxic effect. Oogenesis and oviposition were otherwise unaffected, also suggesting that precocene affects the gland cells directly, rather than indirectly by antagonism of juvenile hormone.     

The effects of “Cell age” upon the lethal effects of physical and chemical mutagens in the yeast, Saccharomyces cerevisiae

Summary SummaryYeast cultures progressing from the exponential to the stationary phase of growth showed changes in cell sensitivity to physical agents such as UV light, heat shock at 52° C and the chemical mutagens ethyl methane sulphonate, nitrous acid and mitomycin C.Exponential phase cells showed maximum resistance to UV light and minimum resistance to heat shock and the three chemicals. The increased resistance of exponential phase cells to UV light was shown to be dependent upon the functional integrity of the RAD ₅₀ gene.Treatment of growing yeast cultures with radioactively labelled ethyl methane sulphonate indicated the preferential uptake of radioactivity during the sensitive exponential stage of growth. The results indicated that the differential uptake of the chemical mutagens was responsible for at least a fraction of the variations in cell sensitivity observed in yeast cultures at different phases of growth.     

The pigs and “Old World peccaries” (Suidae and Palaeochoeridae,Suoidea, Artiodactyla) from the Miocene of Sandelzhausen (southern Germany): phylogeny and an updated classification of the Hyotheriinae and Palaeochoeridae

The fossil remains of two species of Suoidea (Artiodactyla, Mammalia) from the Early/Middle Miocene locality of Sandelzhausen (MN5; Bavaria, Germany) are described. A skull and some isolated teeth and bones reveal hitherto unknown features of Schizoporcus muenzenbergensis, Schizoporcini, Taucanaminae, Palaeochoeridae (Old World peccaries), Suoidea. The phylogeny of the Taucanaminae is discussed and an updated classification of the Palaeochoeridae is presented. The new names Schizoporcus and Schizoporcini replace the junior homonyms Schizochoerus Crusafont and Lavocat (1954) and Schizochoerini Golpe-Posse (1974). Remains of several skulls and mandibles, over 50 associated tooth rows, over 300 isolated teeth, and over 200 bones, constitute one of the largest collections of a Miocene suid known, and are assigned to Hyotherium soemmeringi wylensis, Hyotheriini, Hyotheriinae, Suidae (pigs), Suoidea. Hyotherium is the oldest certain suid genus known and many assumed it to be one of the most primitive. While the postcranial bones of the Suidae and Palaeochoeridae differ in many ways, the bones of Hyotherium are already very similar in morphology to those of living pigs, although they are much more slender, suggesting that the genus was more fleet-footed. Features related to rooting behaviour indicate that Hyotherium was a more efficient rooter than Palaeochoeridae and living Dicotylidae, but not as efficient as living suids. The phylogeny of the Hyotheriinae is discussed. The subfamily is divided into Hyotheriini and Aureliachoerini, new tribe, and an updated classification is presented.     

Cytochrome oxidase activity associated with the inside of the cytoplasmic membrane in swarm cells and sheath-forming cells of Sphaerotilus natans: an ultrastructural study using 3,3′-diaminobenzidine (DAB)

Cytochrome oxidase activity has been demonstrated by electronmicroscopical cytochemical methods in swarm cells as well as in the sheathforming cells of Sphaerotilus natans. This enzyme is associated with the inside of the cytoplasmic membrane. The conditions for demonstrating this cytoplasmic membrane-associated cytochrome oxidase differ from those enabling the demonstration of the cytochrome oxidase in the polar organelle of this bacterial species. The location of this enzyme also contrasted to that generally known from other bacteria and from mitochondria.