Changes in the structure of Nippostrongylus brasiliensis intestinal cells during development from the free-living to the parasitic stages

The ultrastructure of Nippostrongylus brasiliensis intestinal cells was examined in free-living, feeding second-stage larvae, infective, nonfeeding third-stage larvae, and parasitic, feeding third-stage larvae. The intestinal cells of second-stage larvae were characterized by a well-developed microvillar border, large numbers of ribosomes, Golgi complexes, rough endoplasmic reticulum, and nuclei with prominent nucleoli. The intestinal cells of infective, third-stage larvae had very few microvilli and the cells were extremely narrow. Few ribosomes, Golgi complexes, and little rough endoplasmic reticulum were present. Nuclei did not contain nucleoli. When worms were introduced into an in vitro culture system, development of intestinal cells began. By 36 hr, microvilli were well differentiated and the cysoplasm contained numerous ribosomes and Golgi complexes, and rough endoplasmic reticulum, mitochondria, and nucleoli were prominent. These morphological changes were related to changes in the physiology of Nippostrongylus brasiliensis which occur during development from a free-living to parasitic form.     

High cold tolerance through four seasons and all free-living stages in an ectoparasite

Off-host stages of temperate parasites must cope with low temperatures. Cold tolerance is often highest in winter, as a result of diapause and cold acclimation, and low during the active summer stages. In some blood-feeding ectoparasites, offspring provisioning determines cold tolerance through all the non-feeding, off-host stages. Large size increases survival in the cold, but so far seasonal variation in within-female offspring size has not been associated with offspring cold tolerance. The deer ked (Lipoptena cervi) reproduces on cervids from autumn to spring. Newborn pupae drop off the host, facing frosts without any acclimation. We examined cold tolerance through 4 seasons and from birth to adulthood by means of short- and long-term frost exposure. We expected females to produce more tolerant offspring in winter than in spring. Large spring pupae survived prolonged frosts better than did small winter pupae. Thus more tolerant offspring were not produced when the temperature outside the host is at its lowest. Unexpectedly, the freezing points were -20 °C or below all year round. We showed that high cold tolerance is possible without acclimation regardless of life stage, which presumably correlates with other survival characteristics, such as the starvation resistance of free-living ectoparasites.     

Intertidal meiofauna of a high-latitude glacial Arctic fjord (Kongsfjorden, Svalbard) with emphasis on the structure of free-living nematode communities

Meiofauna communities of four intertidal sites, two sheltered and two more exposed, in Kongsfjorden (Svalbard) were investigated in summer 2001 at two different tidal levels (i.e. the low-water line and close below the driftline, referred to as mid-water (MW) level). A total of seven meiofaunal higher taxa were recorded with nematodes, oligochaetes and turbellarians being numerically dominant. Mean total meiofaunal densities ranged between 50 ind. 10 cm⁻² and 903 ind. 10 cm⁻². Our data showed a clear decrease in total meiofaunal densities with increasing coarseness of the sediment. Total meiofaunal biomass varied from 0.2 g dwt m⁻² to 2 g dwt m⁻² and, in general, was high even at low meiofaunal densities, i.e. larger interstitial spaces in coarser sediments supported larger meiofauna, especially turbellarians. The results on the vertical distribution of meiofauna contrasted sharply with typical meiobenthic depth profiles on other beaches, probably in response to ice-scouring and concomitant salinity fluctuations. Oligochaetes were the most abundant taxon, with a peak density of 641 ind. 10 cm⁻² at Breoyane Island. They were mainly comprised of juvenile Enchytraeidae, which prohibited identification to species/genus level. Nematode densities ranged between 4 ind. 10 cm⁻² and 327 ind. 10 cm⁻². Nematodes were identified up to genus level and assigned to trophic guilds. In total, 28 nematode genera were identified. Oncholaimus and Theristus were the most abundant genera. The composition of the nematode community and a dominance of predators and deposit feeders were in agreement with results from other arctic and temperate beaches. Nematode genus diversity was higher at the more sheltered beaches than at the more exposed ones. Low-water level stations also tended to harbour a more diverse nematode communities than stations at the MW level. Differences in nematode community structure between low- and MW stations of single beaches were more pronounced than community differences between different beaches and were mainly related to resources quality and availability.     

