Modulation of the motility of the vagina vera of Ascaris suum in vitro by FMRFamide-related peptides

Ascaris suum contains a large number of FMRFamide-related peptides (FaRPs) of which KNEFIRFamide (AF1), KHEYLRFamide (AF2) and KSAYMRFamide (AF8, also called PF3) have been extensively studied and are known to exert actions on somatic muscle strips of the worm. In the present study, the effects of AF1, AF2 and AF8 on the activity of the vagina vera of female A. suum have been examined in vitro. The vagina vera is a muscular tube connecting the uterus and vagina uteri to the gonopore and is probably involved in regulating egg output. The tissue exhibited spontaneous, rhythmic contractions in vitro, which were modulated by each of the FaRPs tested. The effects of each of the peptides were qualitatively and quantitatively different, and in each case were reversible. AF1 (1 mu M) caused a biphasic response in the form of a transient lengthening of the preparation, followed by a shortening; contractions were initially inhibited but resumed 5 min post-addition of the peptide. Lower concentrations (less than or equal to 0.1 mu M) induced a less marked effect, with rhythmic contractions returning 5 min post-addition. AF2 and AF8 reduced contraction frequency at concentrations greater than or equal to 0.1 mu M. Both peptides also caused the tissue to shorten, although the effects of AF8 on baseline tension were inconsistent. The apparent potencies of AF1 and AF8 on contraction frequency of the vagina vera were 10-fold greater than AF2 and, unlike their actions on A. suum somatic body wall muscles, the actions of AF1 and AF2 were qualitatively different. Indeed, the effects of each of these FaRPs on the vagina vera were markedly different from those observed on the somatic muscle.

COMPARATIVE ANALYSES OF THE NEUROPEPTIDE-F (NPF)-RELATED AND FMRFAMIDE-RELATED PEPTIDE (FARP)-IMMUNOREACTIVITIES IN FASCIOLA-HEPATICA AND SCHISTOSOMA SPP

Immunochemical techniques were used to determine the distribution, chemical characteristics and relative abundance of immunoreactivity (IR) to two native platyhelminth neuropeptides, neuropeptide F (NPF) (Moniezia expansa) and the FMRFamide-related peptide (FaRP), GNFFRFamide, in the trematodes, Fasciola hepatica and Schistosoma mansoni; the larger S. margrebowiei was used in the chemical analysis. Extensive immunostaining for the two peptides was demonstrated throughout the nervous systems of both F. hepatica and S. mansoni, with strong IR also in the innervation of muscular structures, including those associated with the egg-forming apparatus. The patterns of immunostaining were similar to those previously described for the vertebrate neuropeptide Y superfamily of peptides and for FMRFamide. Ultrastructurally, gold labelling of NPF- and GNFFRFamide-IRs was localized exclusively to the contents of secretory vesicles in the axons and somatic cytoplasm of neurones. Double-labelling experiments showed an apparent homogeneity of antigenic sites, in all probability due to the demonstrated cross-reactivity of the FaRP antiserum with NPF. Radioimmunoassay of acid-ethanol extracts of the worms detected 8.3 pmol/g and 4.7 pmol/g equivalents of NPF- and FMRFamide-IRs, respectively, for F. hepatica, and corresponding values of 4.9 pmol/g and 4.3 pmol/g equivalents for S. margrebowiei. Gel-permeation chromatography resolved IR to both peptides in discrete peaks and these eluted in similar positions to synthetic NPF (M. expansa) and GNFFRFamide, respectively.

5-HYDROXYTRYPTAMINE-IMMUNOREACTIVITY IN THE MONOGENEAN PARASITE, ENTOBDELLA-SOLEAE

5-HT-immunoreactivity in Entobdella soleae was found to be extensive throughout both the central and peripheral nervous systems, with the strongest staining occurring in the innervation of the forebody, most notably in the paired cerebral ganglia, pharynx and adhesive pads. In the reproductive system, staining was evident throughout the numerous cell bodies and fibres innervating the musculature of the egg-assembly apparatus. The haptor contained an extensive array of serotoninergic fibres derived from the main longitudinal cords; this array was associated with the haptoral muscles and sclerites, and possibly with the ventral sensory papillae.

