Novel insight into the genetic diversity of strongylid nematodes infecting South-East and East Asian primates.

With many non-human primates (NHPs) showing continued population decline, there is an ongoing need to better understand their ecology and conservation threats. One such threat is the risk of disease, with various bacterial, viral and parasitic infections previously reported to have damaging consequences for NHP hosts. Strongylid nematodes are one of the most commonly reported parasitic infections in NHPs. Current knowledge of NHP strongylid infections is restricted by their typical occurrence as mixed infections of multiple genera, which are indistinguishable through traditional microscopic approaches. Here, modern metagenomics approaches were applied for insight into the genetic diversity of strongylid infections in South-East and East Asian NHPs. We hypothesized that strongylid nematodes occur in mixed communities of multiple taxa, dominated by Oesophagostomum, matching previous findings using single-specimen genetics. Utilizing the Illumina MiSeq platform, ITS-2 strongylid metabarcoding was applied to 90 samples from various wild NHPs occurring in Malaysian Borneo and Japan. A clear dominance of Oesophagostomum aculeatum was found, with almost all sequences assigned to this species. This study suggests that strongylid communities of Asian NHPs may be less species-rich than those in African NHPs, where multi-genera communities are reported. Such knowledge contributes baseline data, assisting with ongoing monitoring of health threats to NHPs.

Comparative evaluation of different molecular methods for DNA extraction from individual Teladorsagia circumcincta nematodes.

BACKGROUND: The purpose of this study was to develop a reliable DNA extraction protocol to use on individual Teladorsagia circumcincta nematode specimens to produce high quality DNA for genome sequencing and phylogenetic analysis. Pooled samples have been critical in providing the groundwork for T. circumcincta genome construction, but there is currently no standard method for extracting high-quality DNA from individual nematodes. 11 extraction kits were compared based on DNA quality, yield, and processing time. RESULTS: 11 extraction protocols were compared, and the concentration and purity of the extracted DNA was quantified. Median DNA concentration among all methods measured on NanoDrop 2000™ ranged between 0.45-11.5 ng/µL, and on Qubit™ ranged between undetectable - 0.962 ng/µL. Median A260/280 ranged between 0.505-3.925, and median A260/230 ranged - 0.005 - 1.545. Larval exsheathment to remove the nematode cuticle negatively impacted DNA concentration and purity. CONCLUSIONS: A Schistosoma sp. DNA extraction method was determined as most suitable for individual T. circumcincta nematode specimens due to its resulting DNA concentration, purity, and relatively fast processing time.

New records of Amphibiophilus spp. (Nematoda: Amphibiophilidae) parasitic in Strongylopus grayii (Smith) and Amietia delalandii (Duméril & Bibron) (Amphibia: Anura: Pyxicephalidae) in South Africa, with a description of Amphibiophilus bialatus n. sp.

Nematodes of the genus Amphibiophilus Skrjabin, 1916 are a small group of parasites restricted to pyxicephalid frogs in southern Africa. In the present study, the new species A. bialatus parasitising the clicking stream frog Strongylopus grayii (Smith) as well as two forms parasitising the common river frog Amietia delalandii (Duméril & Bibron) from two distant localities are described. Amphibiophilus bialatus n. sp. clearly differs from the remaining species of the genus by having wide cervical alae, the dorsal oesophageal tooth not reaching the oral opening, and the presence of extra processes on the spicules. Specimens parasitising Am. delalandii in Mpumalanga Province and Limpopo Province, South Africa, differed from other species and from each other in the shape of the gubernaculum, though were almost identical in other characters. Based on morphological and molecular data, specimens from two localities were assigned to Amphibiophilus sp. 1 and Amphibiophilus sp. 2. Pairwise analyses of ITS-28S and cox1 gene fragments are presented for four Amphibiophilus spp.

Revision of Macroponema Mawson, 1978 (Nematoda: Strongylida) from macropodid marsupials with the description of two new species.