Toxicity of Bacillus thuringiensis to parasitic and free-living life-stages of nematode parasites of livestock

A collection of Bacillus thuringiensis (Bt) strains (Bts) were screened for activity against the free-living larval stages of nematode parasites of livestock. Two strains were identified with significant activity in inhibiting larval development of Haemonchus contortus, Trichostrongylus colubriformis and Ostertagia circumcincta. These strains were also toxic to the adult parasitic stages of these nematode species in vitro. Adult H. contortus and O. circumcincta showed complete cessation of movement within 2 and 4 days, respectively. Trichostrongylus colubriformis adults were less affected, however, movement was still significantly reduced compared with controls. The in vitro activity against the larval stages was of a magnitude similar to or greater than that seen with the anthelmintic drugs thiabendazole and levamisole. N-terminal amino acid sequencing indicated that the two Bts contained either Cry5A and Cry5B proteins, or a Cry13 protein, and the presence of the corresponding cry5A, cry5B and cry13 genes was confirmed by PCR and sequencing. Bacillus thuringiensis spore-crystal suspensions exposed to acidic pH conditions (pH相似文献   

Nippostrongylus brasiliensis and Ascaridia galli: mitochondrial respiration in free-living and parasitic stages

Aerobic respiratory pathways have been delineated and respiratory efficiency has been assessed in mitochondria isolated from embryonated eggs, infective larvae, and adult Nippostrongylus brasiliensis and Ascaridia galli. Mitochondrial respiration in free-living stages of N. brasiliensis is mediated mainly by a mammalian-like antimycin A- and cyanide-sensitive pathway; specific respiratory activity is high and oxidative phosphorylation efficient. In mitochondria of adult N. brasiliensis, antimycin A- and cyanide-sensitive respiration is decreased relative to respiration though an alternative pathway, and specific respiratory activity and mitochondrial efficiency are lower. Respiration in mitochondria from embryonated eggs and tissues of adult A. galli is comparable, and apparently mediated by an antimycin A- and cyanide-insensitive alternative respiratory pathway; no evidence for the presence of a mammalian-like respiratory pathway in embryonated eggs of A. galli was found. The results of this study are compared to mitochondrial respiration in eggs, larvae, and adult body wall muscle of Ascaris suum.     

Adaptations to Stochastic Environmental Variations: The Effects of Seasonal Temperatures on the Migratory Window of Svalbard Arctic Charr

The circumpolar Arctic charr, Salvelinus alpinus, is ideal for studying how environmental factors affect life history in fishes. Charr populations demonstrate a tremendous ecological plasticity and adaptations to harsh environments. Arctic charr is the only freshwater fish on Svalbard, including anadromous, resident, and landlocked stocks. Freshwater lake systems on Svalbard are characterized by very low water temperatures, long-term or even permanent ice cover, and low levels of nutrients. Food is thus limited and may lead to growth stagnation and early maturity in Arctic charr. The individual growth pattern may alternatively follow a sigmoid-shaped curve, caused by a shift to either cannibalistic or anadromous (migration to sea) behaviour. In lake systems that include migratory charr, the population may consist of a mixture of parr, postsmolt, and adult migratory individuals, as well as small-sized resident, large-sized resident (cannibals), and large formerly resident individuals transformed to anadromy. Our study in the Lake Dieset watercourse (79°N), Svalbard, demonstrates that the annual water flow in the outlet river is strongly correlated to air temperatures and provides a passage to the sea, allowing the charr access to the nutrient-rich seawater environment, during at most two months each year. During one of the years studied, the youngest and small-sized part of the sea-going stock was prevented from ascending the river and probably suffered mortality during winter. The migratory window of the Arctic charr in Lake Dieset is therefore highly variable among years and thus unpredictable. We hypothesize that in worst case scenarios (cold years, low water discharge), climatic variations may occasionally prevent charr from migrating upstream in Svalbard lake systems in late autumn, resulting in high mortality in the population.     

Changes in cold tolerance due to a 14-day stay in the Canadian Arctic

Responses to cold exposure tests both locally and of the whole body were examined in subjects who stayed in the Arctic (average maximum and minimum temperatures –11 and –21° C respectively) for 14 days of skiing and sleeping in tents. These changes were compared to responses in subjects living working in Ottawa, Canada (average max. and min. temperatures –5 and –11° C respectively). The tests were done before the stay in the Arctic (Pre), immediately after the return (Post 1) and approximately 32 days after the return (Post 2). For the whole-body cold exposure each subject, wearing only shorts and lying on a rope mesh cot, was exposed to an ambient temperature of 10° C. There was no consistent response in the changes of metabolic or body temperature to this exposure in either of groups and, in addition, the changes over time were variable. Cold induced vasodilatation (CIVD) was determined by measuring temperature changes in the middle finger of the nondominant hand upon immersion in ice water for 30 min. CIVD was depressed after the Arctic exposure whilst during the Post 2 testing, although variable, did not return to the Pre values; the responses of the control group were similar. These results indicate that normal seasonal changes may be as important in adaptation as a stay in the Arctic. Caution is advised in the separation of seasonal effects when examining the changes in adaptation after exposure to a cold environment.     