A novel calmodulin-like protein from the liver fluke, Fasciola hepatica

An 18.2 kDa protein from the liver fluke, Fasciola hepatica has been identified and characterised. The protein shows strongest sequence similarity to egg antigen proteins from Schistosoma mansoni, Schistosoma japonicum and Clonorchis sinensis. The protein is predicted to adopt a calmodulin-like fold; it thus represents the third calmodulin-like protein to be characterised in F. hepatica and has been named FhCaM3. Compared to the classical calmodulin structure there are some variations. Most noticeably, the central, linker helix is disrupted by a cysteine residue. Alkaline native gel electrophoresis showed that FhCaM3 binds calcium ions. This binding event increases the ability of the protein to bind the hydrophobic fluorescent probe 8-anilinonaphthalene-1-sulphonate, consistent with an increase in surface hydrophobicity as seen in other calmodulins. FhCaM3 binds to the calmodulin antagonists trifluoperazine and W7, but not to the myosin regulatory light chain binding compound praziquantel. Immunolocalisation demonstrated that the protein is found in eggs and vitelline cells. Given the critical role of calcium ions in egg formation and hatching this suggests that FhCaM3 may play a role in calcium signalling in these processes. Consequently the antagonism of FhCaM3 may, potentially, offer a method for inhibiting egg production and thus reducing the spread of infection.

Gastric emptying in patients with flatulent dyspepsia, with and without gallbladder disease

It has been suggested that the symptoms of flatulent dyspepsia are caused by a functional disturbance of the upper gastrointestinal tract. The aim of this study was to investigate delayed gastric emptying as the basis of symptoms in patients with and without gallbladder disease and after cholecystectomy. There were 13 dyspeptic patients with gallbladder disease, 12 with normal gallbladders, and 13 post-cholecystectomy patients. Gastric emptying was measured by means of a 99mTc-labelled scrambled egg meal and external scintillation counter. The rate of emptying in the symptomatic groups was compared with that in 24 asymptomatic normal control subjects and 12 non-dyspeptic patients with gallbladder disease. Delayed gastric emptying tended to occur in patients with gallbladder disease with and without dyspepsia and was not specifically associated with symptoms.

Role of Predalors, Habitat Attributes, and Spatial Autocorrelation on the Distribution of Eggs in the Northern Spectacled Salamander (Salamandrina perspicillata)

We predicted that the probability of egg occurrence of salamander Salamandrina perspicillata depended on stream features and predation by native crayfish Austropotamobius fulcisianus and the introduced trout Salmo trutta. We assessed the presence of S. perspicillata at 54 sites within a natural reserve of southern Tuscany, Italy. Generalized linear models with binomial errors were constructed using egg presence/absence and altitude, stream mean size and slope, electrical conductivity, water pH and temperature, and a predation factor, defined according to the presence/absence of crayfish and trout. Some competing models also included an autocovariate term, which estimated how much the response variable at any one sampling point reflected response values at surrounding points. The resulting models were compared using Akaike's information criterion. Model selection led to a subset of 14 models with Delta AIC(c) <7 (i.e., models ranging from substantial support to considerably less support), and all but one of these included an effect of predation. Models with the autocovariate term had considerably more support than those without the term. According to multimodel inference, the presence of trout and crayfish reduced the probability of egg occurrence from a mean level of 0.90 (SE limits: 0.98-0.55) to 0.12 (SE limits: 0.34-0.04). The presence of crayfish alone had no detectable effects (SE limits: 0.86-0.39). The results suggest that introduced trout have a detrimental effect on the reproductive output of S. perspicillata and confirm the fundamental importance of distinguishing the roles of endogenous and exogenous forces that act on population distribution.