BACKGROUND: Species of Macroponema Mawson, 1978 are strongyloid nematodes which occur in the stomachs of macropodid marsupials in Australia. In this study, the genus Macroponema is revised, redescriptions of the two known species are provided, and two new species are added to the genus. METHODS: A molecular characterisation of the internal transcribed spacers of the nuclear ribosomal DNA of representative specimens of Macroponema from all known host species was undertaken to confirm the status of M. cf. comani. This resulted in the identification of a further new species within the genus. Consequently, a review of all available material in museum collections was undertaken. RESULTS: The two known species M. beveridgei Mawson, 1978 from Osphranter antilopinus (Gould) and O. robustus (Gould), and M. comani Mawson, 1978 from Macropus giganteus Shaw are re-described and their geographical distributions expanded. Two new species added to the genus are M. arundeli n. sp. from Ma. giganteus found in Queensland and the north east of New South Wales, and M. obendorfi n. sp. from O. antilopinus and O. robustus in the Northern Territory, the Kimberley Division of Western Australia and eastern Queensland. The latter species was formerly identified as M. cf. comani based on molecular studies. The specific identification of both of the new species is supported by ribosomal DNA sequence data. CONCLUSIONS: Based on the morphological and molecular characterisation of nematodes, this study has revealed the existence of four species within the genus Macroponema. The current phylogenetic data suggest that Macroponema spp. plausibly evolved by host switching; however, further studies are required to test this hypothesis.

Development of amplicon sequencing for the analysis of benzimidazole resistance allele frequencies in field populations of gastrointestinal nematodes.

Anthelmintic resistant gastrointestinal helminths have become a major cause of poor health in sheep and goats. Sensitive and specific molecular markers are needed to monitor the genotypic frequency of resistance in field parasite populations. Gastrointestinal nematode resistance to benzimidazole is caused by a mutation in one of three positions within the isotype 1 ß-tubulin gene. In the absence of markers for resistance to the other broad spectrum anthelmintic classes, these provide a relevant study example. Determination of the prevalence of these single nucleotide polymorphisms in field nematode populations can be impractical using conventional molecular methods to examine individual parasites; which can be laborious and lack sensitivity in determining low levels of resistance in parasite populations. Here, we report the development of a novel method based on an Illumina MiSeq deep amplicon sequencing platform to sequence the isotype 1 ß-tubulin locus of the small ruminant gastrointestinal nematode, Teladorsagia circumcincta, and determine the frequency of the benzimidazole resistance mutations. We validated the method by assessing sequence representation bias, comparing the results of Illumina MiSeq and pyrosequencing, and applying the method to populations containing known proportions of resistant and susceptible larvae. We applied the method to field samples collected from ewes and lambs on over a period of one year on three farms, each highlighting different aspects of sheep management and approaches to parasite control. The results show opportunities to build hypotheses with reference to selection pressures leading to differences in resistance allele frequencies between sampling dates, farms and ewes or lambs, and to consider the impact of their genetic fixation or otherwise. This study provides proof of concept of a practical, accurate, sensitive and scalable method to determine frequency of anthelmintic resistance mutations in gastrointestinal nematodes in field studies and as a management tool for livestock farmers.

New species and new records of species of Cloacina von Linstow, 1898 (Nematoda: Strongylida) parasitic in the western scrub wallaby, Notamacropus irma (Jourdan) (Marsupialia: Macropodidae) from Western Australia.

The helminth parasites of the western scrub wallaby or black-glove wallaby, Notamacropus irma (Jourdan) which occurs in Western Australia are relatively poorly documented. Six new species of the strongyloid genus Cloacina von Linstow, 1898 (Strongylida: Chabertiidae) are described namely C. asymmetrica n. sp., C. brazellei n. sp., C. harriganae n. sp., C. hobbsi n. sp., C. middletoni n. sp. and C. woodi n. sp. A redescription of C. laius Beveridge, 1999 from the same host species is included. Molecular sequence data (ITS1 and ITS2 ribosomal DNA) were obtained for C. asymmetrica, C. brazellei, C. hobbsi, C. middletoni and from the previously described species C. themis Beveridge, 1998 occurring in the same host species. Phylogenetically, C. asymmetrica, C. hobbsi and C. middletoni formed a distinct clade, suggesting the possibility of within-host speciation. Cloacina themis clustered with a group of morphologically distinctive species in a separate clade and C. brazellei clustered in a third clade but with poor support. This pattern of congeners in a single host species occurring in multiple clades mirrors the situation in other kangaroos and wallabies. Species of Cloacina from N. irma reported thus far therefore consist of a series of species found only in this host, with two species (C. brazellei and C. laius) shared with the sympatric macropodid Setonix brachyurus (Quoy & Gaimard).