The cuticular proteins from free-living and parasitic stages of Haemonchus contortus--I. Isolation and partial characterization

1. Cuticles were isolated from the adult males, adult females, the second molt (2M) sheath from the infective larvae (L3(2M)), and the parasitic third stage (L3) of the sheep parasite Haemonchus contortus by a combination of mechanical disruption and detergent treatment. 2. The 2ME soluble cuticular proteins from adult males contained 4 or 5 major protein bands with molecular weights ranging from 100 to 56 kD with the most prominent band at 56 kD. The cuticular proteins from adult females were similar to the male. 3. Cuticular proteins from the larval stages, 2M cuticle, and L3 cuticle, differed from the adults and from each other. The most prominent protein bands were observed with molecular weights on 78 and 39 kD for the L3 cuticle and 100, 91 and 46 kD for the 2M cuticle. The 2ME soluble cuticular proteins from all developmental stages were at least partially digested by bacterial collagenase. 4. The amino acid composition of cuticular proteins was similar for the L3 and 2M, but adults had lesser amounts of glycine and greater amounts of basic amino acids than the larval stages. The amount of the isolated cuticle solubilized by the 2ME treatment was greatest in adults (80%) compared to the L3 (64%) and the 2M (22%). 5. These results support a hypothesis that there are quantitative and qualitative stage specific differences in the cuticular proteins of H. contortus.     

Anthelmintic tolerance in free-living and facultative parasitic isolates of Halicephalobus (Panagrolaimidae)

SUMMARY Studies on anthelmintic resistance in equine parasites do not include facultative parasites. Halicephalobus gingivalis is a free-living bacterivorous nematode and a known facultative parasite of horses with a strong indication of some form of tolerance to common anthelmintic drugs. This research presents the results of an in vitro study on the anthelmintic tolerance of several isolates of Halicephalobus to thiabendazole and ivermectin using an adaptation of the Micro-Agar Larval Development Test hereby focusing on egg hatching and larval development. Panagrellus redivivus and Panagrolaimus superbus were included as a positive control. The results generally show that the anthelmintic tolerance of Halicephalobus to both thiabendazole and ivermectin was considerably higher than that of the closely related Panagrolaimidae and, compared to other studies, than that of obligatory equine parasites. Our results further reveal a remarkable trend of increasing tolerance from fully free-living isolates towards horse-associated isolates. In vitro anthelmintic testing with free-living and facultative parasitic nematodes offers the advantage of observing drug effect on the complete life cycle as opposed to obligatory parasites that can only be followed until the third larval stage. We therefore propose Halicephalobus gingivalis as an experimental tool to deepen our understanding of the biology of anthelmintic tolerance.     

Differential DNA methylation in discrete developmental stages of the parasitic nematode Trichinella spiralis

Background

DNA methylation plays an essential role in regulating gene expression under a variety of conditions and it has therefore been hypothesized to underlie the transitions between life cycle stages in parasitic nematodes. So far, however, 5''-cytosine methylation has not been detected during any developmental stage of the nematode Caenorhabditis elegans. Given the new availability of high-resolution methylation detection methods, an investigation of life cycle methylation in a parasitic nematode can now be carried out.

Results

Here, using MethylC-seq, we present the first study to confirm the existence of DNA methylation in the parasitic nematode Trichinella spiralis, and we characterize the methylomes of the three life-cycle stages of this food-borne infectious human pathogen. We observe a drastic increase in DNA methylation during the transition from the new born to mature stage, and we further identify parasitism-related genes that show changes in DNA methylation status between life cycle stages.

Conclusions

Our data contribute to the understanding of the developmental changes that occur in an important human parasite, and raises the possibility that targeting DNA methylation processes may be a useful strategy in developing therapeutics to impede infection. In addition, our conclusion that DNA methylation is a mechanism for life cycle transition in T. spiralis prompts the question of whether this may also be the case in any other metazoans. Finally, our work constitutes the first report, to our knowledge, of DNA methylation in a nematode, prompting a re-evaluation of phyla in which this epigenetic mark was thought to be absent.     

