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Relationships of Nematodirus Species and Nematodirus battus Isolates (Nematoda: Trichostrongyloidea) Based on Nuclear Ribosomal DNA Sequences
Abstract
Nuclear ribosomal sequence data from the internal transcribed spacers (ITS-1 and ITS-2), 5.8S subunit, and regions of the 18S and 28S genes were used to investigate sequence diversity among geographic samples of Nematodirus battus, and to infer phylogenetic relationships among Nematodirus species. Phylogenetic analysis of these data yielded strong support for relationships among species, depicting Nematodirus helvetianus and Nematodirus spathiger as sister-taxa and a clade of these 2 species and Nematodirus filicollis. This tree is consistent with caprine bovids as ancestral hosts, with a subsequent host shift to Bovinae in N. helvetianus. Eleven of 14 N. battus sequences were unique, with 19 variable sites among sequences representing 5 geographic samples. The lowest number of variable nucleotide sites was observed in samples representing apparently recent introductions to the United States and Canada, which is consistent with a population bottleneck concomitant with translocation. Comparison of directly sequenced polymerase chain reaction products and clones revealed evidence for intraindividual variation at some of the sequence sites, and this pattern of variation and that within geographic samples indicates incomplete rDNA repeat homogenization within species. This pattern of variation is not conducive for inferring phylogenetic relationships among sequences representing N. battus or addressing the putative history of introduction.
... Entomopathogenic nematodes (EPNs), which occur naturally in soils, are obligate parasites of soilinhabiting insects. EPNs were first described in 1923 with the identification of Aplectana kraussei Steiner (now known as Steinernema kraussei) (Nguyen and Hunt, 2007). Steinernematidae and Heterorhabditidae are two major families of EPNs with potential for managing insect populations (Kaya and Gaugler, 1993;Georgis et al., 2006). ...
... A part of rDNA comprising the internal transcribed spacer regions (ITS), ITS1 and ITS2 including 5.8 S were sequenced using two sets of primers. Primer set ITS-F (5'-TTGAACCGGGTAAAAGTCG-3 and ITS-R (5'-TTAGTTTCTTTTCCTCCGCT-3') was used to sequence the entire ITS1, 5.8 S and ITS2 regions (Nadler et al., 2000) while primer set Fnema18S (5'-TTGATTACGTCCCTGCCCTTT-3') and rDNA1.58 S (rev) (5'-ACGAGCCGAGTGATCCACCG-3') pair targeted the ITS1 region (Cherry et al., 1997). ...
... The reason behind the successful EPN reproduction in this study can be the already freeze-killed insect cadavers. The low recovery rate of nematodes might also be due to the failure of the EPN extraction method (Mráček et al., 2005), unsuitability of the laboratory environment for EPN culture and reproduction (Grewal et al., 1996), or the unsuitability of G. mellonella as a host because some EPNs are known to infect only certain hosts (Mráček et al., 2005;Klingen and Haukeland, 2006). In addition, there is a possibility that symbiotic bacterial cells may have been unable to effectively help EPNs reproduce in the hosts. ...
A total of 30 different agricultural fields in the Golden Triangle Region of Montana, USA were surveyed, and 150 soil samples were evaluated for the presence of entomopathogenic nematodes (EPNs). The authors isolated EPNs from 10% of the collected samples. The recovered isolates were identified as Steinernema feltiae and Heterorhabditis bacteriophora by using morphological and molecular analysis. Steinernema feltiae was found from two fields, Kalispell ( S. feltiae 1) and Choteau ( S. feltiae 2). Steinernema feltiae (1 and 2) differed significantly from each other in terms of morphological characters for infective juveniles (distance from anterior end to excretory pore and nerve ring) and 1st generation males (body length, spicule length, gubernaculum length, oesophagus, tail, and anal body diameter). Steinernema feltiae 2 and H. bacteriophora were recovered from the same field in Choteau. All these species were recovered from wheat fields with sandy clay loam and loam soils with 3.3 to 3.4% organic matter content and pH 8.
... 93 (forward, 5′-TTGAACCGGGTAAAAGTCG) and 264 (reverse, 5′-CGTTTTTCATCGATACGCG) for ITS-1; primers no. 623 (forward, 5′-ACGTCTGGTTCAGGGTTGTT) and 94 (reverse, 5′-TTAGTTTCTTTTCCTCCGCT) for ITS-2 (Nadler et al. 2000a(Nadler et al. , 2000b. Amplifications were performed in a 25µl reaction mixture containing 2.5µl 10X PCR buffer, 3mM MgCl 2 , 200µM deoxynucleoside triphosphates (dNTPs), 0.5µM of each primer, 1.25µl of bovine serum albumin (New England Biolabs, Ipswich, MA), 0.2µl Taq DNA polymerase (5000units/ml), and 2µl DNA template. ...
... (HQ262104 and HQ262132) from pinniped hosts (Table 3.4). (Nadler et al. 2000b). b GenBank accession no. ...
... rauschi and U. yukonensis, the analysis of the ITS regions confirmed their species status, and the presence of female polymorphism within both taxa. The ITS regions were further demonstrated to be reliable genetic markers to discriminate among nematode species; their low level (typically ≤ 1%) of intra-specific variability, along with higher rates of evolution than functional rRNA genes, makes ITS-1 and ITS-2 valuable molecular markers for species delineation (Baldwin et al. 1995;Stevenson et al. 1995Stevenson et al. , 1996Dallas et al. 2000;Gasser and Newton 2000;Nadler et al. 2000aNadler et al. , 2000bNadler 2002;Chilton 2004;Ngui et al. 2012;Ramos et al. 2013). ...
Classical and molecular parasitology are powerful tools for clinical diagnostics, for disease transmission surveys, and for designing strategies to control infections and outbreaks. Parasite communities have been demonstrated to strongly affect host population dynamics and viability. The absence of baseline data, and the potential detrimental effects on host health, supported the investigation of the gastrointestinal parasite fauna of free-living grizzlies (Ursus arctos horribilis) and black bears (Ursus americanus) from Alberta and British Columbia, Canada.
The study provided new insights into parasite biodiversity and infection patterns in Canadian bears. For the first time, the cestode species Diphyllobothrium dendtriticum, Diphyllobothrium nihonkaiense, and Taenia arctos have been unequivocally identified in North American bears. The present research also elucidated the systematics of the ursine hookworm species Uncinaria rauschi and Uncinaria yukonensis, determining their place within the family Ancylostomatidae.
... digitatusand H. contortus) clustered together. N. oiratianus and N. spathiger samples in the present study clustered together and were highly closely related to the family Dictyocaulidae with high clade support (Bpp = 1.00; Bf = 100), confirming the results of previous studies using the morphological features and ITS rDNA as the genetic marker [2,9,42]. However, amino acid sequence-based inferences of the relationship between the two Nematodirus species gave slightly different results to those using ITS rDNA data in which they clustered in sister clades with high support, possibly due to the different taxa inclusions and types of analysis performed [2,11-13,46]. ...
... To date, more than 45 Nematodirus species have been described based on their morphological features [1,2], however, no information is yet available about their mt genomes. Although previous studies have indicated that ITS rDNA provides a useful marker for identification and differentiation of Nematodirus species [2,11,13], mtDNA in nematodes is usually more variable in sequences within a species than the nuclear ribosomal DNA [14]. ...
... Nematodirus spp. are among the most common nematodes of ruminants and more than 45 species have been described in the genus Nematodirus Ransom, 1907 [1,2]. Of these, Nematodirus oiratianus and N. spathiger are widely distributed as highly prevalent gastrointestinal nematodes, mainly inhabiting the small intestines of sheep and goats [3-5]. ...
Background
Nematodirus spp. are among the most common nematodes of ruminants worldwide. N. oiratianus and N. spathiger are distributed worldwide as highly prevalent gastrointestinal nematodes, which cause emerging health problems and economic losses. Accurate identification of Nematodirus species is essential to develop effective control strategies for Nematodirus infection in ruminants. Mitochondrial DNA (mtDNA) could provide powerful genetic markers for identifying these closely related species and resolving phylogenetic relationships at different taxonomic levels.
Methods
In the present study, the complete mitochondrial (mt) genomes of N. oiratianus and N. spathiger from small ruminants in China were obtained using Long-range PCR and sequencing.
Results
The complete mt genomes of N. oiratianus and N. spathiger were 13,765 bp and 13,519 bp in length, respectively. Both mt genomes were circular and consisted of 36 genes, including 12 genes encoding proteins, 2 genes encoding rRNA, and 22 genes encoding tRNA. Phylogenetic analyses based on the concatenated amino acid sequence data of all 12 protein-coding genes by Bayesian inference (BI), Maximum likelihood (ML) and Maximum parsimony (MP) showed that the two Nematodirus species (Molineidae) were closely related to Dictyocaulidae.
Conclusions
The availability of the complete mtDNA sequences of N. oiratianus and N. spathiger not only provides new mtDNA sources for a better understanding of nematode mt genomics and phylogeny, but also provides novel and useful genetic markers for studying diagnosis, population genetics and molecular epidemiology of Nematodirus spp. in small ruminants.
... This homogenisation of the rDNA is referred to as concerted evolution (Anderson, Blouin & Beech, 1998). However, in parasitic nematodes multiple sequences of ITS are frequently found within individual worms (Hoste et al., 1995; Nadler et al., 2000b; Troell et al., 2003). Both direct sequencing and cloning of PCR products can be used for ITS analysis. ...
... Advantages with this approach are that the Pyrosequencing assay allows the estimation of SNP allele frequencies using peak height estimates (Berg, Sanders & Alderborn, 2002) while at the same time enabling high throughput of samples. This is important, as incomplete rRNA repeat homogenization, which may lead to intraindividual differences, has been observed in related trichostrongylids (Nadler et al., 2000b). Pyrosequencing is a real-time sequencing method designed for the identification of SNPs (Ahmadian et al., 2000; Fakhrai-Rad, Pourmand & Ronaghi, 2002). ...
... The two positions analyzed with Pyrosequencing technology both represent positions with intraindividual differences, i.e. heterogenetic sites that differ between the different copies in the multigene family. This incomplete rRNA repeat homogenization has been observed in other related trichostrongylid nematodes (Hoste et al., 1995; Nadler et al., 2000b). ...
Haemonchus contortus is a blood-sucking nematode of the abomasum in small ruminants. It is responsible for extensive losses and huge animal welfare problems globally. This thesis is based on four publications with the overall aim to characterize H. contortus from Sweden, and includes both genotypic and phenotypic aspects of this parasite.
This nematode has by tradition been considered a tropical parasite due to the climatic requirements for its preparasitic lifestages. Its development and survival is dependent on temperature and humidity, and the window of opportunity to complete its life cycle in Sweden is limited.