Phylogenetic relationships of three tribes of cloacinine nematodes (Strongylida: Chabertiidae) from macropodid marsupials.

The phylogenetic relationships of 42 species of cloacinine nematodes belonging to three tribes (Coronostrongylinea, Macropostrongylinea and Zoniolaiminea) were examined based on sequence data of the first and second internal transcribed spacers (ITS-1 and ITS-2) of the nuclear ribosomal DNA. All nematodes examined are parasites of Australian macropodid marsupials. None of the three nematode tribes was monophyletic. Paraphyly was also encountered in three genera: Papillostrongylus, Monilonema and Wallabinema. Species within the genus Thallostonema were limited to a single host genus (i.e. Thylogale), whereas species within the five principal genera (Coronostrongylus, Macropostrongylus, Popovastrongylus, Wallabinema and Zoniolaimus) were found to occur in multiple host genera. Potential modes of evolution among these nematodes are discussed.

Metabarcoding analysis of strongylid nematode diversity in two sympatric primate species.

Strongylid nematodes in large terrestrial herbivores such as great apes, equids, elephants, and humans tend to occur in complex communities. However, identification of all species within strongylid communities using traditional methods based on coproscopy or single nematode amplification and sequencing is virtually impossible. High-throughput sequencing (HTS) technologies provide opportunities to generate large amounts of sequence data and enable analyses of samples containing a mixture of DNA from multiple species/genotypes. We designed and tested an HTS approach for strain-level identification of gastrointestinal strongylids using ITS-2 metabarcoding at the MiSeq Illumina platform in samples from two free-ranging non-human primate species inhabiting the same environment, but differing significantly in their host traits and ecology. Although we observed overlapping of particular haplotypes, overall the studied primate species differed in their strongylid nematode community composition. Using HTS, we revealed hidden diversity in the strongylid nematode communities in non-human primates, more than one haplotype was found in more than 90% of samples and coinfections of more than one putative species occurred in 80% of samples. In conclusion, the HTS approach on strongylid nematodes, preferably using fecal samples, represents a time and cost-efficient way of studying strongylid communities and provides a resolution superior to traditional approaches.

Molecular and morphological characterisation of Pharyngostrongylus kappa Mawson, 1965 (Nematoda: Strongylida) from Australian macropodid marsupials with the description of a new species, P. patriciae n. sp.

BACKGROUND: Pharyngostrongylus kappa Mawson, 1965 is a nematode (Strongyloidea: Cloacininae), endemic to the sacculated forestomachs of Australian macropodid marsupials (kangaroos and wallaroos). A recent study revealed genetic variation within the internal transcribed spacer region of the nuclear ribosomal DNA among P. kappa specimens collected from Macropus giganteus Shaw and Osphranter robustus (Gould). This study aimed to characterise the genetic and morphological diversity within P. kappa from four macropodid host species, including M. giganteus, O. robustus, O. antilopinus (Gould) and O. bernardus (Rothschild). METHODS: Specimens of P. kappa from M. giganteus and Osphranter spp. from various localities across Australia were examined. The first and second internal transcribed spacers (ITS1 and ITS2, respectively) were amplified using polymerase chain reaction and sequenced. Phylogenetic methods were used to determine the interspecific diversification within P. kappa and its evolutionary relationship with other congeners. RESULTS: Morphological examination revealed that P. kappa from M. giganteus, the type-host, can be distinguished from those in Osphranter spp. by the greater length and number of striations on the buccal capsules. DNA sequences showed that P. kappa from M. giganteus was genetically distinct from that in Osphranter spp., thereby supporting the morphological findings. Based on these finding, a new species from Osphranter spp., Pharyngostrongylus patriciae n. sp., is described. CONCLUSION: Pharyngostrongylus patriciae n. sp. from Osphranter spp. is distinguished from P. kappa based on molecular and morphological evidence. The study highlights the importance of combining molecular and morphological techniques for advancing the nematode taxonomy. Although ITS genetic markers have proven to be effective for molecular prospecting as claimed in previous studies, future utilisation of mitochondrial DNA to validate ITS data could further elucidate the extent of speciation among macropodid nematodes.