Bioinformatic identification of cytochrome b5 homologues from the parasitic nematode Ascaris suum and the free-living nematode Caenorhabditis elegans highlights the crucial role of A. suum adult-specific secretory cytochrome b5 in parasitic adaptation

We previously reported that adult Ascaris suum possesses NADH-metmyoglobin and NADH-methaemoglobin reductase systems that are located in the cells of the body wall and in the extracellular perienteric fluid, respectively, which helps them adapt to environmental hypoxia by recovering the differential functions of myoglobin and haemoglobin. A. suum cytochrome b₅, an adult-specific secretory protein and an essential component of the NADH-metmyo (haemo) globin reductase system, has been extensively studied, and its unique nature has been determined. However, the relationship between A. suum cytochrome b₅ and the canonical cytochrome b₅ proteins, from the free-living nematode Caenorhabditis elegans is unclear. Here, we have characterised four cytochrome b₅-like proteins from C. elegans (accession numbers: CAB01732, CCD68984, CAJ58492, and CAA98498) and three from A. suum (accession numbers: ADY48796, ADY46277, and ADY48338) and compared them with A. suum cytochrome b₅ in silico. Bioinformatic and molecular analyses showed that CAA98498 from C. elegans is equivalent of A. suum cytochrome b₅, which was not expressed as a mature mRNA. Further, the CAA98498 possessed no secretory signal peptide, which occurs in A. suum cytochrome b₅ precursor. These results suggest that this free-living nematode does not need a haemoprotein such as the A. suum cytochrome b₅ and highlight the crucial function of this A. suum adult-specific secretory cytochrome b₅ in parasitic adaptation.     

Cold tolerance and dehydration in Enchytraeidae from Svalbard

When cooled in contact with moisture, eight species of arctic Enchytraeidae from Svalbard were killed by freezing within minutes or hours at −3 and −5 °C; an exception was Enchytraeus kincaidi which survived for up to 2 days. When the temperature approached 0 °C the enchytraeids apparently tried to escape from the moist soil. The supercooling capacity of the enchytraeids was relatively low, with mean supercooling points of −5 to −8 °C. In contrast, specimens of several species were extracted from soil cores that had been frozen in their intact state at −15 °C for up to 71 days. Compared to freezing in a moist environment, higher survival rates were obtained during cooling at freezing temperatures in dry soil. Survival was recorded in species kept at −3 °C for up to 35 days, and in some species kept at −6 °C for up to 17 days. Slow warming greatly increased survival rates at −6 °C . The results strongly suggest that arctic enchytraeids avoid freezing by dehydration at subzero temperatures. In agreement with this, weight losses of up to ca. 42% of fresh weight were recorded in Mesenchytraeus spp. and of up to 55% in Enchytraeus kincaidi at water vapour pressures above ice at −3 to −6 °C. All specimens survived dehydration under these conditions. Accepted: 12 December 1997     

Nitrogen fixation in Arctic vegetation and soils from Svalbard,Norway

Nitrogen fixation was measured by the acetylene reduction method in a high Arctic ecosystem at Kongsfjorden, Spitsbergen (79°N, 12°E). The most important source of biologically fixed nitrogen was found in cyanobacteria either as free living colonies ofNostoc sp. in wet unvegetated or sparsely vegetated grounds or growing as epiphytes on bryophytes. Fixation associated with plant roots or in soil and peat samples had little or no significance for nitrogen input to the ecosystem. The ability to support an epiphytic flora of nitrogen-fixing cyanobacteria varied greatly between bryophyte species.Calliergon richardsonii andSanionia uncinata seemed especially well adapted for harbouring epiphytic cyanobacteria, but the extent of nitrogen fixation varied with the growing location. The rate of nitrogen fixation was greatly influenced by grazing by geese. In a geese-grazing area values were found with a maximum of 693.6±1.5 nmol C₂H₄ h⁻¹ g (dry weight)⁻¹ while the maximum value for ungrazed areas was 65.3±16.6 nmol C₂H₄ h⁻¹ g (dry weight)⁻¹. In the grazed area cyanobacteria were also found fixing nitrogen epiphytically on grass. The high plant productivity, supporting heavy grazing, clearly indicates an effective transfer of fixed nitrogen to the plant community. Under cliffs harbouring colonies of birds, the biological nitrogen fixation was inhibited by bird droppings.