The species status of Haemonchus in Sweden was investigated by comparing the genetic differences in the internal transcribed spacers (ITS-1 and ITS-2) of the ribosomal DNA (rDNA) in worms from Sweden and Kenya. As no fixed differences were found between these isolates they are considered to be distinct populations of the same species, H. contortus. The population structure of H. contortus at a global level was studied by analysing the genetic variability using both amplified fragment length polymorphism (AFLP) and nad4 sequences of the mitochondrial genome. The results show that both the genetic variation and the genetic structure were high between the different isolates, and that populations from each continent mostly formed monophyletic groups in the phylogenetic analysis.
The thesis also includes experimental infection studies performed with both fresh and cold- treated infective larvae of either Kenyan or Swedish origin. These investigations covered a range of phenotypic traits, to search for any potential adaptations the parasite might have in order to survive Sweden’s cold temperate climate. In addition, several studies were performed on the development and long-term survival of the parasite in climatic chambers, as well as studies on the overwinter survival of H. contortus on pastures in Sweden. The results show that this parasite can survive winters on pastures. However, the survival capacity was very low and is of limited significance in the transmission between grazing-seasons.
Taken together, the results presented in this thesis indicate that H. contortus has a large phenotypic plasticity rather than having undergone any evolutionary adaptation.
... There is still a question of how many different hookworm species infect pinnipeds [29,34] based on morphological and morphometric characteristics of two species of pinniped hookworms that had been fully described previously: Uncinaria lucasi [35] and Uncinaria hamiltoni [36,37]. Further phylogenetic analyses in other specimens that had been isolated from nine different pinniped species showed seven independent evolutionary lineages or species, including the above-described species and five undescribed ones [30]. ...
... From the NCBI (GenBank®-www.ncbi.nlm.nih.gov/ (accessed on 30 September 2021) database, the only deposited genomic regions for identified Uncinaria species included one in MtDNA (Cytochrome oxidase subunit I (COI)-after) [107], one in ITS-rDNA, and two regions in 28SrDNA(D2/D3 and D18/D19), including the 18S 3'-end, ITS-1, and ITS-2, 5.8S subunit, and 28S 5'-end [34] (Table 2) The PCR amplification products were separated in 1.5% (wt/vol) agarose gels using 1X Tris Acetate EDTA (TAE) and photographed on a UV transilluminator. PCR amplification products were purified by using the NucleoSpin Extract Kit (Macherey Nagel, Duren, Germany) in order to remove secondary metabolites prior to sequencing. ...
The Mediterranean monk seal (Monachus monachus) is classified by the IUCN as "endan-gered," with a global population estimated to number fewer than 800 individuals. Our understanding of the biology and health status of the species is still limited, rendering every medical case a challenge for conservationists and veterinary clinicians. Although studying and managing disease in wild marine hosts is complex and challenging, studying and mitigating the effects of any disease to the Mediterranean monk seal is of utmost importance for conservation. The aim of this study was to document for the first time the presence of the hookworm Uncinaria hamiltoni in rehabilitated Mediterranean monk seal pups in Greece. A detailed examination protocol was followed for all pups that live-stranded over 30 years in 22 different locations, including physical, parasitological, and other examinations. Hookworms (adults and/or eggs) were detected in all the fecal samples, from all animals. Molecular identification using MtDNA (COI) and ribosomal DNA (D2/D3 28S and internal transcribed spacer [ITS] regions) identified the nematode species as Uncinaria hamiltoni. The clinical impacts and the benefits of anthelmintic treatment as a tool for the conservation management of the species are discussed.
... The coding regions are usually conserved even between distantly related species, while ITS spacers show considerable variability between different animal genomes. The level of ITS sequence variation observed among individuals of the same species is about the same as that observed among ITS repeats within individuals , typically £1% (Heise et al., 1999; Nadler et al., 2000). In Pratylenchus spp., ITS regions are one of the most variable nuclear loci so far identified . ...
... Most of the variations in length in these genes are accounted for by D expansion segments (Clark et al., 1984), which are believed to be redundant structures tolerated during evolution because they do not interfere with ribosome functions (Hancock and Dover, 1988). In particular, the D3 region is used to investigate sequence diversity among samples of different nematode species of different geographic origins (Al-Banna et al., 1997; Duncan et al., 1999; Nadler et al., 2000; Carta et al., 2001; Handoo et al., 2001). Although the low level of similarity detected in Pratylenchus spp. of different geographically isolated samples suggested that the Pratylenchus genome is highly variable (Duncan et al., 1999; Waeyenberge et al., 2002), the extent of variability within and between individuals in the different species has not received much attention. ...
Plant parasitic nematodes belonging to the genus Pratylenchus, also known as root lesion nematodes, cause serious economic damage to different crop plants. In order to explore genetic structures in different isolates, we investigated several P. thornei,
P. neglectus and P. penetrans populations of different geographic origins. The analysis at the species level was also extended to P. penetrans, P. pinguicaudatus, P. vulnus and P. mediterraneus. Sequence analysis of a specific portion of DNA was carried out. In particular, the sequences of the D3 region of the 26S gene were obtained and compared with similar sequences available in databases. The results support the hypothesis that P. penetrans may represent a species complex, while in P. neglectus the intra-species heterogeneity observed is due to intra-individual variability. Furthermore, the specific conservation of some nucleotides in different P. thornei populations indicates their fixation in the rDNA repeats in this species. The presence of these nucleotides, the molecular signature of P. thornei, may assist in determining the nature of nematode infections.
... Only 10% of isolated N. spathiger represented a low intraspecific variation by a single nucleotide difference (0·34%). The observed number of variation site is in agreement with the proportion of polymorphic ITS2 sites (0-0·9%) in Nematodirus (Audebert et al. 2000) but less than that reported by Nadler et al. (2000) within N. spathiger, N. filicollis and N. battus (12; 15 and 19 sites, respectively). ...
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.
... Ideally, parasite invasion scenarios that are indirectly inferred from population genetic data should be matched with known information on host movements. The invasion pathway of the nematode Nematodirus battus into western North America and northern Europe could be tied to known importation routes of domestic sheep from central Europe ( Nadler et al., 2000). Timing of the establishment of the liver fluke, F. magna, in ungulate populations in eastern Europe coincided with the importation of elk from western North America to support local hunting economies ( Králová-Hromadová et al., 2011). ...
Parasite distributions are constantly changing due to climate change, local and global movement of animals and humans, as well as land use and habitat change. The trematode D. dendriticum is a relatively recent invader into Canada being first reported in Eastern Canada in the 1930s and Western Canada in the 1970s. However, historical records are scarce and its emergence is poorly understood. The establishment of this parasite in Canada provides an interesting opportunity to explore the use of population genetic approaches to help elucidate the invasion history of a relatively recently established helminth parasite. In this study, we compare the genetic diversity and population structure of a number of D. dendriticum populations from Western and Eastern Canada and compare these with much longer established European populations. Two independent genetic marker systems were used; a microsatellite marker panel and a COX1 mtDNA sequence marker. We found distinct differences in both genetic diversity and population structure of the different Canadian populations that provide insights into their invasion histories when compared to the European populations. Two populations from British Columbia - Salt Spring and Vancouver Islands - are of low diversity, show evidence of a
population bottleneck, and are closely related to each other suggesting a shared recent history of establishment. These west coast populations are otherwise most closely related to those from Eastern Canada and Western Europe and in contrast are genetically divergent from those in Cypress Hills, Alberta. Although the Alberta parasite population is the most recently reported in Canada, being first identified there in the earl 1990s, it was the most genetically diverse of those examined and showed a strong pattern of admixture of genotypes present in western and Eastern Europe. Overall, our results are consistent with a model in which Western
Europe is likely the source of flukes on the east coast of Canada which were then subsequently translocated to the west coast of Canada. The most recently reported D. dendriticum population in Canada appears to have a
different history and likely has multiple origins.
... using scanning electron microscopy. Molecular characterization and phylogeny of some Thelazia species have been studied by Nadler et al. [5], Otranto et al. [6], and Traversa et al. [7]. Owing to the localization of the nematode, thelaziasis can be treated topically by direct application of drugs into the eyes. ...
A cross sectional study was conducted from April 2014 to June 2014 in Mersa Town of South Wollo Zone, Amhara Regional Stat to determine the prevalence of thelaziasis of the disease in cattle associated risk factors responsible for the occurrence of the disease. A total of 384 cattle of both ages (98 young and 286 adult) sexes (203 male and 181 females) were examined using visual observation of the eyes by flushing the conjunctiva sac and lachrymal duct with sterile saline solution. The overall prevalence for thelaziasis was 18.23% (70 cases). Age, sex, body condition scores and management systems did not show any statistical significant difference (P>0.05) in the prevalence for thelaziasis. The prevalence was higher in male (24.12%) than female (11.6%), age groups of young (26.53%) were higher than adult (18.38%) in animals kept in extensive (20.69%) than those kept in semi-intensive system (13.01%). The prevalence of bovine thelaziasis was recorded highest it in poor (30.19%) than medium (17.79%) and lowest in good (14.48%) body conditioned score cattle. The results of the present study showed that bovine thelaziasis requires special attention considering its impact on cattle production and productivity.
... Additional internal primers , 4R, 22F, 13R and 4F, were also used for sequencing the 18S rRNA (Bert et al. 2008). The 5.8S rRNA gene and its flanking regions ITS-1 and ITS-2 were amplified using primers N93 and N94 (Nadler et al. 2000). All PCRs were 25 lL made of 5 lL of DNA template, 0.2 lL of each primer (20 lM) and 19.6 lL of PCR purified water in combination with Pure Taq-Ready to Go kit (GE Health Care â , Buckinghamshire, UK). ...
The phylogenetic position of Cephalenchus is enigmatic with respect to other tylench nematodes. In this study, Cephalenchus populations representing 11 nominal species were sampled worldwide for molecular and morphological characterization. Species identification was based on light microscopy (LM) and scanning electron microscopy (SEM). Molecular analyses were based on the genes (i.e. 18S, 28S, 5.8S) and internal transcribed spacers (ITS-1 and ITS-2) of the ribosomal RNA (rRNA). Phylogenetic analyses (i.e. full and reduced alignments) of either concatenated or single genes always supported the monophyly of Cephalenchus. A sister relationship between Cephalenchus and Eutylenchus excretorius was recovered by most analyses, although branch support varies depending on the dataset used. The position of Cephalenchus + E. excretorius within Tylenchomorpha nevertheless remains ambiguous, thus highlighting the importance of sampling additional genes as well as taxa. Placement of Cephalenchus + E. excretorius as sister of Tylenchinae or Boleodorinae could not be rejected on the basis of 18S and 28S rRNA genes. Within Cephalenchus, amphidial opening morphology shows congruence with molecular-based phylogenetic relationships, whereas the number of lines in the lateral field is likely to be a convergent trait. Morphometric analyses clearly distinguished short tail from medium–long tail species, and SEM observations seem to suggest a relation between tail length and amphidial opening. In addition, molecular phylogenies support the non-monophyly of Cephalenchus cephalodiscus, Cephalenchus cylindricus, Cephalenchus daisuce and Cephalenchus leptus. The known extent of Cephalenchus diversity is increased with the inclusion of two new species, and the biogeography of the genus is discussed.