The size and shape of shells used by hermit crabs: A multivariate analysis of Clibanarius erythropus

Shell attributes Such as weight and shape affect the reproduction, growth, predator avoidance and behaviour of several hermit crab species. Although the importance of these attributes has been extensively investigated, it is still difficult to assess the relative role of size and shape. Multivariate techniques allow concise and efficient quantitative analysis of these multidimensional properties, and this paper aims to understand their role in determining patterns of hermit crab shell use. To this end, a multivariate approach based on a combination of size-unconstrained (shape) PCA and RDA ordination was used to model the biometrics of southern Mediterranean Clibanarius erythropus Populations and their shells. Patterns of shell utilization and morphological gradients demonstrate that size is more important than shape, probably due to the limited availability of empty shells in the environment. The shape (e.g. the degree of shell elongation) and weight of inhabited shells vary considerably in both female and male crabs. However, these variations are clearly accounted for by crab biometrics in males only. Oil the basis of statistical evidence and findings from past studies. it is hypothesized that larger males of adequate size and strength have access to the larger, heavier and relatively more available shells of the globose Osilinus turbinatus, which cannot be used by average-sized males or by females investing energy in egg production. This greater availability allows larger males to select more Suitable Shapes. (C) 2009 Elsevier Masson SAS. All rights reserved.

The effects of climate change on ovine parasitic gastroenteritis determined using veterinary surveillance and meteorological data for Northern Ireland over the period 1999-2009

While the influence of temperature and moisture on the free-living stages of gastrointestinal nematodes have been described in detail, and evidence for global climate change is mounting, there have been only a few attempts to relate altered incidence or seasonal patterns of disease to climate change. Studies of this type have been completed for England Scotland and Wales, but not for Northern Ireland (NI). Here we present an analysis of veterinary diagnostic data that relates three categories of gastrointestinal nematode infection in sheep to historical meteorological data for NI. The infections are: trichostrongylosis/teladorsagiosis (Teladorsagia/Trichostrongylus), strongyloidosis and nematodirosis. This study aims to provide a baseline for future climate change analyses and to provide basic information for the development of nematode control programmes. After identifying and evaluating possible sources of bias, climate change was found to be the most likely explanation for the observed patterns of change in parasite epidemiology, although other hypotheses could not be refuted. Seasonal rates of diagnosis showed a uniform year-round distribution for Teladorsagia and Trichostrongylus infections, suggesting consistent levels of larval survival throughout the year and extension of the traditionally expected seasonal transmission windows. Nematodirosis showed a higher level of autumn than Spring infection, suggesting that suitable conditions for egg and larval development occurred after the Spring infection period. Differences between regions within the Province were shown for strongyloidosis, with peaks of infection falling in the period September-November. For all three-infection categories (trichostrongylosis/teladorsagiosis, strongyloidosis and nematodirosis), significant differences in the rates of diagnosis, and in the seasonality of disease, were identified between regions. (C) 2012 Elsevier B.V. All rights reserved.

Single-laboratory validation of a multiplex flow cytometric immunoassay for the simultaneous detection of coccidiostats in eggs and feed

Coccidiostats are authorized in the European Union (EU) to be used as poultry feed additives. Maximum (residue) levels (M(R)Ls) have been set within the EU for consumer and animal protection against unintended carry-over, and monitoring is compulsory. This paper describes the single-laboratory validation of a previously developed multiplex flow cytometric immunoassay (FCIA) as screening method for coccidiostats in eggs and feed and provides and compares different approaches for the calculation of the cut-off levels which are not described in detail within Commission Decision 2002/657/EC. Comparable results were obtained between the statistical (reference) approach and the rapid approaches. With the most rapid approach, the cut-off levels for narasin/salinomycin, lasalocid, diclazuril, nicarbazin (DNC) and monensin in egg, calculated as percentages of inhibition (%B/B0), were 60, 32, 76, 80 and 84, respectively. In feed, the cut-off levels for narasin/salinomycin, lasalocid, nicarbazin (DNC) and monensin were 70, 64, 72 and 78, respectively, and could not be determined for diclazuril. For all analytes, except for diclazuril in feed, the rate of false positives (false non-compliant) in blank samples was lower than 1 %, and the rate of false negatives (false compliant) at the M(R)Ls was below 5 %. Additionally, very good correlations (r ranging from 0.994 to 0.9994) were observed between two different analysers, a sophisticated flow cytometer (FlexMAP 3D(®)) and a more cost-efficient and transportable planar imaging detector (MAGPIX(®)), hence demonstrating adequate transferability.

