Comparative transcriptomic analysis of candidate effectors to explore the infection and survival strategy of Bursaphelenchus xylophilus during different interaction stages with pine trees.

BACKGROUND: The pine wood nematode (PWN), Bursaphelenchus xylophilus, is a devastating pathogen of many Pinus species in China. The aim of this study was to understand the interactive molecular mechanism of PWN and its host by comparing differentially expressed genes and candidate effectors from three transcriptomes of B. xylophilus at different infection stages. RESULTS: In total, 62, 69 and 46 candidate effectors were identified in three transcriptomes (2.5 h postinfection, 6, 12 and 24 h postinoculation and 6 and 15 d postinfection, respectively). In addition to uncharacterized pioneers, other candidate effectors were involved in the degradation of host tissues, suppression of host defenses, targeting plant signaling pathways, feeding and detoxification, which helped B. xylophilus survive successfully in the host. Seven candidate effectors were identified in both our study and the B. xylophilus transcriptome at 2.5 h postinfection, and one candidate effector was identified in all three transcriptomes. These common candidate effectors were upregulated at infection stages, and one of them suppressed pathogen-associated molecular pattern (PAMP) PsXEG1-triggered cell death in Nicotiana benthamiana. CONCLUSIONS: The results indicated that B. xylophilus secreted various candidate effectors, and some of them continued to function throughout all infection stages. These various candidate effectors were important to B. xylophilus infection and survival, and they functioned in different ways (such as breaking down host cell walls, suppressing host defenses, promoting feeding efficiency, promoting detoxification and playing virulence functions). The present results provide valuable resources for in-depth research on the pathogenesis of B. xylophilus from the perspective of effectors.

Human Migration and the Spread of the Nematode Parasite Wuchereria bancrofti.

The human disease lymphatic filariasis causes the debilitating effects of elephantiasis and hydrocele. Lymphatic filariasis currently affects the lives of 90 million people in 52 countries. There are three nematodes that cause lymphatic filariasis, Brugia malayi, Brugia timori, and Wuchereria bancrofti, but 90% of all cases of lymphatic filariasis are caused solely by W. bancrofti (Wb). Here we use population genomics to reconstruct the probable route and timing of migration of Wb strains that currently infect Africa, Haiti, and Papua New Guinea (PNG). We used selective whole genome amplification to sequence 42 whole genomes of single Wb worms from populations in Haiti, Mali, Kenya, and PNG. Our results are consistent with a hypothesis of an Island Southeast Asia or East Asian origin of Wb. Our demographic models support divergence times that correlate with the migration of human populations. We hypothesize that PNG was infected at two separate times, first by the Melanesians and later by the migrating Austronesians. The migrating Austronesians also likely introduced Wb to Madagascar where later migrations spread it to continental Africa. From Africa, Wb spread to the New World during the transatlantic slave trade. Genome scans identified 17 genes that were highly differentiated among Wb populations. Among these are genes associated with human immune suppression, insecticide sensitivity, and proposed drug targets. Identifying the distribution of genetic diversity in Wb populations and selection forces acting on the genome will build a foundation to test future hypotheses and help predict response to current eradication efforts.

Small RNAs in parasitic nematodes - forms and functions.

Small RNAs are important regulators of gene expression. They were first identified in Caenorhabditis elegans, but it is now apparent that the main small RNA silencing pathways are functionally conserved across diverse organisms. Availability of genome data for an increasing number of parasitic nematodes has enabled bioinformatic identification of small RNA sequences. Expression of these in different lifecycle stages is revealed by small RNA sequencing and microarray analysis. In this review we describe what is known of the three main small RNA classes in parasitic nematodes - microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs) and small interfering RNAs (siRNAs) - and their proposed functions. miRNAs regulate development in C. elegans and the temporal expression of parasitic nematode miRNAs suggest modulation of target gene levels as parasites develop within the host. miRNAs are also present in extracellular vesicles released by nematodes in vitro, and in plasma from infected hosts, suggesting potential regulation of host gene expression. Roles of piRNAs and siRNAs in suppressing target genes, including transposable elements, are also reviewed. Recent successes in RNAi-mediated gene silencing, and application of small RNA inhibitors and mimics will continue to advance understanding of small RNA functions within the parasite and at the host-parasite interface.