... DNA was extracted from single individuals using proteinase K protocol and Worm Lysis Buffer (WLB) as described in Pereira et al. (2010). The D2–D3 domains of the 28S gene were amplified with primers D2Ab and D3B (De Ley et al., 2005) and the ITS region (including ITS-1, 5.8S, and ITS-2) with primers N93 and N94 (Nadler et al., 2000). All PCR reactions were 25 ll made as follows: 5 ll of DNA template, 0.2 ll of each primer (20 lM) and 19.6 ll of PCR purified water in combination with Pure Taq-Ready to Go kit (GE Health Care Ò ). ...
Concerted evolution is often assumed to be the evolutionary force driving multi-family genes, including those from ribosomal DNA (rDNA) repeat, to complete homogenization within a species, although cases of non-concerted evolution have been also documented. In this study, sequence variation of 28S and ITS ribosomal RNA (rRNA) genes in the genus Cephalenchus is assessed at three different levels, intragenomic, intraspecific, and interspecific. The findings suggest that not all Cephalenchus species undergo concerted evolution. High levels of intraspecific polymorphism, mostly due to intragenomic variation, are found in Cephalenchus sp1 (BRA-01). Secondary structure analyses of both rRNA genes and across different species show a similar substitution pattern, including mostly compensatory (CBC) and semi-compensatory (SBC) base changes, thus suggesting the functionality of these rRNA copies despite the variation found in some species. This view is also supported by low sequence variation in the 5.8S gene in relation to the flanking ITS-1 and ITS-2 as well as by the existence of conserved motifs in the former gene. It is suggested that potential cross-fertilization in some Cephalenchus species, based on inspection of female reproductive system, might contribute to both intragenomic and intraspecific polymorphism of their rRNA genes. These results reinforce the potential implications of intragenomic and intraspecific genetic diversity on species delimitation, especially in biodiversity studies based solely on metagenetic approaches. Knowledge of sequence variation will be crucial for accurate species diversity estimation using molecular methods.
... Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase (an enzyme originally isolated from the bacterium The ribosomal internal transcribed spacer 2 (ITS.2) are considered to be the most reliable and practical indicators to evaluate and differential diagnosis of various nematode species (Gasser et al., 2000). These structures are considered as the best tool as they present specific areas where maximum inconstant nuclear loci and are under intensive development (Nadler et al., 2000). Thus the ITS-2 gene is an important and unique gene which is used to identify within the genus Haemonchus (Gasser, 1999; Heise et al., 1999). ...
Haemonchus populations were collected from cattle from Punjab province of Pakistan (four populations) to determine the relative species prevalence of Haemonchus placei. This work is the first documentation on the rDNA internal transcribed spacer-2 (ITS-2) sequences of H. placei from cattle and the results provide a preliminary insight on the genetic variation of H. placei in this region. Total of 78 individual adult worms were genotyped at position 24 of the rDNA ITS-2 from four populations to determine species identity. Analysis of the ITS-2 confirmed that all samples investigated belonged to 100% H. placei. Within the species of H. placei few intraspecific differences in the ITS-2 has been reported. For Instance, rDNA ITS-2 sequences identified 4 sites that showed intraspecific variations (positions 65, 111, 125, 148). Further detailed and large size samples strategy will be required to identified the co-infection and interspecies hybridization between H. placei (common parasite of cattle) and H. contortus (common parasite of sheep/goat) present in samples extracted from cattle in Pakistan.
Keywords: Haemonchus placei, ITS-2, Pakistan
... DNA was eluted in 20 L TE buffer and stored at −20°C. DNA extracts from hookworm specimens were amplified for the ITS-1 and ITS-2 genes using primers 93 and 264 for ITS-1, and primers 623 and 94 for ITS-2 (Nadler et al. 2000a(Nadler et al. , 2000b. DNA extracts from cestode specimens were amplified for a segment of the cox1 gene using primers 2575 and 3021 for isolates of the genus Taenia L., 1758 (Bowles et al. 1992), and primers BW3 and BW4.5 for isolates of the genus Diphyllobothrium Cobbold, 1858 (Wicht et al. 2007). ...
Between May 2011 and June 2013, we collected the carcasses and gastrointestinal tracts of 40 American black bears (Ursus americanus Pallas, 1780) and 13 grizzly bears (Ursus arctos L., 1758) from populations of Alberta and British Columbia, Canada. Specimens were examined for helminths, which were identified to the species level by applying an integrated morphological and molecular approach. Our goal was to investigate parasite biodiversity and infection parameters in the sampled grizzly and black bears. We found seven parasite taxa:. The statistical significance of infection prevalence, intensity, and abundance for each helminth species was assessed relative to host species, gender, age class, sampling season, and location. This is the first unequivocal report of the potentially zoonotic tapeworms D. dendriticum and D. nihonkaiense in North American bears. Furthermore, we provide insight into the biology and ecology of the nematodes B. transfuga, D. ursi, and species of Uncinaria Frölich, 1789, and enrich the information available on the recently described tapeworm T. arctos.
... (Thorne) Boström & De Ley) pooled samples of adults (six to 100 individuals) were extracted using the ID Pure method (S. similis) or by Proteinase K digestion followed by phenol-chloroform enrichment, ethanol precipitation and RNAse A treatment (Nadler et al., 2000b). DNA from phenol-chloroformextracts was quanti ed by spectrophotometry and 100-200 ng used per PCR ampli cation. ...
Acromoldavicus (Cephalobina, Cephaloboidea) with its highly distinctive lip region has only a single species, Acromoldavicus skrjabini Nesterov & Lisetskaya, originally described from Moldova and subsequently also detected at sites in the Middle East and near the Mediterranean. Herein, Acromoldavicus mojavicus n. sp. is described from sandy soil surrounding a Joshua tree (Yucca brevifolia) in a remote area of the Mojave Desert, California, USA. The lip region of A. mojavicus n. sp. is bilaterally symmetrical with three triangular probolae surrounded by three pairs of plate-like lips. The lip region is organised along similar lines as that of A. skrjabini, but differs in several respects, such as its larger size, presence of elongate posterior processes on each lip and division of the lateral lips into two lobes (excluding the dorso-sublateral guard processes). In addition, phylogenetic interpretation of sequence data from the large-subunit of ribosomal DNA provides further evidence for autapomorphies and separate species status for A. skrjabini and A. mojavicus n. sp. Characteristics shared with Cephaloboidea include the offset spermatheca and males with eight pairs of genital papillae. Both species of Acromoldavicus have a buccal capsule with a reduced gymnostom, a character that seems to be shared with the cephalobid Elaphonema and in part is a basis for placement of both genera in Elaphonematidae. The species A. mojavicus n. sp. exhibits additional similarities with Elaphonema spp. that further support this placement.
... 93, 5¢ TTGAACCGGGTAAAAGTCG and no. 94, 5¢ TTAGTTT CTTTTCCTCCGCT) designed by Nadler et al. (2000) and the restriction enzymes Sau3AI, AluI, and DdeI (for more detailed protocols see Hodson, 2010). ...
Entomopathogenic nematodes (EPNs) can kill and regulate populations of soil-inhabiting insects, but studies evaluating these interactions in native ecosystems are rare. The objective of this study was to examine the effects of EPNs on a non-agricultural caterpillar, Platyprepia virginalis (Boisduval) (Lepidoptera: Arctiidae), under natural conditions. Platyprepia virginalis caterpillars live in litter on the soil surface feeding beneath bush lupine during summer, autumn, and winter. Initial laboratory assays revealed that the caterpillars were vulnerable to at least two species of EPNs with which they co-occur in the coastal prairie in northern California (USA). In contrast to laboratory assays, caterpillars survived exposure to prairie soil containing EPNs under natural conditions in field assays. To better understand the divergence between laboratory and field results for this native caterpillar, we used sentinel insects [Galleria mellonella L. (Lepidoptera: Pyralidae)] to identify particular locations where EPNs were present in the field. Platyprepia virginalis caterpillars were caged at these sites but again showed no evidence of susceptibility to EPNs. Platyprepia virginalis caterpillars reduce their exposure to EPNs by spending their time in and above the litter rather than contacting the soil when given the choice in nature. We conclude that P. virginalis is unlikely to serve as a reservoir for EPNs and that nematodes are unlikely to be important mortality factors for P. virginalis in this natural system.
... The pairwise comparison between these sequences showed a percentage of dissimilarity varying from 0 to 5%. The level of ITS sequence variation observed among individuals of the same species is about the same as that observed among ITS repeats within individuals, typically # 1% (Heise et al., 1999; Nadler et al., 2000). This finding suggests that P. lentis n. sp. is characterized by substantial intra-specific variation in the ITS region. ...
Pratylenchus lentis n. sp. parasitizing roots of lentil in Sicily, Italy, is described and illustrated. The new species is characterized by a relatively high lip region with three annuli, mean stylet length of 16 mum, with anteriorly flattened knobs, cylindrical body with a relatively anterior vulva, large and ovoid spermatheca full of sperm, plump tail with truncate, irregularly annulated terminus, and by the presence of males. Molecular ITS-RFLP and sequencing analyses of the new species showed clear differences from other most morphologically similar species, such as P. thornei and P. mediterraneus. Preliminary host range tests revealed that chickpea, pea, faba bean and durum wheat are good hosts of P. lentis n. sp., whereas common bean, alfalfa and barley are less robust hosts and tomato, bell pepper, eggplant, melon and sunflower are poor hosts for the nematode.
... A region of nuclear ribosomal DNA (rDNA), including the 18S 3-end, ITS-1 and ITS-2, 5.8S subunit, and 28S 5-end was amplified using the polymerase chain reaction (PCR). The strategy for design of ITS PCR primers (sequence positions according to Caenorhabditis elegans numbering, Ellis et al, 1986; GenBank X03680) was described previously (Nadler et al., 2000). These primers anneal to the 3-end of the 18S rDNA (forward [] primer no. ...