Molecular identification of parasitic nematodes (Nematoda: Strongylida) in feces of wild ruminants from Tunisia.

In Tunisia and other North African countries, there is a lack of knowledge about parasite biodiversity within threatened wild ruminants and there are not any studies on their gastrointestinal nematodes. Thus the aim of this study was to identify gastrointestinal fauna in the faecal samples of Tunisian wild ruminants. A total of 262 faecal samples were collected from domestic sheep and goat, and wild ruminants (Addax, Barbary sheep, Barbary red deer, Dorcas gazelle, Slender-horned gazelle and Scimitar-horned Oryx) living in protected areas. Samples were examined with floatation (saturated sodium chloride solution), polymerase chain reaction and sequencing of the second internal transcribed spacer region of the rDNA. Microscopic analysis allowed the identification of only Nematodirus genus or molecular tools allowed a first identification of five gastrointestinal nematode species in North African wild ruminants: Chabertia ovina (1.6%), Camelostrongylus mentulatus (1.6%), Marshallagia marshalli (4.7%), Nematodirus helvetianus (62.5%) and Nematodirus spathiger (29.7%). This study reported the first records of C. mentulatus and M. marshalli in Addax and of M. marshalli in Dorcas gazelle and it was the first reported record of N. helvetianus and M. marshalli in Tunisia.

Nematode Species Identification-Current Status, Challenges and Future Perspectives for Cyathostomins.

Human and animal health is globally affected by a variety of parasitic helminths. The impact of co-infections and development of anthelmintic resistance requires improved diagnostic tools, especially for parasitic nematodes e.g., to identify resistant species or attribute pathological effects to individual species or particular species combinations. In horses, co-infection with cyathostomins is rather a rule than an exception with typically 5 to 15 species (out of more than 40 described) per individual host. In cyathostomins, reliable morphological species differentiation is currently limited to adults and requires highly specialized expertize while precise morphological identification of eggs and early stage larvae is impossible. The situation is further complicated by a questionable validity of some cyathostomins while others might actually represent cryptic species complexes. Several molecular methods using different target sequences were established to overcome these limitations. For adult worms, PCR followed by sequencing of mitochondrial genes or external or internal ribosomal RNA spacers is suitable to genetically confirm morphological identifications. The most commonly used method to differentiate eggs or larvae is the reverse-line-blot hybridization assay. However, both methods suffer from the fact that target sequences are not available for many species or even that GenBank® entries are unreliable regarding the cyathostomin species. Recent advances in proteomic tools for identification of metazoans including insects and nematodes of the genus Trichinella will be evaluated for suitability to diagnose cyathostomins. Future research should focus on the comparative analysis of morphological, molecular and proteomic data from the same cyathostomin specimen to optimize tools for species-specific identification.

PULMONARY LESIONS CAUSED BY THE LUNGWORM (DIDELPHOSTRONGYLUS HAYESI) IN THE OPOSSUM (DIDELPHIS VIRGINIANA) IN COLIMA, MEXICO.