Gastrointestinal helminth parasites of the common wallaroo or euro, Osphranter robustus (Gould) (Marsupialia: Macropodidae) from Australia.

The gastrointestinal helminth parasites of 170 common wallaroos or euros, Osphranter robustus (Gould), collected from all mainland states in which the species occurs as well as the Northern Territory, are presented, including previously published data. A total of 65 species of helminths were encountered, including four species of anoplocephalid cestodes found in the bile ducts and small intestine, and 61 species of strongylid nematodes, all but two of which occurring in the stomach, and with the remainder occurring in the terminal ileum, caecum and colon. Among the mainland subspecies of O. robustus, 52 species of helminths were encountered in O. r. robustus, compared with 30 species in O. r. woodwardi and 35 species in O. r. erubescens. Of the parasite species encountered, only 17 were specific to O. robustus, the remaining being shared with sympatric host species. Host-specific species or species occurring in O. robustus at a high prevalence can be classified as follows: widely distributed; restricted to northern Australia; restricted to the northern wallaroo, O. r. woodwardi; found only in the euro, O. r. erubescens; found essentially along the eastern coast of Australia, primarily in O. r. robustus; and species with highly limited regional distributions. The data currently available suggest that the acquisition of a significant number of parasites is due to co-grazing with other macropodids, while subspeciation in wallaroos as well as climatic variables may have influenced the diversification of the parasite fauna.

Decreased nematode clearance and anti-phosphorylcholine-specific IgM responses in mannose-binding lectin-deficient mice.

Brugia malayi is a nematode that causes human lymphatic filariasis. Previously, we showed that mannose-binding lectin (MBL)-A is necessary for clearance of B. malayi microfilariae in mice and presence of MBL-A is linked with maximal levels of parasite-specific IgM. Common human MBL gene polymorphisms result in low MBL expression and lead to recurring bacterial infections. Furthermore, these low-expressing human MBL polymorphisms result in greatly increased susceptibility to lymphatic filarial infection. Indeed, gain of new filarial infections over a 30-year period are 10-fold higher in people with low, compared to high, MBL-expression phenotypes. Human MBL closely resembles mouse MBL-C, rather than MBL-A; therefore, we examined the role of mouse MBL-C in clearance of microfilariae. Absence of MBL-C alone, or both MBL-A and -C, resulted in delayed clearance of microfilariae and reduced parasite-specific IgM in mice. There were few profound changes in B cell sub-populations or in the ability of MBL-deficient mice to respond to T-dependent or T-independent antigens. However, absence of MBL-A and/or MBL-C resulted in reduced IgM to phosphorylcholine, a constituent of filarial and bacterial antigens, suggesting that inability to form proficient antibody responses to this moiety leads to lack of microfilarial clearance and overall susceptibility to filariasis.

Molecular detection and quantification of slug parasitic nematodes from the soil and their hosts.

Terrestrial gastropod molluscs are widely distributed and are well known as pests of many types of plants that are notoriously difficult to control. Many species of nematodes are able to parasitize land snails and slugs, but few of them are lethal to their host. Species and/or populations of mollusc-parasitic nematodes (MPNs) that kill their hosts are promising for biological control purposes. The recent discovery of new nematode species of the genus Phasmarhabditis in Europe and the associations between Alloionema spp. and slugs are expanding the possibilities of using MPNs as control agents. However, very little is known about the distribution and ecology of these species. Using molecular techniques based on qPCR methods for quick identification and quantification of various species of MPN isolated directly from the soil or from infected hosts can assist in providing information on their presence and persistence, as well as the composition of natural assemblages. Here, we developed new primers and probes for five species of the genus Phasmarhabditis and one species of the genus Alloionema. We employed these novel molecular techniques and implemented a published molecular set to detect MPN presence in soil samples coming from natural and agricultural areas in Switzerland. We also developed a method that allows the detection and quantification of Phasmarhabditis hermaphrodita directly from the tissues of their slug host in a laboratory experiment. The new molecular approaches were optimized to a satisfactory limit of detection of the species, with only few cross-amplifications with closely related species in late cycles (>32). Using these tools, we detected MPNs in 7.5% of sampled sites, corresponding to forest areas (P. hermaphrodita and Alloionema appendiculatum) and wheat-oriented agricultural areas (Phasmarhabditis bohemica). Moreover, we confirmed that the method can be used to detect the presence of P. hermaphrodita inside slug hosts, with more detections in the susceptible slug Deroceras larvae compared to the resistant Arion vulgaris. These primers/probe sets provide a novel and quick tool to identify MPNs from soil samples and infected slugs without having to culture and retrieve all nematode life stages, as well as a new tool to unravel the ecology of nematode-slug complexes.