Patterns of synchrony in natterjack toad breeding activity and reproductive success at local and regional scales

Empirical studies of the spatiotemporal dynamics of populations are required to better understand natural fluctuations in abundance and reproductive success, and to better target conservation and monitoring programmes. In particular, spatial synchrony in amphibian populations remains little studied. We used data from a comprehensive three year study of natterjack toad Bufo calamita populations breeding at 36 ponds to assess whether there was spatial synchrony in the toad breeding activity (start and length of breeding season, total number of egg strings) and reproductive success (premetamorphic survival and production of metamorphs). We defined a novel approach to assess the importance of short-term synchrony at both local and regional scales. The approach employs similarity indices and quantifies the interaction between the temporal and spatial components of populations using mixed effects models. There was no synchrony in the toad breeding activity and reproductive success at the local scale, suggesting that populations function as individual clusters independent of each other. Regional synchrony was apparent in the commencement and duration of the breeding season and in the number of egg strings laid (indicative of female population size). Regional synchrony in both rainfall and temperature are likely to explain the patterns observed (e.g. Moran effect). There was no evidence supporting regional synchrony in reproductive success, most likely due to spatial variability in the environmental conditions at the breeding ponds, and to differences in local population fitness (e.g. fecundity). The small scale asynchronous dynamics and regional synchronous dynamics in the number of breeding females indicate that it is best to monitor several populations within a subset of regions. Importantly, variations in the toad breeding activity and reproductive success are not synchronous, and it is thus important to consider them both when assessing the conservation status of pond-breeding amphibians. © 2012 The Authors. Ecography © 2012 Nordic Society Oikos.

In Vitro and In Vivo Anti-Schistosomal Activity of the Alkylphospholipid Analog Edelfosine

Background: Schistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. Five species of Schistosoma are known to infect humans, out of which S. haematobium is the most prevalent, causing the chronic parasitic disease schistosomiasis that still represents a major problem of public health in many regions of the world and especially in tropical areas, leading to serious manifestations and mortality in developing countries. Since the 1970s, praziquantel (PZQ) is the drug of choice for the treatment of schistosomiasis, but concerns about relying on a single drug to treat millions of people, and the potential appearance of drug resistance, make identification of alternative schistosomiasis chemotherapies a high priority. Alkylphospholipid analogs (APLs), together with their prototypic molecule edelfosine (EDLF), are a family of synthetic antineoplastic compounds that show additional pharmacological actions, including antiparasitic activities against several protozoan parasites.

Methodology/Principal Findings: We found APLs ranked edelfosine> perifosine> erucylphosphocholine> miltefosine for their in vitro schistosomicidal activity against adult S. mansoni worms. Edelfosine accumulated mainly in the worm tegument, and led to tegumental alterations, membrane permeabilization, motility impairment, blockade of male-female pairing as well as induction of apoptosis-like processes in cells in the close vicinity to the tegument. Edelfosine oral treatment also showed in vivo schistosomicidal activity and decreased significantly the egg burden in the liver, a key event in schistosomiasis.

Conclusions/Significance: Our data show that edelfosine is the most potent APL in killing S. mansoni adult worms in vitro. Edelfosine schistosomicidal activity seems to depend on its action on the tegumental structure, leading to tegumental damage, membrane permeabilization and apoptosis-like cell death. Oral administration of edelfosine diminished worm and egg burdens in S. mansoni-infected CD1 mice. Here we report that edelfosine showed promising antischistosomal properties in vitro and in vivo.