Didelphostrongylus hayesi is an important and prevalent pulmonary nematode in the opossum ( Didelphis virginiana ). An in-depth description of the pulmonary lesions caused by this nematode is lacking. The objective of this investigation was to make a detailed account of the gross, subgross, and microscopic changes that occur in the lungs of opossums naturally infected with D. hayesi. Forty-four opossums trapped in the state of Colima, Mexico, were euthanized by an overdose of barbiturates. Following a postmortem examination, the right lung was cut from the main bronchi and placed in a Petri dish containing a saline solution for the detection and identification of live parasites. The left lung was fixed and cut serially for subgross microscopic examination and sections of lung were cut and stained for histopathologic examination. The most remarkable gross change in parasitized lungs was a poorly collapsible pulmonary parenchyma and mild emphysema. The right lung tested positive for lungworms on gross examination in 20/44, and 11/44 (25%) of the left lungs showed tan nodules on the pleural surface. Microscopically, the bronchi of 20/44 animals harbored adult and larval stages of D. hayesi (left lung), the same 20 opossums from which nematodes were grossly evident at necropsy (right lung). Adults and larvae were present in bronchi, bronchioles, and alveoli mixed with desquamated cells and many eosinophils, and to a lesser extent neutrophils, alveolar macrophages, and giant cells. Bronchi and bronchioles exhibited goblet cell hyperplasia and metaplasia respectively, and infiltration of lymphoplasmacytic cells in the interstitium and lamina propria. The tan nodules consisted of focal alveolar endogenous lipidosis, which likely resulted from parasitic airway obstruction. The lungs of 3/20 parasitized opossums also showed alveolar bronchiolization (Lambertosis). The absence of Eucoleus aerophilus or bacterial pneumonia incriminates D. hayesi as the putative cause of pulmonary lesions in these opossums.

Genetic variation between and within Triodontophorus brevicauda and Triodontophorus nipponicus revealed by analyses of mtDNA and rDNA gene sequences.

Triodontophorus spp. parasitizes the large intestine of equine, causing strongylid diseases. The present study assessed genetic variation in five gene regions within and between Triodontophorus brevicauda and Triodontophorus nipponicus from Heilongjiang Province and the Inner Mongolia Autonomous region. The five gene markers were three mitochondrial (mt) genes, cytochrome c oxidase subunit I (cox1), NADH dehydrogenase subunit 5 (nad5), cytochrome b (cytb); and two ribosomal RNA genes, the internal transcribed spacer 1 (ITS1) and the internal transcribed spacer 2 (ITS2). Partial (p) sequences of cox1, nad5, cytb and the complete ITS rDNA region were PCR amplified from individual nematodes, and the amplicons were subjected to sequencing in both directions. The size of the three mt genes is identical in both species: 761 bp (p cox1), 505 bp (pnad5) and 562 bp (pcytb); the length of the two ribosomal genes is different: 376 bp and 370 bp (ITS1), and 333 bp and 322 bp (ITS2), respectively. Intraspecific variation between T. brevicauda and T. nipponicus was 0-1.5% and 0-1.1% for pcox1, 0-2.0% and 0-2.0% for pnad5, 0-1.4% and 0-2.2% for pcytb, 0-0.8% and 0-1.1% for ITS1 and 0-0.9% and 0-2.2% for ITS2. Interspecific variation within the nematodes was 13.5-14.3% for pcox1, 15.5-18.7% for pnad5, 16.7-18.6% for pcytb, 11.5-13.1% for ITS1 and 16.0-18.4% for ITS2. Phylogenetic analyses based on the combined mt gene sequences, as well as with the ITS sequences, show each species forming a monophyletic group of individuals. However, samples of different species from the same geographical origin did not always cluster together. These results provide valuable information for further studies of systematics and population genetics of the genus Triodontophorus.

The occurrence of the strongylid nematodes Kalicephalus brachycephalus, K. bungari and K. indicus in snake species from Hunan Province, China.

Kalicephalus is a genus of strongylid nematodes infecting snakes and causing serious diseases and even death when it is complicated with secondary bacterial infections. The infection of snakes with Kalicephalus has been reported in many countries in the world. However, little information is available on the prevalence of Kalicephalus in snakes in China. In the present study, the prevalence of Kalicephalus in snakes was investigated. The worms were examined, counted and identified to species according to existing keys and descriptions. Three species of Kalicephalus, namely K. indicus, K. bungari and K. brachycephalus, were found in six species of snakes (Elaphe carinata, Zaocys dhumnade, Naja najaatra, Elaphe taeniura, Bungarus multicinctus and Dinodon rufozonatum). The total prevalence of Kalicephalus in snakes in Hunan Province was 39.7%. The most common species was K. indicus, with the highest prevalence 72.8%, followed by K. bungari (24.0%). The prevalence of K. brachycephalus was 0.9%. This is the first report on the prevalence of Kalicephalus species in snakes in China, and the findings have important implications for the control of Kalicephalus infections in snakes in China.