Comparison of direct and indirect techniques for evaluating endoparasite infections in wild-caught newts (Taricha torosa and T. granulosa).

Studies of amphibian parasites have increased over the past 20 yr, in part because of their role in amphibian population declines and deformities. Such patterns underscore the importance of non-lethal methods for detecting and quantifying endoparasitic infections. The goal of this study was to compare results of indirect methods (fecal smears and fecal floats) with quantitative necropsies to detect endoparasitic infections in adult newts. In 2015, we collected fecal samples from 68 adult newts (Taricha granulosa and T. torosa) in the East Bay region of California and used fecal smears, sodium nitrate fecal flotation solution, and Sheather's sugar flotation solution to assess infection (i.e. the presence and/or abundance of a parasite). Across all methods, we detected 3 protozoans (Eimeria tarichae, Tritrichomonas sp., and Balantidium sp.) and 3 nematodes (Rhabdias tarichae, Cosmocercoides variabilis, and Chabaudgolvania sp.). Based on generalized linear mixed models, the likelihood of detection varied between hosts (with T. torosa showing more overall infection relative to T. granulosa) and by assessment method: while fecal smears were more sensitive in detecting protozoans, comprehensive necropsies were the most reliable for quantifying infections of R. tarichae. Nonetheless, both the likelihood of R. tarichae detection within fecal samples as well as the number of infectious stages observed correlated strongly with infection intensity from necropsy, highlighting the utility of non-lethal assessment methods. The overall congruence between indirect methods and gross necropsy helps to validate the use of less-invasive methods for parasite detection and abundance, especially for sensitive or protected host taxa such as amphibians.

Comparative evolutionary histories of fungal proteases reveal gene gains in the mycoparasitic and nematode-parasitic fungus Clonostachys rosea.

BACKGROUND: The ascomycete fungus Clonostachys rosea (order Hypocreales) can control several important plant diseases caused by plant pathogenic fungi and nematodes. Subtilisin-like serine proteases are considered to play an important role in pathogenesis in entomopathogenic, mycoparasitic, and nematophagous fungi used for biological control. In this study, we analysed the evolutionary histories of protease gene families, and investigated sequence divergence and regulation of serine protease genes in C. rosea. RESULTS: Proteases of selected hypocrealean fungal species were classified into families based on the MEROPS peptidase database. The highest number of protease genes (590) was found in Fusarium solani, followed by C. rosea with 576 genes. Analysis of gene family evolution identified non-random changes in gene copy numbers in the five serine protease gene families S1A, S8A, S9X, S12 and S33. Four families, S1A, S8A, S9X, and S33, displayed gene gains in C. rosea. A gene-tree / species-tree reconciliation analysis of the S8A family revealed that the gene copy number increase in C. rosea was primarily associated with the S08.054 (proteinase K) subgroup. In addition, regulatory and predicted structural differences, including twelve sites evolving under positive selection, among eighteen C. rosea S8A serine protease paralog genes were also observed. The C. rosea S8A serine protease gene prs6 was induced during interaction with the plant pathogenic species F. graminearum. CONCLUSIONS: Non-random increases in S8A, S9X and S33 serine protease gene numbers in the mycoparasitic species C. rosea, Trichoderma atroviride and T. virens suggests an involvement in fungal-fungal interactions. Regulatory and predicted structural differences between C. rosea S8A paralogs indicate that functional diversification is driving the observed increase in gene copy numbers. The induction of prs6 expression in C. rosea during confrontation with F. graminearum suggests an involvement of the corresponding protease in fungal-fungal interactions. The results pinpoint the importance of serine proteases for ecological niche adaptation in C. rosea, including a potential role in the mycoparasitic attack on fungal prey.