California sea lions (Zalophus californianus) and northern fur seals (Callorhinus ursinus) are each believed to host distinct hookworm species (Uncinaria spp.). However, a recent morphometric analysis suggested that a single species parasitizes multiple pinniped hosts, and that the observed differences are host-induced. To explore the systematics of these hookworms and test these competing hypotheses, we obtained nucleotide sequences of nuclear ribosomal DNA (D2/D3 28S, D18/D19 28S, and internal transcribed spacer [ITS] regions) from 20 individual hookworms parasitizing California sea lion and northern fur seal pups where their breeding grounds are sympatric. Five individuals from an allopatric population of California sea lions were also sampled for ITS-1 and D18/D19 28S sequences. The 28S D2/D3 sequences showed no diagnostic differences among hookworms sampled from individual sea lions and fur seals, whereas the 28S D18/D19 sequences had one derived (apomorphic) character demarcating hookworms from northern fur seals. ITS sequences were variable for 7 characters, with 4 derived (apomorphic) states in ITS-1 demarcating hookworms from California sea lions. Multivariate analysis of morphometric data also revealed significant differences between nematodes representing these 2 host-associated lineages. These results indicate that these hookworms represent 2 species that are not distributed indiscriminately between these host species, but instead exhibit host fidelity, evolving independently with each respective host species. This evolutionary approach to analyzing sequence data for species delimitation is contrasted with similarity-based methods that have been applied to numerous diagnostic studies of nematode parasites.
Members of the genus Microphallus Ward, 1901, are endoparasites mainly of birds and mammals distributed worldwide. Unencysted metacercariae of Microphallus sp., were collected from the mesoglea of ctenophores of the genus Pleurobrachia Fleming; adult digeneans were recovered from the intestines of Eudocimus albus Linnaeus (Threskiornithidae) and Buteogallus urubitinga Gmelin (Accipitridae), in four locations from southeastern Mexico. Adult specimens were identified as M. basodactylophallus (Bridgman, 1969) based on the following features: body pyriform entirely covered by minute spines, prepharynx short, oesophagus very long, caeca short and widely divergent, testes slightly symmetrical and excretory vesicle short and V-shaped. Sequences from D1–D3 domain of the large subunit of ribosomal DNA (LSU) were generated, aligned, and compared with those of congeneric species available in GenBank. Phylogenetic analyses indicated that the metacercariae and adults formed a clade together with an isolate identified as M. basodactylophallus from Florida, USA (GenBank: AY220628). The intraspecific genetic divergence among isolates was low ranged from 0.0% to 0.6%, allowing the link between the two stages of the life cycle. We observed phenotypic plasticity in the morphological traits of M. basodactylophallus adults in definitive hosts (mammals and birds) throughout the distribution, which ranged from the USA to southeastern Mexico. Finally, the unencysted metacercariae identified as M. basodactylophallus represent the first report of a microphallid in ctenophores.
Members of the genus Strigea Abildgaard, 1790 are endoparasites of birds distributed worldwide. Adults of an undescribed species of the genus Strigea were collected from the intestines of two hawk species (Rupornis magnirostris and Accipiter coperii). Other species identified as Parastrigea macrobursa that were described in Argentina were also recovered from two hawk species (Buteogallus urubitinga and Buteogallus anthracinus) in three localities along the coasts of Mexico. Specimens of the two species were sequenced for three molecular markers, the internal transcribed spacers locus (ITS1-5.8S rDNA- ITS2) and the domains D1-D3 from the large subunit from nuclear ribosomal DNA and the cytochrome c oxidase subunit 1 from mitochondrial DNA. The newly sequenced specimens were aligned with other strigeids sequences downloaded from GenBank. Maximum likelihood and Bayesian analyses inferred with each molecular marker revealed that our specimens of Strigea sp. formed an independent lineage, which is recognized herein as a new species, Strigea magnirostris n. sp., representing the first species in Mexico and the 16th in the Neotropical region. Morphologically, the new species is distinguished from other congeneric species from the Americas by having an oral sucker with several papillae around it, well-developed pseudosuckers (118-248 μm), a tegument covered with tiny spines, a larger cone genital (193-361 × 296-637) and a larger copulatory bursa (247-531 × 468-784). Our phylogenetic analyses revealed that P. macrobursa is not closely related to other members of the genus Parastrigea and is nested within Strigea, suggesting that P. macrobursa should be transferred to Strigea to form Strigea macrobursa n. comb., expanding its distribution range from Mexico to Argentina. Finally, the analyses also revealed that the taxonomy and systematics of Strigea should be re-evaluated, combining morphological and molecular characteristics.
Attraction sites are important for environmental pathogen transmission and spillover. Yet, their role in wildlife disease dynamics is often poorly substantiated. Herein, we study the role of salt licks as potential attraction sites for the spillover of gastrointestinal parasites from domestic sheep to wild reindeer. Eggs from the introduced sheep nematode Nematodirus battus were found in faecal samples of both species, suggestive of spillover. DNA metabarcoding of soil, collected at salt licks, revealed that N. battus, in addition to Teladorsagia circumcincta, were the most frequently occurring parasitic nematodes, with a significantly higher prevalence of nematodal DNA in salt lick soil compared to soil from control sites nearby. The finding of similar DNA haplotypes of N. battus in sheep, reindeer, and salt lick soil supports the hypothesis of spillover to reindeer via salt licks. More detailed investigation of the genetic diversity of N. battus across these hosts is needed to draw firm conclusions. Infection with these sheep nematodes could potentially explain a recently observed decline in the calf recruitment rate of the Knutshø reindeer herd. This study also supports the hypothesized role of artificial salt licks as hot spots for the transmission of environmentally persistent pathogens and illustrates the importance of knowledge about such attraction points in the study of disease in free-roaming animals.
A parasitological survey of terrestrial slugs and snails was conducted at popular dog walking locations across the city of Nottingham, with the intensions of finding gastropods infected with parasites of medical (or veterinary) importance such as lungworm (metastrongyloid nematodes) and trematodes. A total of 800 gastropods were collected from 16 sites over a 225 km2 area. The extracted nematodes and trematodes were identified by molecular barcoding. Of the 800 gastropods collected, 227 were infected (172 had nematode infections, 37 had trematode infections and 18 had both nematode and trematode infections). Of the nematode infected gastropods genotyped, seven species were identified, Agfa flexilis, Angiostoma gandavense, Angiostoma margaretae, Cosmocerca longicauda, Phasmarhabditis hermaphrodita, Phasmarhabditis neopapillosa and an unknown Cosmocercidae species. Of the trematode infected gastropods genotyped, four species were identified, Brachylaima arcuate, Brachylaima fuscata, Brachylaima mesostoma and an unknown Plagiorchioidea species. No lungworm species were found within the city of Nottingham. To our knowledge, this study represents the first survey of gastropod-associated nematodes and trematodes in the East midlands of the United Kingdom.
Background
Steinernema feltiae is an entomopathogenic nematode used in biological control programs with a global distribution. Populations of this species show phenotypic plasticity derived from local adaptation and vary in different traits, such as location and host penetration. The aim of this work was to describe a Chilean isolate of this nematode species, using integrative approaches.
Methods
Nematode morphological and morphometric studies were conducted along with molecular analysis of nuclear genes. The symbiotic bacterium was also identified by sequencing the 16S rRNA gene. Some ecological characteristics were described, including the temperature requirements for the nematode life cycle and the effect of soil water content for optimal reproduction.
Results
Morphometric characterization revealed a large intra-specific variability. The isolate identity was also corroborated with the analysis of nuclear genes. Based on the 16S gene, its symbiont bacteria, Xenorhabdus bovienii, was identified. The lowest, optimal and highest temperatures found to limit the infestation and reproduction on Galleria mellonella were 10, 20 and 30 °C, respectively; the emergence from the host larvae occurred approximately 10 days after inoculation. Differences were observed in offspring, and 120 infective juveniles (IJ)/larva was the most prolific dose at 20 °C. The soil water content did not affect the number of IJ invaders, penetration efficacy and IJ emergence time or offspring per larva, but it caused a delay in achieving full mortality at the permanent wilting point with respect to saturation and field capacity.
Conclusions
For the first time, a Chilean isolate of S. feltiae is described in detail considering morphological, molecular and ecological aspects. The isolate was shown to be efficient in soil containing water, with optimal temperatures ranging from 15 to 25 °C for host infestation and production of an abundant offspring; these characteristics would allow its potential use as control agents in a wide geographical area of the country.
In Fiji, little or no attention has been given to entomopathogenic nematodes (EPNs) in biocontrol programs due to the lack of awareness about their occurrence and distribution in Fiji. A survey of EPNs was conducted for the first time in Fiji Islands in 2012 and 2013, throughout the eight provinces in Viti Levu to determine the occurrence and distribution across different habitat in Viti Levu. The soil samples from various habitats were collected and assayed for the presence of EPN using Galleria mellonella as baits. EPNs were recovered from five out of seven provinces with 35 positive sites (7.3%) out of 478 sites sampled. The only EPN genera encountered was Heterorhabditis. Steinernema was not isolated from any of the samples. Characterization of isolates was done by using morphometric and molecular examinations and isolates were identified as Heterorhabditis indica. H. indica isolates were primarily recovered from leeward side of the Viti Levu Island along the coastline and riversides, being more prevalent in lighter soil with pH > 6. Further, this study found significant association between habitat type, soil type, soil pH, average annual rainfall and EPN occurrence. This is the first record of naturally occurring EPNs in Fiji. The found nematodes will serve as the basis for efficacy screening with the ultimate aim of delivering effective, more sustainable and environmentally safe control for agricultural pests in Fiji.
Ribosomal RNA genes have long been a favored locus in phylogenetic and metabarcoding studies. Within a genome, rRNA loci are organized as tandem repeated arrays and the copies are homogenized through the process of concerted evolution. However, some level of rRNA variation (intragenomic polymorphism) is known to persist and be maintained in the genomes of many species. In nematode worms, the extent of rRNA polymorphism (RP) across species and the evolutionary and life history factors that contribute to the maintenance of intragenomic RP is largely unknown. Here we present an extensive analysis across 30 terrestrial nematode species representing a range of free‐living and parasitic taxa isolated worldwide. Our results indicate that RP is common and widespread, ribosome function appears to be maintained despite mutational changes, and intragenomic variants are stable in the genome and neutrally evolving. However, levels of variation were varied widely across rRNA locus and species, with some taxa observed to lack RP entirely. Higher levels of RP were significantly correlated with shorter generation time and high reproductive rates, and population‐level factors may play a role in the geographic and phylogenetic structuring of rRNA variants observed in genera such as Rotylenchulus and Pratylenchus. Although RP did not dramatically impact the clustering and recovery of taxa in mock metabarcoding analyses, the present study has significant implications for global biodiversity estimates of nematode species derived from environmental rRNA amplicon studies, as well as our understanding of the evolutionary and ecological factors shaping genetic diversity across the nematode Tree of Life.