Pharyngostrongylus thylogale n. sp. (Nematoda: Strongylida) from the stomachs of macropodid marsupials defined by morphological and molecular criteria.

Pharyngostrongylus thylogale n. sp. (Nematoda: Strongylida) is described from the stomach of the red-legged pademelon, Thylogale stigmatica (Gould) (Marsupialia: Macropodidae) from north-eastern Queensland and Papua New Guinea, having formerly been confused with P. iota Johnston & Mawson, 1939. Pharyngostrongylus thylogale n. sp. differs from all congeners in having 12 labial crown elements rather than eight or 16. Pharyngostrongylus iota was found in T. stigmatica, but only in southern Queensland and northern New South Wales, in the subspecies T. s. wilcoxi, compared with P. thylogale n. sp. which was found in T. s. stigmatica in northern Queensland and T. s. oriomo in Papua New Guinea. Differences in the sequences of the first and second internal transcribed spacers (ITS-1 and ITS-2) of the nuclear ribosomal DNA of P. thylogale n. sp. and ten congeners support the erection of the new species, and the validity of the morphospecies examined. However, results of the phylogenetic analyses of the molecular data also provide evidence for the existence of cryptic species within P. kappa Mawson, 1965. No obvious co-evolutionary relationships were observed between parasite species and their macropodid marsupial hosts.

Three Nematode Species Recovered from Terrestrial Snakes in Republic of Korea.

The majority of parasitological studies of terrestrial snakes in Korea have focused on zoonotic parasites. However, in the present study, we describe 3 unrecorded nematode species recovered from 5 species of snakes (n=6) in Korea. The examined snakes, all confiscated from illegal hunters, were donated by the Chungnam Wild Animal Rescue Center and Korean Broadcasting System in July 2014 and February 2015. Light and scanning electron microscopies on the shapes of spicules that are either bent or straight (kalicephalids) and the presence of the intestinal cecum (ophidascarids) figured out 3 nematodes; Kalicephalus brachycephalus Maplestone, 1931, Kalicephalus sinensis Hsü, 1934, and Ophidascaris excavata Hsü and Hoeppli, 1934. These 3 species of nematode faunas are recorded for the first time in Korea.

Real-time PCR/DNA melting curve-based assay to identify individual strongylid larvae recovered from ovine faecal cultures.

A closed-tube real-time PCR (RT PCR) method was developed to identify individual strongylid nematode larvae recovered from ovine faecal cultures. The method builds on an earlier conventional PCR assay established by our group and similarly targets species-specific sequence motifs in the ITS-2 region of ribosomal DNA. The new procedure combines RT PCR with DNA melting curve analyses to identify species-specific amplicons, thus avoiding the need to undertake gel electrophoresis. As with the earlier method, it involves two sets of species-specific reactions. The first targets Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, Nematodirus spathiger and Oesophagostomum venulosum while the second targets Trichostrongylus axei, Trichostrongylus vitrinis, Cooperia curticei and Chabertia ovina. With two exceptions, all the DNA primers employed in the new assay were among those described and tested in developing the earlier assay. The exceptions are the forward "generic" primer, which was re-designed to generate smaller amplicon sizes more suitable for melting curve analyses, and the T. axei-specific primer, which was modified to achieve a higher amplicon melt temperature to enable larvae of this species to be more readily differentiated from those of C. curticei. The melt temperature range for amplicons representing each of the species targeted was determined using lysates derived from both microscopically identified adult male worms (2-12/species), as well as 30 larvae of each of the species which were derived from at least 6 different geographical locations throughout New Zealand. The new assay potentially provides a simpler, faster method to identify individual ovine strongylid larvae for downstream applications than was provided by the earlier conventional PCR assay.