Insect Immunity to Entomopathogenic Nematodes and Their Mutualistic Bacteria.

Entomopathogenic nematodes are important organisms for the biological control of insect pests and excellent models for dissecting the molecular basis of the insect immune response against both the nematode parasites and their mutualistic bacteria. Previous research involving the use of various insects has found distinct differences in the number and nature of immune mechanisms that are activated in response to entomopathogenic nematode parasites containing or lacking their associated bacteria. Recent studies using model insects have started to reveal the identity of certain molecules with potential anti-nematode or antibacterial activity as well as the molecular components that nematodes and their bacteria employ to evade or defeat the insect immune system. Identification and characterization of the genes that regulate the insect immune response to nematode-bacteria complexes will contribute significantly to the development of improved practices to control insects of agricultural and medical importance, and potentially nematode parasites that infect mammals, perhaps even humans.

Infected host macerate enhances entomopathogenic nematode movement towards hosts and infectivity in a soil profile.

Vertical dispersal and infectivity of the infective juveniles (IJs) of three entomopathogenic nematodes (EPNs), Steinernema carpocapsae, S. feltiae, and Heterorhabditis bacteriophora, were tested in the presence or absence of cadaver macerate of nematode-infected Galleria mellonella. Infected host macerate applied on the top of column surface induced higher numbers of IJs to move to the bottom of the column for all three species, indicating a dispersal-enhancing effect of host cadaver on IJs. Among the three EPNs, H. bacteriophora was the most responsive to host macerate, followed by S. feltiae, and S. carpocapsae was the least. Also, more IJs of H. bacteriophora invaded Tenebrio molitor hosts at the bottom of soil columns in the presence of host macerate compared with the treatment without cadaver macerate. These findings suggest enhanced dispersal and/or infectivity of all three EPNs may be leveraged toward superior biocontrol efficacy.

Using the fungus Arthrobotrys cladodes var. macroides as a sustainable strategy to reduce numbers of infective larvae of bovine gastrointestinal parasitic nematodes.

Research in the area of sanitation in ruminant production has focused on discovery of potential agents for biological control of helminths with nematophagous fungi and has provided evidence of success. The antagonistic potential of the fungus Arthrobotrys cladodes var. macroides on infective larvae of bovine gastrointestinal parasitic nematodes was evaluated by scanning electron microscopy. Additionally, an in vivo test of the resistance to digestive processes and viability of the fungus was carried out using a formulation based on sodium alginate administered orally in cattle. Production of conidia and chlamydospores was high. In in vitro tests, the number of infective nematode larvae was reduced 68.7% by the fungus in the treated group compared to the control group. The interaction between the fungus and the nematodes was confirmed by scanning electron microscopy. Plates containing fecal samples collected after oral administration of 100 g of pellets containing the A. cladodes fungus showed that the fungus survived passage through the gastrointestinal tract of ruminants, grew on agar, formed traps and preyed on L3 larvae of gastrointestinal parasites. The results of the present study provide a new opportunity for alternative, environmentally safe control of ruminant nematodes.

Pack hunting by a common soil amoeba on nematodes.

Soils host the most complex communities on Earth, including the most diverse and abundant eukaryotes, i.e. heterotrophic protists. Protists are generally considered as bacterivores, but evidence for negative interactions with nematodes both from laboratory and field studies exist. However, direct impacts of protists on nematodes remain unknown. We isolated the soil-borne testate amoeba Cryptodifflugia operculata and found a highly specialized and effective pack-hunting strategy to prey on bacterivorous nematodes. Enhanced reproduction in presence of prey nematodes suggests a beneficial predatory life history of these omnivorous soil amoebae. Cryptodifflugia operculata appears to selectively impact the nematode community composition as reductions of nematode numbers were species specific. Furthermore, we investigated 12 soil metatranscriptomes from five distinct locations throughout Europe for 18S ribosomal RNA transcripts of C. operculata. The presence of C. operculata transcripts in all samples, representing up to 4% of the active protist community, indicates a potential ecological importance of nematophagy performed by C. operculata in soil food webs. The unique pack-hunting strategy on nematodes that was previously unknown from protists, together with molecular evidence that these pack hunters are likely to be abundant and widespread in soils, imply a considerable importance of the hitherto neglected trophic link 'nematophagous protists' in soil food webs.