The present study reports the occurrence of the genus Belonolaimus in the state of Sinaloa, Mexico, associated with native plants (i.e., Ziziphus amole and Stenocereus alamosensis) in a natural coastal ecosystem. Both morphological and molecular approaches were employed to characterize the Sinaloa population. Notwithstanding of some morphological and morphometric variation between Belonolaimus from Sinaloa and other valid species, the characterization indicates that this population might belong to the Belonolaimus longicaudatus species complex. Molecular analyses based on the 28S gene and ITS1-5.8S-ITS2 regions of the ribosomal RNA (rRNA) identified four major clades within Belonolaimus; however, none of the species including B. longicaudatus, B. gracilis, and B. euthychilus were supported as monophyletic; yet monophyly is argued to be a basic requirement of species status. Sequence divergence among different Belonolaimus populations and species varied according to the rRNA dataset (i.e., ITS1-5.8S-ITS2 . 28S . 18S) used, thus showing the importance of using genes with different rates of evolution to estimate species relationships. The fact that Belonolaimus has not been found in other cultivated (including on suitable hosts) areas in Sinaloa and that this population is relatively distant from the common B. longicaudatus groups (i.e., clades A and B) suggests that its appearance was not due to a recent introduction associated with the local agriculture.
Genetic markers (ribosomal DNA and mitochondrial DNA) were used for molecular dissection of the Anisakis simplex s.l. complex populations. Host fish were caught off Moroccan coasts, where only A. pegreffii is present, the sympatric area comprising Spanish coasts, and the Little Sole Bank fishing area from Nordeast Atlantic Ocean where the only present species is A. simplex s.s. Sequence variations in the amplification products were then assessed indirectly by digestion with restriction endonucleases or directly by sequencing for 623 L3 larvae. The sequences were used to infer the relationships between the two species under study using various methodological approaches. We reveal the high genetic diversity of Anisakis simplex s.s. and Anisakis pegreffii in both mitochondrial and nuclear genes. We detected 10 and 2 fixed differences between A. simplex s.s and A. pegreffii in the Cox2 and ITS1, respectively. We found a proportion of putative hybrids below 20% with similar figures on the Atlantic and Mediterranean coasts. Moroccan hybrids were more similar to Anisakis pegreffii reflecting backcrosses between these mixed genotypes and his ancestor Anisakis pegreffii. We discuss the possible interpretation of these putative hybrids.
Haemonchus populations were collected from cattle from Punjab province of Pakistan (four populations) to determine the relative species prevalence of Haemonchus placei. This work is the first documentation on the rDNA internal transcribed spacer-2 (ITS-2) sequences of H. placei from cattle and the results provide a preliminary insight on the genetic variation of H. placei in this region. Total of 78 individual adult worms were genotyped at position 24 of the rDNA ITS-2 from four populations to determine species identity. Analysis of the ITS-2 confirmed that all samples investigated belonged to 100% H. placei. Within the species of H. placei few intraspecific differences in the ITS-2 has been reported. For Instance, rDNA ITS-2 sequences identified 4 sites that showed intraspecific variations (positions 65, 111, 125, 148). Further detailed and large size samples strategy will be required to identified the co-infection and interspecies hybridization between H. placei (common parasite of cattle) and H. contortus (common parasite of sheep/goat) present in samples extracted from cattle in Pakistan.
Keywords: Haemonchus placei, ITS-2, Pakistan
Haemonchus populations were collected from cattle from Punjab province of Pakistan (four populations) to determine the relative species prevalence of Haemonchus placei. This work is the first documentation on the rDNA internal transcribed spacer-2 (ITS-2) sequences of H. placei from cattle and the results provide a preliminary insight on the genetic variation of H. placei in this region. Total of 78 individual adult worms were genotyped at position 24 of the rDNA ITS-2 from four populations to determine species identity. Analysis of the ITS-2 confirmed that all samples investigated belonged to 100% H. placei. Within the species of H. placei few intraspecific differences in the ITS-2 has been reported. For Instance, rDNA ITS-2 sequences identified 4 sites that showed intraspecific variations (positions 65, 111, 125, 148). Further detailed and large size samples strategy will be required to identified the co-infection and interspecies hybridization between H. placei (common parasite of cattle) and H. contortus (common parasite of sheep/goat) present in samples extracted from cattle in Pakistan.
Haemonchus populations were collected from cattle from Punjab province of Pakistan (four populations) to determine the relative species prevalence of Haemonchus placei. This work is the first documentation on the rDNA internal transcribed spacer-2 (ITS-2) sequences of H. placei from cattle and the results provide a preliminary insight on the genetic variation of H. placei in this region. Total of 78 individual adult worms were genotyped at position 24 of the rDNA ITS-2 from four populations to determine species identity. Analysis of the ITS-2 confirmed that all samples investigated belonged to 100% H. placei. Within the species of H. placei few intraspecific differences in the ITS-2 has been reported. For Instance, rDNA ITS-2 sequences identified 4 sites that showed intraspecific variations (positions 65, 111, 125, 148). Further detailed and large size samples strategy will be required to identified the co-infection and interspecies hybridization between H. placei (common parasite of cattle) and H. contortus (common parasite of sheep/goat) present in samples extracted from cattle in Pakistan.
Plant parasitic nematodes are major pests for crops. They significantly damage agriculture worldwide. It has been estimated that plant parasitic nematodes reduce the yield of the world's 40 major food staples and cash crop by about 12.5 % (Sasser, 1980). Furthermore, much of the crop damage caused by nematodes is unrecognized. However in spite of their impact on world agriculture, the basic biology of plant parasitic nematodes is poorly understood (Bird Mck. D., 1996). This can be explained by the difficulties in accumulating sufficient biological material to undertake biochemical and molecular biology studies. Most plant parasitic nematodes are microscopic, obligate root-parasites. One of the problems of working with plant parasitic nematodes is that they are difficult to grow: even when it is possible to collect enough material to perform a biochemical experiment, the eagerness to repeat the experiment is frustrated by the need to wait until new material is accumulated again! The control of plant parasitic nematodes relies on the application of nematicides to nematode infested fields, crop rotation and the use of resistant plant varieties as well. Only recently the development of new techniques has allowed researchers to address questions that can be answered in terms of biochemistry and molecular biology.
Abstract The hookworms Uncinaria rauschi Olsen, 1968 and Uncinaria yukonensis (Wolfgang, 1956) were formally described from grizzly (Ursus arctos horribilis) and black bears (Ursus americanus) of North America. We analyzed the intestinal tracts of 4 grizzly and 9 black bears from Alberta and British Columbia, Canada, and isolated Uncinaria specimens with anatomical traits never documented previously. We applied morphological and molecular techniques to investigate the taxonomy and phylogeny of these Uncinaria parasites. The morphological analysis supported polymorphism at the vulvar region for females of both U. rauschi and U. yukonensis. The hypothesis of morphological plasticity for U. rauschi and U. yukonensis was confirmed by genetic analysis of the internal transcribed spacers (ITS-1 and ITS-2) of the nuclear ribosomal DNA. Two distinct genotypes were identified, differing at 5 fixed sites for ITS-1 (432 base pairs) and 7 for ITS-2 (274 base pairs). Morphometric data for U. rauschi revealed host-related size differences: adult U. rauschi were significantly larger in black bears than grizzly bears. Interpretation of these results considering the historical biogeography of North American bears suggests a relatively recent host-switching event of U. rauschi from black bears to grizzly bears, which likely occurred after the end of the Wisconsin glaciation. Phylogenetic Maximum Parsimony (MP) and Maximum Likelihood (ML) analyses of the concatenated ITS-1 and ITS-2 datasets strongly supported monophyly of U. rauschi and U. yukonensis, and their close relationship with Uncinaria stenocephala (Railliet, 1884), the latter a parasite primarily of canids and felids. Relationships among species within this group, although resolved by ML, were unsupported by MP and bootstrap resampling. The clade of U. rauschi, U. yukonensis, and U. stenocephala was recovered as sister to the clade represented by Uncinaria spp. from otariid pinnipeds. These results support the absence of strict host-parasite co-phylogeny for Uncinaria spp. and their carnivore hosts. Phylogenetic relationships among Uncinaria spp. provided a framework to develop the hypothesis of similar transmission patterns for the closely related U. rauschi, U. yukonensis, and U. stenocephala.
Background:
Varestrongylus alces, a lungworm in Eurasian moose from Europe has been considered a junior synonym of Varestrongylus capreoli, in European roe deer, due to a poorly detailed morphological description and the absence of a type-series.
Methods:
Specimens used in the redescription were collected from lesions in the lungs of Eurasian moose, from Vestby, Norway. Specimens were described based on comparative morphology and integrated approaches. Molecular identification was based on PCR, cloning and sequencing of the ITS-2 region of the nuclear ribosomal DNA. Phylogenetic analysis compared V. alces ITS-2 sequences to these of other Varestrongylus species and other protostrongylids.
Results:
Varestrongylus alces is resurrected for protostrongylid nematodes of Eurasian moose from Europe. Varestrongylus alces causes firm nodular lesions that are clearly differentiated from the adjacent lung tissue. Histologically, lesions are restricted to the parenchyma with adult, egg and larval parasites surrounded by multinucleated giant cells, macrophages, eosinophilic granulocytes, lymphocytes. The species is valid and distinct from others referred to Varestrongylus, and should be separated from V. capreoli. Morphologically, V. alces can be distinguished from other species by characters in the males that include a distally bifurcated gubernaculum, arched denticulate crura, spicules that are equal in length and relatively short, and a dorsal ray that is elongate and bifurcated. Females have a well-developed provagina, and are very similar to those of V. capreoli. Morphometrics of first-stage larvae largely overlap with those of other Varestrongylus. Sequences of the ITS-2 region strongly support mutual independence of V. alces, V. cf. capreoli, and the yet undescribed species of Varestrongylus from North American ungulates. These three taxa form a well-supported crown-clade as the putative sister of V. alpenae. The association of V. alces and Alces or its ancestors is discussed in light of host and parasite phylogeny and host historical biogeography.
Conclusions:
Varestrongylus alces is a valid species, and should be considered distinct from V. capreoli. Phylogenetic relationships among Varestrongylus spp. from Eurasia and North America are complex and consistent with faunal assembly involving recurrent events of geographic expansion, host switching and subsequent speciation.
The guanaco (Lama guanicoe) is the major inhabitant and the largest wild artiodactyl in Patagonia. The introduction of invasive species into its ecological niche poses ecological risks, since invasive species may introduce harmful parasites to this native species. In this work, filariform larvae of the Nematodirus genus were found in feces of guanacos from the Perito Moreno National Park in Argentina. All species were characterized according to morphological features and molecular analyses using ribosomal DNA (rDNA). For the molecular analysis, rDNA fragments were amplified by PCR and then sequenced. The results of the BLASTN comparison threw a 99 % of identity with Nematodirus spathiger and 97 % with N. helvetianus, suggesting that N. spathiger is the infecting parasite. Nematodirus spathiger together with N. filicollis and N. battus causes diarrhea and deaths in sheep and, in some cases, in South American camelids. The availability of more accurate diagnostic methods such as PCR could improve the control measures for gastrointestinal helminthiasis.