Nematodes of Heligmonellidae (Strongylida) of Pogonomys championi Flannery and Pogonomys sylvestris Thomas (Rodentia: Muridae) from Papua New Guinea with descriptions of five new species.

Eight species of heligmonellid nematodes including five new species and a putative new species were identified from the digestive tracts of 12 Pogonomys championi Flannery and 27 P. sylvestris Thomas (Murinae: Hydromyini) from Papua Indonesia. Hasanuddinia pogonomyos Smales, 2014 had been previously described from P. loriae Thomas and P. macrourus (Milne-Edwards) and Odilia mackerrasae (Mawson, 1961) from several endemic rodent species. Bunomystrongylus ilami n. sp. can be distinguished from its congeners by the number of rounded ridges in the synlophe; Hasanuddinia hasegawai n. sp. by the number of ridges, three ventral being hypertrophied, in the synlophe; Montistrongylus kaindiensis n. sp. by the number of ridges in the synlophe and the length of the spicules; Odilia helgeni n. sp. in the characters of the synlophe ridges; Odilia whittingtoni n. sp. in the characters of the synlophe ridges and the length of the spicules. Species richness of the nematode assemblage of P. sylvestris, nine species and a juvenile heligmonellid, was comparable to those of P. loriae and P. macrourus but that of P. championi, five species, was considered depauperate. Species composition showed both commonalities between the host species as well as distinctive features. Three of five species in the assemblage were unique to P. championi and five of nine species to P. sylvestris.

Biased sex ratio among worms of the family Heligmosomidae--searching for a mechanism.

According to Fisher's principle, an equal sex ratio is an evolutionary stable strategy. However, biased sex ratios have been reported in many metazoan parasite species, although the causes and mechanisms of the observed bias are still poorly understood. In the present study, we analysed sex ratios in long-term datasets from three populations of bank voles (Clethrionomys (=Myodes) glareolus) infected with Heligmosomum mixtum and Heligmosomoides glareoli. The overall sex ratios of both species were female-biased but in contrast to previous studies we did not find a relationship between the proportion of females and infection intensity. A higher female bias was observed in older hosts, suggesting that the sex ratio changes over time; the lifespan of nematodes in the family Heligmosomidae is known to be comparable with that of their hosts. We also compared the distributions of sexes in voles infected with two, three, four or five worms and we found significant differences from the expected values in both parasite species. In infections with four and five H. glareoli we observed more single-sex infections than expected, both female- and male-dominated, whereas in the case of H. mixtum female-dominated infections were more frequent.

Spatial distribution of infectious stages of the nematode Syngamus trachea within pheasant (Phasianus colchicus) release pens on estates in the South West of England: Potential density dependence?

The spatial distribution of the infectious stages of parasites with a direct life cycle is one of the most important factors influencing infectious disease dynamics, and acquisition rates will generally increase as the contact time between parasite and host increases. For animal species that are constrained by feeding opportunities, one might expect disease patterns to be highly skewed within confined systems. The aim of the present study was to identify to what extent, if any, eggs of avian parasites are aggregated within the release pen, and to evaluate what effect, if any, this aggregation had on the distribution of the adult stages within the host species. The abundance of Syngamus trachea eggs were highly aggregated within pens, with high levels of contamination driven by a combination of feeder placement, soil moisture and host-mediated heterogeneities in immuno-competence. The log mean and log variance of egg abundance was highly linear (R(2)=0.97-0.99), with an estimated slope (b) of between 1.79 and 1.97 for individual sites, and 2.11 when sites were combined, which indicated aggregation relative to an estimated Poisson slope of unity. Although the placement of feeders and environmental moisture could be contributing to parasite aggregation, density-dependent processes appear to be ensuring the population does not become too over or under-dispersed, in order to maintain the transmission-virulence equilibrium. To the best of our knowledge, this is the first paper to explicitly demonstrate the high spatial aggregation of eggs around feeding sites and the first to suggest possible density-dependent regulatory mechanisms stabilising disease dynamics between S. trachea and ring necked Pheasants (Phasianus colchicus).