[Efficacy of a new fenbendazole formulation produced by nanotechnology-based drug delivery system against nematodosis].

The efficacy of a new fenbendazile formulation produced by nanotechnology-based drug delivery system was investigated in45 sheep naturally infected with gastrointestinal nematodes. The formulation showed 95.6% efficacy against Nematodes spp. at a dose of 1.0 mg/kg dw of its active ingredient and 100% efficacy against other species of gastrointestinal nematodes. Given at a dose of 10 mg/kg dw, the basic drug--fenbendazole (substance) displayed 96.39 and 100% efficacy, respectively.

Crystallization and preliminary crystallographic studies of a cysteine protease inhibitor from the human nematode parasite Ascaris lumbricoides.

The cysteine protease inhibitor from Ascaris lumbricoides, a roundworm that lives in the human intestine, may be involved in the suppression of human immune responses. Here, the molecular cloning, protein expression and purification, preliminary crystallization and crystallographic characterization of the cysteine protease inhibitor from A. lumbricoides are reported. The rod-shaped crystal belonged to space group C2, with unit-cell parameters a = 99.40, b = 37.52, c = 62.92 Å, ß = 118.26°. The crystal diffracted to 2.1 Šresolution and contained two molecules in the asymmetric unit.

Kinetics of capture and infection of infective larvae of trichostrongylides and free-living nematodes Panagrellus sp. by Duddingtonia flagrans.

Duddingtonia flagrans, a nematode-trapping fungus, has been investigated as an agent for biological control against infective larvae of gastrointestinal nematode parasites of production animals. The initial process of nematode-trapping fungi infection is based on an interaction between the trap structure of the fungus and the surface of the nematode cuticle. This report investigates by light and scanning electron microscopy the kinetics of capture and infection during the interaction of D. flagrans with the infective larvae (L(3)) of trichostrongylides and the free-living nematode Panagrellus sp. D. flagrans was cultivated for 7 days in a Petri dish containing agar-water. L(3) and Panagrellus sp. were inoculated in the Petri dishes and the samples consisting of agar-L(3)-fungi and agar-Panagrellus sp.-fungi were collected after 10, 20, 30, 40, 50, 60, and 70 min and 3, 4, 5, 10, 15, 20, and 25 h of interaction. All samples were observed by light microscopy. The samples with 1, 5, 15, and 25 h of interaction were also analyzed by scanning electron microscopy. The interaction was monitored up to 25 h. An initial differentiation of predation structures was observed after 30 min of interaction. The presence of traps and of captured L(3) or Panagrellus sp. occurred after 70 min. The live captured nematodes were observed up to 3 h of interaction. However, after 4 h, all Panagrellus sp. were dead. It took 15 h of interaction for the fungus to invade the L(3), and the presence of hyphae inside the nematode near the region of penetration was evident. At this time, the hyphae had filled the whole body of Panagrellus sp. The complete occupation of the body of L(3) occurred at 20 h of interaction and with 25 h the nematode was completely damaged except for the cuticle. Although the double cuticle of L(3) slows the penetration of D. flagrans, it was possible to verify that the process of trap formation and capture occurs quickly when both nematodes were tested, suggesting that the organisms would eventually be killed once in contact with the fungi encouraging the use of the fungus as a biological control agent.

Excretion of Histomonas meleagridis following experimental co-infection of distinct chicken lines with Heterakis gallinarum and Ascaridia galli.