Internal transcribed spacer (ITS) rDNA sequences of three Nematodirus species from naturally infected goats or sheep in two endemic provinces of China were analysed to establish an effective molecular approach to differentiate Nematodirus species in small ruminants. The respective intra-specific genetic variations in ITS1 and ITS2 rDNA regions were 0.3-1.8% and 0-0.4% in N. spathiger, 0-6.5% and 0-5.4% in N. helvetianus, and 0-4.4% and 0-6.1% in N. oiratianus from China. The respective intra-specific variations of ITS1 and ITS2 were 1.8-4.4% and 1.6-6.1% between N. oiratianus isolates from China and Iran, 5.7-7.1% and 6.3-8.3% between N. helvetianus samples from China and America. For N. spathiger, compared with samples from China, sequence differences in ITS1 rDNA were 0.3-2.4% in isolates from America, 0.3-2.9% in New Zealand and 2.1-2.4% in Australia. Genetic variations in ITS2 rDNA of N. spathiger were 0-0.4% between samples from China and America, and 0-0.8% between samples from China and New Zealand. Using mutation sites, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and specific PCR techniques were developed to differentiate these three Nematodirus species. The specific PCR assay allowed the accurate identification of N. oiratianus from other common nematodes with a sensitivity of 0.69 pg and further examination of Nematodirus samples demonstrated the reliability of these two molecular methods.
Entomopathogenic nematodes (EPNs) are lethal insect parasites that develop inside the body of their insect host. The EPN Steinernema riobrave infects many different types of soil insects, including larvae of the invasive citrus root weevil, Diaprepes abbreviatus, which is a key pest in citrus production. Soil characteristics have a great impact on nematode foraging efficacy and therefore on the ability of EPNs to be used successfully in biological control programs. Our specific objectives were to: (1) survey for the presence of native EPNs in California citrus groves; and (2) identify soil parameters that would allow us to predict EPN efficacy. The long-term objective of this study is to develop biological control methods to address the invasion of the citrus root weevil, D. abbreviatus, into California using EPNs.
Libyostrongylus sp. are nematodes that infect ostriches. Libyostrongylus douglassii was first described in ostriches from several countries in the world. Later Libyostrongylus dentatus was morphologically identified in ostriches in the USA and Brazil, and mixed infection is common in the latter country. The internal transcribed spacer (ITS) region of the ribosomal DNA gene is used for genetic variability assessment and phylogenetic reconstruction for many organisms. Through genetic analysis the status of different species morphologically defined was confirmed and a molecular method was developed to differentiate both species. ITS1, 5.8S, ITS2 regions of L. douglassii and L. dentatus were characterized. Regarding complete ITS region, the K2-p genetic distance between the species was 0.060 (SE 0.008) and the intra-specific distance was 0.002 (SE 0.001) for L. dentatus and 0.006 (SE 0.002) for L. douglassii. NJ and MP phylogenetic analysis of ITS1 and ITS2 regions indicated that both species belong to the Trichostrongylidae family, and are evolutionarily different, suported by high bootstrap value. Based on ITS DNA polymorphisms, a molecular approach was designed to detect both species. These results are the first molecular characterization of L. douglassii and L. dentatus, and provide new tools for the identification of these parasites of veterinary importance.
Restriction analysis of amplified ribosomal ITS sequences has provided species-specific fragment patterns for nematodes of several genera, including Bursaphelenchus. We used restriction enzymes RsaI, HaeIII, MspI, HinfI and AluI to produce ITS-RFLP reference profiles of 44 Bursaphelenchus species, including two intraspecific types in each of B. mucronatus and B. leoni. In addition, reference profiles of Aphelenchoides stammeri and Ruehmaphelenchus asiaticus were produced. Reference profiles of six species are shown here for the first time. Identical ITS-RFLP patterns were usually obtained from different isolates and from individual specimens of the same species. However, in the case of B. 'corneolus', B. lini, B. singaporensis and B. sexdentati, additional bands in the patterns of certain isolates or individual nematodes were observed which may be explained by ITS sequence microheterogeneity, i.e., the presence of ITS sequence variants within the number of rDNA tandem repeats. Since these 'extra' bands appeared only with one out of the five restriction enzymes employed, they did not seriously impair identification of species based on the overall reference patterns. ITS-RFLP analysis has proved valuable for differentiation of the pathogenic pine wood nematode, B. xylophilus, from related species. In many recent descriptions of new Bursaphelenchus species, ITS-RFLP profiles have been used as additional species identification criteria. Comparison of profiles from isolates of many different origins has provided new information on intraspecific types or genetically distinct provenances of several Bursaphelenchus species.
Entomopathogenic nematodes (EPNs) in the genera Steinernema and Heterorhabditis and their associated bacteria (Xenorhabdus spp. and Photorhabdus spp., respectively) are lethal parasites of soil dwelling insects. We collected 168 soil samples from five provinces, all located in southern Thailand. Eight strains of EPNs were isolated and identified to species using restriction profiles and sequence analysis. Five of the isolates were identified as Heterorhabditis indica, and one as Heterorhabditis baujardi. Two undescribed Steinernema spp. were also discovered which matched no published sequences and grouped separately from the other DNA restriction profiles. Behavioral tests showed that all Heterorhabditis spp. were cruise foragers, based on their attraction to volatile cues and lack of body-waving and standing behaviors, while the Steinernema isolates were more intermediate in foraging behavior. The infectivity of Thai EPN strains against Galleria mellonella larvae was investigated using sand column bioassays and the LC(50) was calculated based on exposures to nematodes in 24-well plates. The LC(50) results ranged from 1.99-6.95 IJs/insect. Nine centimeter columns of either sandy loam or sandy clay loam were used to determine the nematodes' ability to locate and infect subterranean insects in different soil types. The undescribed Steinernema sp. had the greatest infection rate in both soil types compared to the other Thai isolates and three commercial EPNs (Heterorhabditis bacteriophora, Steinernema glaseri and Steinernema riobrave).
Species-specific ITS-RFLP patterns have been established for more than 30 Bursaphelenchus species including 5 species of the ‘sexdentati’ group (B. sexdentati, B. vallesianus, B. pinophilus, B. poligraphi and B. borealis). Morphological species differentiation in the ‘sexdentati’ group is based on the shape of female tail and the spicules.
However, observations on different isolates of B. sexdentati have revealed considerable variability of these features suggesting the existence of intraspecific genetic types which could
not be differentiated by ITS-RFLP analysis using five enzymes. To improve intraspecific differentiation, we have determined
ITS1/2 sequences of 17 isolates belonging to five species of the ‘sexdentati’ group. In some isolates, sequence heterogeneity
at a few sites of ITS2 was detected. In the sequence-based phylogenetic tree, branching of clusters confirmed the affiliation
of isolates to their respective species. In addition, isolates of B. sexdentati were separated in two groups suggesting the existence of a Central European and a South European type of this species or
two separate species. This was supported by the differences in shape of female tails and spicules between the two types. The
information obtained from sequencing was used to select three additional enzymes for extending the scope of ITS-RFLP analysis.
In this way, improved distinction of species and differentiation of the two types of B. sexdentati was achieved.
Nematodes of the family Heligmonellidae (Heligmosomoidea; Trichostrongylina) reside in the digestive tracts of rodents and lagomorphs. Although this family contains large numbers of genera and species, genetic information on the Heligmonellidae is very limited. We collected and isolated adult worms of three species in Japan that belong to the family Heligmonellidae, namely Heligmonoides speciosus (Konno, 1963) Durette-Desset, 1970 (Hs) from Apodemus argenteus, Orientostrongylus ezoensis Tada, 1975 (Oe) from Rattus norvegicus and Lagostrongylus leporis (Schulz, 1931) (Ll) from Pentalagus furnessi, and sequenced the entire internal transcribed spacer regions, ITS-1 and ITS-2 of ribosomal DNA. ITS-1 of Hs, Oe and Ll was 426, 468 and 449 bp in length, and had a G+C content of about 41, 41 and 37 %, respectively. ITS-2 of Hs, Oe and Ll was 297, 319 and 276 bp in length and had a G+C content of about 38, 40 and 28%, respectively. The data of Hs, Oe and Ll were compared with those of two other known species within the family Heligmonellidae, Calorinensis minutus (Dujardin, 1845) (Cm) and Nippostrogylus brasiliensis (Travassos, 1914) (Nb), and with those of two species of Heligmosomidae (Heligmosomoidea), Heligmosomoides polygyrus bakeri and Ohbayashinema erbaevae. Phylogenetic analysis placed Hs, Oe and Ll in the same clade with Cm and Nb, forming a Heligmonellidae branch in both ITS-1 and ITS-2, separate from the Heligmosomoidea branch. These results demonstrated that the ITS-1 and ITS-2 sequences are useful for differentiating the Heligmonellidae nematode species. This study is the first to describe the ITS-1 and ITS-2 sequences of Hs, Oe and Ll.
Nematodirosis was diagnosed in the south-east of Scotland during two consecutive autumns in lambs which were grazed on the same field. The problem was unpredicted based on the knowledge of the pasture and animal management, and rudimentary understanding of the behaviour of free-living stages of Nematodirus battus in the region. Unlike the epidemiology that has been described in the south of England, whereby autumn infection of lambs is believed to arise from autumn hatching of eggs shed during the previous spring without prior chilling, it is concluded that the autumn nematodirosis in a particular sheep flock in Scotland most likely arose following prolonged survival of larvae hatched during the spring from eggs shed during the previous summer, following periods of cold exposure over the previous winter. The infective larvae survived in large numbers in a small, sheltered strip of rough grazing, where they would have been protected from harmful ultraviolet radiation and heavy rainfall, before infecting lambs during the autumn. Understanding of the evolutionary potential, nematode parasites to adapt to changing environmental conditions depends on a thorough clinical investigative approach, and is a prerequisite for future preventive management.