Supplementary feeding increases Common Buzzard Buteo buteo productivity but only in poor-quality habitat

Temporal heterogeneity in the effects of food supply during the breeding season on the productivity of the Common Buzzard Buteo buteo was investigated in a supplementary feeding experiment. Pairs were fed artificially (1) before egg-laying, (2) after chicks hatched and (3) continuously throughout the season, and compared with (4) unfed controls. Pairs fed before egg-laying had marginally larger clutches than those not fed, but lay date, egg volume and weight, brood size and hatching success were unaffected. Territorial quality had far greater effects, with pairs nesting in low-quality habitats (bog, scrub and semi-natural grassland) laying later and having lower hatching success, smaller broods and fewer fledglings than those in more productive agricultural landscapes. Supplementary feeding after egg hatching neutralized the negative effect of poor habitat, resulting in fed birds having significantly more fledglings. This study emphasizes the importance of food availability when provisioning chicks in suboptimal habitats and has implications for the success of diversionary feeding in reducing game losses to Buzzards.

Biological control of the fish louse in a rainbow trout fishery

Novel egg-laying boards were found to be effective in the biological control of the freshwater fish louse Argulus foliaceus in a 12.9 ha rainbow trout Oncorhynchus mykiss fishery which had a high prevalence and intensity of infection of juvenile parasites in the early spring of 1999. Approximately 228 000d during an extensive 14 week period of egg laying which peaked in June 1999. In contrast, only 1566 clutches were harvested in 2000, when egg laying activity showed a bi-modal distribution, peaking in May and again in July and August. iaceus on rainbow trout in consecutive years was 2.9 : 1 and 2.1 : 1. Estimates of the size of the female A. foliaceus population based on egg-laying activity in 1999 exceeded that derived from measurements of prevalence and intensity of infection, whereas in 2000, this was more in balance. A minimum temperature of 10 degree C was identified for egg laying, which occurred continuously from May to October in a broadly synchronous manner.. Copyright 2002 The Fisheries Society of the British Isles

The Fasciola hepatica genome: gene duplication and polymorphism reveals adaptation to the host environment and the capacity for rapid evolution

BACKGROUND: The liver fluke Fasciola hepatica is a major pathogen of livestock worldwide, causing huge economic losses to agriculture, as well as 2.4 million human infections annually.

RESULTS: Here we provide a draft genome for F. hepatica, which we find to be among the largest known pathogen genomes at 1.3 Gb. This size cannot be explained by genome duplication or expansion of a single repeat element, and remains a paradox given the burden it may impose on egg production necessary to transmit infection. Despite the potential for inbreeding by facultative self-fertilisation, substantial levels of polymorphism were found, which highlights the evolutionary potential for rapid adaptation to changes in host availability, climate change or to drug or vaccine interventions. Non-synonymous polymorphisms were elevated in genes shared with parasitic taxa, which may be particularly relevant for the ability of the parasite to adapt to a broad range of definitive mammalian and intermediate molluscan hosts. Large-scale transcriptional changes, particularly within expanded protease and tubulin families, were found as the parasite migrated from the gut, across the peritoneum and through the liver to mature in the bile ducts. We identify novel members of anti-oxidant and detoxification pathways and defined their differential expression through infection, which may explain the stage-specific efficacy of different anthelmintic drugs.

CONCLUSIONS: The genome analysis described here provides new insights into the evolution of this important pathogen, its adaptation to the host environment and external selection pressures. This analysis also provides a platform for research into novel drugs and vaccines.

Genetic structure of the lumpfish Cyclopterus lumpus across the North Atlantic

Lumpfish, or lumpsucker, Cyclopterus lumpus (Linnaeus, 1758) is widely distributed in the North Atlantic Ocean. It has a considerable economic value and substantial fisheries occur in several North Atlantic regions owing to the use of its fully ripe internal egg masses in the ovaries as an alternative to sturgeon caviar. Despite being intensively fished in several locations, biological knowledge is limited and no genetic structure information is available. In this study, the stock structure of C. lumpus was investigated across the North Atlantic using ten microsatellite loci. Out of ten loci, two exhibited higher level of differentiation but their inclusion/ exclusion from the analyses did not drastically change the observed genetic pattern. A total of three distinct genetic groups were detected: Maine-Canada-Greenland, Iceland-Norway and Baltic Sea. These results, discussed in terms of origin of differentiation, gene flow, and selection, showed that gene flow was rather limited among the detected groups, and also between Greenland and Maine-Canada.