BACKGROUND: Histomonosis is a severe re-emerging disease of poultry caused by Histomonas meleagridis, a protozoan parasite which survives in the environment via the cecal worm Heterakis gallinarum. Following infection, the parasites reside in the ceca and are excreted via host feces. In the present work, male birds of conventional broiler (Ross 308, R), layer (Lohmann Brown Plus, LB) and a dual-purpose (Lohmann Dual, LD) chicken line were infected with 250 embryonated eggs of Ascaridia galli and Heterakis gallinarum, respectively, with the latter nematode harboring Histomonas meleagridis, to investigate a co-infection of nematodes with the protozoan parasite in different host lines. METHODS: In weekly intervals, from 2 to 9 weeks post infection (wpi), individual fecal samples (n = 234) from the chickens were collected to quantify the excretion of H. meleagridis by real-time PCR and to determine the number of nematode eggs per gram (EPG) in order to elucidate excretion dynamics of the flagellate and the nematodes. This was further investigated by indirect detection using plasma samples of the birds to detect antibodies specific for H. meleagridis and worms by ELISA. The infection with H. meleagridis was confirmed by histopathology and immunohistochemistry to detect the flagellate in the cecum of representing birds. RESULTS: The excretion of H. meleagridis could already be observed from the 2nd wpi in some birds and increased to 100% in the last week of the experiment in all groups independent of the genetic line. This increase could be confirmed by ELISA, even though the number of excreted H. meleagridis per bird was generally low. Overall, histomonads were detected in 60% to 78% of birds with temporary differences between the different genetic lines, which also showed variations in the EPG and worm burden of both nematodes. CONCLUSIONS: The infection with H. gallinarum eggs contaminated with H. meleagridis led to a permanent excretion of the flagellate in host feces. Differences in the excretion of H. meleagridis in the feces of genetically different host lines occurred intermittently. The excretion of the protozoan or its vector H. gallinarum was mostly exclusive, showing a negative interaction between the two parasites in the same host.

First Occurrence of Eustrongylides spp. (Nematoda: Dioctophymatidae) in a Subalpine Lake in Northwest Italy: New Data on Distribution and Host Range.

The genus Eustrongylides includes nematodes that infect fish species and fish-eating birds inhabiting freshwater ecosystems. Nematodes belonging to the genus Eustrongylides are potentially pathogenic for humans; infection occurs after the consumption of raw or undercooked fish. In the two-year period 2019-2020, a total of 292 fish belonging to eight species were examined for the occurrence of Eustrongylides spp. from Lake San Michele, a small subalpine lake in northwest Italy. The prevalence of infestation was 18.3% in Lepomis gibbosus, 16.7% in Micropterus salmoides, and 10% in Perca fluviatilis. The other five fish species (Ameiurus melas, Ictalurus punctatus, Squalius cephalus, Carassius carassius, and Scardinius erythrophthalmus) were all negative for parasite presence. There were no significant differences in prevalence between the three fish species (Fisher's exact test; p = 0.744). The mean intensity of infestation ranged from 1 (M. salmoides and P. fluviatilis) to 1.15 (L. gibbosus), and the mean abundance ranged from 0.1 (P. fluviatilis) to 0.28 (L. gibbosus). There were significant differences in the infestation site between the four muscle quadrants (anterior ventral, anterior dorsal, posterior ventral, and posterior dorsal) and the visceral cavity (Kruskal-Wallis test; p = 0.0008). The study findings advance our knowledge about the distribution and host range of this parasite in Italy.

Odds ratios and hurdle models: a long-term analysis of parasite infection patterns in endangered young-of-the-year suckers from Upper Klamath Lake, Oregon, USA.

We used odds ratios and a hurdle model to analyze parasite co-infections over 25 years on >20,000 young-of-the year of endangered Shortnose and Lost River Suckers. Host ecologies differed as did parasite infections. Shortnose Suckers were more likely to be caught inshore and 3-5 times more likely to have Bolbophorus spp. and Contracaecum sp. infections, and Lost River Suckers were more likely to be caught offshore and approximately three times more likely to have Lernaea cyprinacea infections. An observed peak shift seems likely to be due to a lower host size limit for Bolbophorus spp. (13.6 mm) compared with L. cyprinacea (23.4 mm). The large data set allowed us to generate strong hypotheses: (i) that a major marsh restoration project had unintended consequences that resulted in an increase in infections; (ii) that co-infection with Bolbophorus spp. increased the odds of infection by L. cyprinacea and Contracaecum sp.; (iii) that significant declines in the odds of infection over approximately 25 days were due to parasite-induced host mortality; (iv) that the fish's small size relative to L. cyprinacea and Contracaecum sp. might be directly lethal; (v) that the absence of L. cyprinacea infections in the early 1990s was associated with good year-class production of the suckers; and (vi) that parasites might increase the odds of vagrancy from the nursery ground.