The biosphere in evolutionary and ecological time has been structured by episodes of geographic and host colonisation that have determined distributions of complex assemblages of microparasites and macroparasites, including helminths circulating among vertebrates. Biological invasion is an intricate phenomenon often involving 'extra-range dispersal' and establishment of exotic (non-indigenous) species and populations substantially beyond their native range. Invasion may also involve the expansion or shifting of host and geographic distributions of an endemic (indigenous) species or fauna under changing environmental conditions. Invasions result in faunal interchange occurring under influences from both natural and anthropogenic forces where expansion on spatial/temporal continua bridges continents, regions and landscapes. Drivers for invasion are idiosyncratic, multifactorial, interactive, and opportunistic, with a powerful role for historical contingency. The life history patterns of helminths interact with invasion pathways to determine the potential for introduction. Human-mediated events, such as the global expansion of pathogens linked to development of agriculture, domestication of food animals, and European exploration have had a pervasive influence on the distribution of helminths. Globalisation, broad transport networks and environmental perturbation linked to climate change, along with other drivers, have accelerated these processes. A consequence of invasion and establishment of exotic species is that faunal structure will be a mosaic that includes admixtures of indigenous and non-indigenous species and populations; exemplified by helminth faunas among domestic and free-ranging ungulates and a diversity of host-parasite systems among vertebrates. Contemporary mosaics are evident where human-mediated events have brought assemblages of new invaders and relatively old endemic species into sympatry, highlighting interactions at ecotones, particularly those at borderlands between managed and natural ecosystems. Understanding the historical origins and complex components of mosaics is essential in formulating predictions about future responses to environmental change. Powerful tools are available which support the study of invasive species, the most important being systematics and our capacity to accurately identify parasites and to define evolutionary and biogeographic history. Faunal baselines derived from arrays of biological specimens, integrated surveys and informatics are a permanent record of the biosphere when archived in museum collections. The absence of comprehensive taxonomic inventories of parasites, including molecular-genetic data, limits our ability to recognise the introduction of non-indigenous parasites, and to document patterns of expansion for local faunas under a regime of environmental perturbation.
The development and survival of larvae of Nematodirus filicollis, an important nematode parasite of ruminants, were extensively investigated in the laboratory for the first time, using eggs harvested from sheep grazing in the UK in winter. Third-stage larvae (L3s) developed within the eggs between 12 and 25 degrees C, with the proportion of eggs developing successfully being highest at the upper end of this range. Eggs required chilling below 10 degrees C before being able to hatch at all, and maximum hatch was observed only following chilling to 4 degrees C. Hatching occurred above 6 degrees C, and peaked at 13 degrees C, with the proportion of eggs hatching decreasing to 17-20 degrees C, which appears to form an upper threshold temperature for hatching. When fully developed eggs were placed at 11-13 degrees C, L3 emerged very rapidly with hatching being completed within 6 days. Mortality of emerged L3s increased with temperature, particularly above 20 degrees C. For this parasite, given the low minimum hatching thresholds and the rapid emergence of L3, the existence of an upper hatching threshold is likely to have limited impact on patterns of larval availability on pasture. The strict requirement for chilling is surprising, being at odds with both previous observations and the wide geographical distribution of this species. It is possible that the proportion of eggs requiring chilling is an adaptable trait, enabling early infection of naïve hosts in the face of competition from N. battus, yet also persistence in regions that do not provide good conditions for the chilling of eggs. Further study of selectable variation in this trait could help us to understand the contrasting geographical ranges and disease patterns within the genus, and how different species might respond to climate change.
Total DNA was isolated from individual nematodes of the species Longidorus helveticus, L. macrosoma, L. arthensis, L. profundorum, L. elongatus, and L. raskii collected in Switzerland. The ITS region and D1-D2 expansion segments of the 26S rDNA were amplified and cloned. The sequences obtained were aligned in order to investigate sequence diversity and to infer the phylogenetic relationships among the six Longidorus species. D1-D2 sequences were more conserved than the ITS sequences that varied widely in primary structure and length, and no consensus was observed. Phylogenetic analyses using the neighbor-joining, maximum parsimony and maximum likelihood methods were performed with three different sequence data sets: ITS1-ITS2, 5.8S-D1-D2, and combining ITS1-ITS2+5.8S-D1-D2 sequences. All multiple alignments yielded similar basic trees supporting the existence of the six species established using morphological characters. These sequence data also provided evidence that the different regions of the rDNA are characterized by different evolution rates and by different factors associated with the generation of extreme size variation.
The partial mitochondrial cytochrome c-oxidase subunit 1 gene (cox 1) and partial mitochondrial 16S ribosomal DNA of Trichuris skrjabini (Baskakov 1924) isolated from Capra hircus have been amplified and sequenced. The analyses of multiple sequence alignments of mitochondrial 16S rDNA and cox 1 of T. skrjabini revealed high homology with those of Trichinella species. For the first time, the mitochondrial DNA gene sequences of one species of trichurid nematode have been cited.
Entomopathogenic nematodes in Steinernema, together with their symbiont bacteria Xenorhabdus, are obligate and lethal parasites of insects that can provide effective biological control of some important lepidopteran, dipteran, and coleopteran pests of commercial crops. Phylogenetic relationships among 21 Steinernema species were estimated using 28S ribosomal DNA (rDNA) sequences and morphological characters. Sequences of the rDNA internal transcribed spacers were obtained to provide additional molecular characters to resolve relationships among Steinernema carpocapsae, Steinernema scapterisci, Steinernema siamkavai, and Steinernema monticolum. Four equally parsimonious trees resulted from combined analysis of 28S sequences and 22 morphological characters. Clades inferred from analyses of molecular sequences and combined datasets were primarily reliably supported as assessed by bootstrap resampling, whereas those inferred from morphological data alone were not. Although partially consistent with some traditional expectations and previous phylogenetic studies, the hypotheses inferred from molecular evidence, and those from combined analysis of morphological and molecular data, provide a new and comprehensive framework for evaluating character evolution of steinernematids. Interpretation of morphological character evolution on 6 trees inferred from sequence data and combined evidence suggests that many structural features of these nematodes are highly homoplastic, and that some structures previously used to hypothesize relationships represent ancestral character states.
DNA sequence divergence at internal transcribed spacer regions (ITS-1 and ITS-2) was compared with divergence at mitochondrial cox1 or nad4 loci in pairs of congeneric nematode species. Mitochondrial sequences accumulate substitutions much more quickly than internal transcribed spacer, the difference being most striking in the most closely related species pairs. Thus, mitochondrial DNA may be the best choice for applications in which one is using sequence data on small numbers of individuals to search for potential cryptic species. On the other hand, internal transcribed spacer remains an excellent tool for DNA diagnostics (quickly distinguishing between known species) owing to its lower level of intraspecific polymorphism.
It has been 50 years since the parasitic nematode of lambs, Nematodirus battus, was first described. This parasite has several interesting features; in particular, it induces a rapid, protective immune response in infected young lambs (< 3 months of age), which is not observed if lambs are infected with other trichostrongyle nematodes. Indeed, protection against most gastrointestinal nematodes only develops once lambs are over five to six months old. In this article I suggest that N. battus offers an opportunity to improve our understanding of protective immune responses in young lambs, and could therefore hold a key to rational anti-nematode vaccine developments, based on natural antigens.
The ITS region from a wide taxonomic range of nematodes, including secernentean and adenophorean taxa, and free-living, entomopathogenic, and plant-parasitic species, was evaluated as a taxonomic marker. Size of the amplified product aided in the initial determination of group membership, and also suggested groups that may require taxonomic reevaluation. Congeneric species often displayed identically sized ITS regions, but genera such as Pratylenchus and Tylenchorynchus had species with large differences in size. ITS heterogeneity in individuals and populations was identified in several nematode taxa. PCR-RFLP of ITS1 is advocated as a method of taxonomic analysis in genera such as Helicotylenchus that contain numerous species with few diagnostic morphological characteristics.
The use of comparative methods to test evolutionary hypotheses has become more common at both the macro- and microevolutionary levels. The application of such techniques is especially troublesome at the interface of these levels because phylogenetic relationships are often difficult to estimate. The use of a technique developed to estimate intraspecific cladograms combined with more traditional methods of phylogenetic estimation can improve the estimate in data sets containing a range of diversity when the lower bound of the range approaches 0% divergence. For nucleotide sequence data from the 16S region of the mitochondrial DNA from 72 individuals representing 37 species, this combined-procedures approach improved the estimate of phylogenetic relationships using maximum-parsimony, maximum-likelihood, and neighbor-joining methods. The estimated trees were used to examine systematic hypotheses relating to the crayfish genus Orconectes and species relationships within the subgenus Procericambarus . The monophyly of Procericambarusm is not supported by the mitochondrial data, and the hypotheses of unique origins of various morphological features previously used in determining crayfish relationships is unsupported.
The unusual arrangement of the 5S ribosomal gene within the intergenic sequence (IGS) of the ribosomal cistron, previously reported for Meloidogyne arenaria, was also found in the ribosomal DNA of two other economically important species of tropical root-knot nematodes, M, incognita and M. javanica. This arrangement also was found in M. hapla, which is important in temperate regions, and M. mayaguensis, a virulent species of concern in West Africa. Amplification of the region between the 5S and 18S genes by PCR yielded products of three different sizes such that M. mayaguensis could be readily differentiated from the other species in this study. This product can be amplified from single juveniles, females, or egg masses. The sequences obtained in this region for one line of each of M. incognita, M. arenaria, and M. javanica were very similar, reflecting the close relationships of these lineages. The M. mayaguensis sequence for this region had a number of small deletions and insertions of various sizes, including possible sequence duplications.
Ribosomal DNA (rDNA) sequences have been aligned and compared in a number of living organisms, and this approach has provided a wealth of information about phylogenetic relationships. Studies of rDNA sequences have been used to infer phylogenetic history across a very broad spectrum, from studies among the basal lineages of life to relationships among closely related species and populations. The reasons for the systematic versatility of rDNA include the numerous rates of evolution among different regions of rDNA (both among and within genes), the presence of many copies of most rDNA sequences per genome, and the pattern of concerted evolution that occurs among repeated copies. These features facilitate the analysis of rDNA by direct RNA sequencing, DNA sequencing (either by cloning or amplification), and restriction enzyme methodologies. Constraints imposed by secondary structure of rRNA and concerted evolution need to be considered in phylogenetic analyses, but these constraints do not appear to impede seriously the usefulness of rDNA. An analysis of aligned sequences of the four nuclear and two mitochondrial rRNA genes identified regions of these genes that are likely to be useful to address phylogenetic problems over a wide range of levels of divergence. In general, the small subunit nuclear sequences appear to be best for elucidating Precambrian divergences, the large subunit nuclear sequences for Paleozoic and Mesozoic divergences, and the organellar sequences of both subunits for Cenozoic divergences. Primer sequences were designed for use in amplifying the entire nuclear rDNA array in 15 sections by use of the polymerase chain reaction; these "universal" primers complement previously described primers for the mitochondrial rRNA genes. Pairs of primers can be selected in conjunction with the analysis of divergence of the rRNA genes to address systematic problems throughout the hierarchy of life.