Phenotypic plasticity and local adaptation in freeze tolerance: Implications for parasite dynamics in a changing world.

Marshallagia marshalli is a multi-host gastrointestinal nematode that infects a variety of artiodactyl species from temperate to Arctic latitudes. Eggs of Marshallagia are passed in host faeces and develop through three larval stages (L1, L2, and L3) in the environment. Although eggs normally hatch as L1s, they can also hatch as L3s. We hypothesised that this phenotypic plasticity in hatching behaviour may improve fitness in subzero and highly variable environments, and this may constitute an evolutionary advantage under current climate change scenarios. To test this, we first determined if the freeze tolerance of different free-living stages varied at different temperatures (-9 °C, -20 °C and -35 °C). We then investigated if there were differences in freeze tolerance of M. marshalli eggs sourced from three discrete, semi-isolated, populations of wild bighorn and thinhorn sheep living in western North America (latitudes: 40°N, 50°N, 64°N). The survival rates of eggs and L3s were significantly higher than L1s at -9 °C and -20 °C, and survival of all three stages decreased significantly with increasing freeze duration and decreasing temperature. The survival of unhatched L1s was significantly higher than the survival of hatched L1s. There was no evidence of local thermal adaptation in freeze tolerance among eggs from different locations. We conclude that developing to the L3 in the egg may result in a fitness advantage for M. marshalli, with the egg protecting the more vulnerable L1 under freezing conditions. This phenotypic plasticity in life-history traits of M. marshalli might be an important capacity, a potential exaptation capable of enhancing parasite fitness under temperature extremes.

Trace element concentrations in Proteocephalus macrocephalus (Cestoda) and Anguillicola crassus (Nematoda) in comparison to their fish host, Anguilla anguilla in Ria de Aveiro, Portugal.

The use of some fish parasites as bioindicators of heavy metal pollution has been demonstrated as particularly adequate due to their capacity of bioconcentration. This study evaluated the effect of Proteocephalus macrocephalus on the accumulation of trace elements in the edible fish, Anguilla anguilla, in a contaminated area in Portugal (Ria de Aveiro). Also, the model P. macrocephalus/A. anguilla was assessed as a bioindicator system in the presence of the highly prevalent nematode Anguillicola crassus. Samples (kidney, liver, muscle, A. crassus and P. macrocephalus) of 20 eels harbouring A. crassus and another 20 harbouring both A. crassus and P. macrocephalus were selected for element analysis by ICP-MS. The highest concentrations of Cr, Ni and Zn were detected in P. macrocephalus. However, there was a higher liver and muscle Cr concentration in eels not infected by P. macrocephalus. Also, the nematode A. crassus presented higher Cr concentrations in those eels harbouring P. macrocephalus. Results suggest that P. macrocephalus individuals accumulate Cr and Ni while levels of Cr in eel livers and Ni levels in eel kidney are reduced. The system P. macrocephalus/A. anguilla yielded bioaccumulation factors for Cr, Ni, Pb and Zn, whereas bioaccumulation of Cu, Cr and Pb in A. crassus varied according to eel co-infection with P. macrocephalus, thus emphasising the possible role of cestode infection in metal metabolization/storage processes in host tissues. Results suggest that heavy metal pollution in Ria de Aveiro has been decreasing although it is still higher than in other contaminated areas in Europe. Nevertheless, eel consumption in Ria de Aveiro represents no risk for humans although they may represent a real contamination risk for wildlife. The system P. macrocephalus/A. anguilla is proposed as another promising bioindicator system to evaluate environmental Cr, Ni, Pb and Zn exposure in estuarine areas where both species co-occur.