Mitochondrial DNA (mtDNA) sequence data were used to compare the population genetic structures of five species of parasitic nematodes from three different hosts: Ostertagia ostertagi and Haemonchus placei from cattle, H. contortus and Teladorsagia circumcincta from sheep, and Mazamastrongylus odocoilei from white-tailed deer. The parasites of sheep and cattle showed a pattern consistent with high gene flow among populations. The parasite of deer showed a pattern of substantial population subdivision and isolation by distance. It appears that host movement is an important determinant of population genetic structure in these nematodes. High gene flow in the parasites of livestock also indicates great opportunity for the spread of rare alleles that confer resistance to anthelmintic drugs. All species, including the parasite of deer, had unusually high within-population diversities (averages of 0.019-0.027 substitutions per site between pairs of individuals from the same population). Large effective population sizes (Ne), perhaps in combination with rapid mtDNA evolution, appear to be the most likely explanation for these high within-population diversities.
Although nuclear ribosomal DNA (rDNA) repeats evolve together through concerted evolution, some genomes contain a considerable diversity of paralogous rDNA. This diversity includes not only multiple functional loci but also putative pseudogenes and recombinants. We examined the occurrence of divergent paralogues and recombinants in Gossypium, Nicotiana, Tripsacum, Winteraceae, and Zea ribosomal internal transcribed spacer (ITS) sequences. Some of the divergent paralogues are probably rDNA pseudogenes, since they have low predicted secondary structure stability, high substitution rates, and many deamination-driven substitutions at methylation sites. Under standard PCR conditions, the low stability paralogues amplified well, while many high-stability paralogues amplified poorly. Under highly denaturing PCR conditions (i.e., with dimethylsulfoxide), both low- and high-stability paralogues amplified well. We also found recombination between divergent paralogues. For phylogenetics, divergent ribosomal paralogues can aid in reconstructing ancestral states and thus serve as good outgroups. Divergent paralogues can also provide companion rDNA phylogenies. However, phylogeneticists must discriminate among families of divergent paralogues and recombinants or suffer from muddled and inaccurate organismal phylogenies.
CLUSTAL X is a new windows interface for the widely-used progressive multiple sequence alignment program CLUSTAL W. The new
system is easy to use, providing an integrated system for performing multiple sequence and profile alignments and analysing
the results. CLUSTAL X displays the sequence alignment in a window on the screen. A versatile sequence colouring scheme allows
the user to highlight conserved features in the alignment. Pull-down menus provide all the options required for traditional
multiple sequence and profile alignment. New features include: the ability to cut-and-paste sequences to change the order
of the alignment, selection of a subset of the sequences to be realigned, and selection of a sub-range of the alignment to
be realigned and inserted back into the original alignment. Alignment quality analysis can be performed and low-scoring segments
or exceptional residues can be highlighted. Quality analysis and realignment of selected residue ranges provide the user with
a powerful tool to improve and refine difficult alignments and to trap errors in input sequences. CLUSTAL X has been compiled
on SUN Solaris, IRIX5.3 on Silicon Graphics, Digital UNIX on DECstations, Microsoft Windows (32 bit) for PCs, Linux ELF for
x86 PCs, and Macintosh PowerMac.
The multigene family of rDNA in Drosophila reveals high levels of within-species homogeneity and between-species diversity. This pattern of mutation distribution is known as concerted evolution and is considered to be due to a variety of genomic mechanisms of turnover (e.g., unequal crossing over and gene conversion) that underpin the process of molecular drive. The dynamics of spread of mutant repeats through a gene family, and ultimately through a sexual population, depends on the differences in rates of turnover within and between chromosomes. Our extensive molecular analysis of the intergenic spacer (IGS) and internal transcribed spacer (ITS) spacer regions within repetitive rDNA units, drawn from the same individuals in 10 natural populations of Drosophila melanogaster collected along a latitudinal cline on the east coast of Australia, indicates a relatively fast rate of X-Y and X-X interchromosomal exchanges of IGS length variants in agreement with a multilineage model of homogenization. In contrast, an X chromosome-restricted 24-bp deletion in the ITS spacers is indicative of the absence of X-Y chromosome exchanges for this region that is part of the same repetitive rDNA units. Hence, a single lineage model of homogenization, coupled to drift and/or selection, seems to be responsible for ITS concerted evolution. A single-stranded exchange mechanism is proposed to resolve this paradox, based on the role of the IGS region in meiotic pairing between X and Y chromosomes in D. melanogaster.
The six species of Nematodirus parasitic in domestic ruminants of North America have been identified previously on the basis of characteristics of the bursa and tips of the spicules, and females could not be identified. In an effort to find additional diagnostic characteristics of both sexes, cuticular ridges were studied with light and scanning electron microscopy and in whole mounts and cross sections. After the cuticular ridges of males were characterized, females were matched with males by means of cuticular ridges, except for the rare species N. davtiani. Five of the six species have variations of an 18-ridge bilaterally symmetrical system in the cervical region. The sixth species has 26 cervical ridges. Two groups of species were recognized on the basis of cuticular characteristics correlated with other morphological characters. The two species in Group I, Nematodirus filicollis and N. dalviani, lose ridges laterally in the postcervical region and have 14 ridges at midbody. They can be identified by their anteriorly extended pattern of ridges in the cervical region. These two species also share the characteristics of finlike ridges, a small number (30-35) of perioral denticles, a short cephalic expansion, and a large bursa without a separate dorsal lobe. Nematodirus davtiani can be distinguished from N. filicollis by its prominent dorsalmost and ventralmost ridges and its distinctive dorsal ray. In contrast, the four species of Group II, N. helvetianus, N. oiratianus interruptus ssp. n., N. abnormalis, and N. spathiger, share the characteristics of a more posteriorly distributed pattern of ridges in the cervical region, 18 or more ridges near midbody, smaller dorsal and ventral ridges, a larger number (50-65) of perioral denticles, a longer cephalic expansion, and a smaller bursa with separate dorsal lobes. Nematodirus helvetianus and N. oiratianus interruptus add ridges in the cervical and postcervical regions, and are characterized by having more than 18 ridges for most of their length; they do not add ventral ridges in the last quarter of the males. Nematodirus helvetianus has more ridges (30-36 at midbody) than any of the other species. Nematodirus oiratianus interruptus can be easily separated from all other species by its discontinuous ridges in the cervical region. Nematodirus oiratianus oiratianus from Asia and South America have continuous ridges. Nematodirus spathiger and S. abnormalis have 18 ridges for most of their length; they lose all dorsal ridges and add a few ventral ridges in the last quarter of the males. Nematodirus abnormalis can be distinguished from N. spalhiger by the cervical discontinuities in ridges numbered 2 and 8, by spicular and bursal characteristics, and a more anterior vulva position. Possible evolutionary relationships among the six species are described in a cladogram and a key to species.
The estimation procedure utilizes a compatibility analysis between enzyme data sets of the most parsimonious trees constructed from the restriction enzyme. Next, a non-parametric test is given for comparing alternative phylogenies. A 2nd non-parametric test is developed for testing the molecular clock hypothesis. To illustrate the power of these procedures, data derived from the mitochondrial DNA and globin DNA of man and the apes are analyzed. Although previous analyses of these data led to the speculation that 10 times more information would be required to resolve the evolutionary relationships between man with chimps and gorillas, this algorithm resolved these relationships at the 5% level of significance. The molecular clock hypothesis was rejected at the 1% level. The implications of this phylogenetic inference when coupled with other types of data lead to the conclusion that knuckle-walking - not bipedalism - is the evolutionary novelty in mode of locomotion in the primates and that many other hominid features are primitive whereas their African ape counterparts are derived.-from Author
The nucleotide sequence of the 5′ ends of the 28S-like rRNA molecules of five species of helminths was determined directly, using a variation on the dideoxynucleotide chain-termination method which requires only 10 μg of total cellular RNA for analysis. Nucleotide sequence comparisons over 208 bases allowed the phylogeny of these organisms to be determined. The data show that the rDNA sequence of Nematospiroides dubius, a nematode, is as divergent from that of two platyhelminths, Hymenolepis diminuta and Schistosoma mansoni, as it is from the rDNA sequence of the two nematodes Onchocerca gibsoni and Brugia pahangi. The latter two appear to be very closely related, whereas the two platyhelminths are more distant from each other. The study demonstrates the usefulness and generality of rRNA sequencing for the systematic phylogenetic classification of parasitic organisms whose tissues are only available in relatively small amounts.
Genetic differences among Nematodirus spathiger, Nematodirus filicollis, Nematodirus helvetianus and Nematodirus battus in the nucleotide sequence of the second internal transcribed spacer (ITS-2) of ribosomal DNA ranged from 3.9 to 24.7%. Pairwise comparisons of their ITS-2 sequences indicated that the most genetically similar species were N. spathiger and N. helvetianus. N. battus was the most genetically distinct species, with differences ranging from 22.8 to 24.7% with respect to the other three species. Some of the nucleotide differences among species provided different endonuclease restriction sites that could be used in restriction fragment length polymorphism studies. The ITS-2 sequence data may prove useful in studies of the systematics of molineid nematodes.
The reliability of phylogenies reconstructed from data on multigene families is investigated via simulation. The evolutionary scenario used is a character-based model of a twogene family in four species in which clocklike divergence is postulated but neither convergence nor reversal is allowed except as a result of recombination and gene conversion. Thus, any homoplasy emerging from parsimony reconstructions from the simulated data matrices can be attributed to concerted evolution. The probabilities of correctly reconstructing two standard trees are estimated by replicate runs of the simulation. One standard tree (the OP or “orthology/paralogy” tree) reflects the true gene genealogy in the absence of concerted evolution; the other (the CE or “concerted evolution” tree) depicts gene relationships under complete homogenization of the gene family. The probability of correct reconstruction of the OP tree declines quickly as concerted evolution increases, but above an intermediate level of concerted evolution the probability of correctly inferring the CE tree increases rapidly. Trees similar but not identical to the correct trees can be reconstructed above or below the critical intermediate level of concerted evolution. Levels of homoplasy and numbers of equally parsimonious minimal trees are maximized, and bootstrap confidence levels are minimized, near this intermediate level of concerted evolution. When reconstructing the correct gene tree is the goal, both consistency indices and bootstrap levels will show misleadingly high values when concerted evolution is high. However, because the correct species tree can be inferred from either the OP or CE tree (in the absence of homoplasy from sources other than concerted evolution), these same measures correlate well with fidelity of reconstructing the species tree.
In early 1989, two-thirds of the Soay sheep population on St Kilda died over 12 weeks. Post-mortem examinations revealed emaciated carcasses and considerable nematode burdens, with protein-energy malnutrition as the probable cause of death. Haematological and blood biochemical changes in the sheep, as well as fecundity of gastrointestinal nematodes, suggested the hosts were immunosuppressed. In parallel, laboratory experiments in which Soay sheep on a high plane of nutrition were artificially infected with Ostertagia circumcincta, showed no clinical signs or mortality when supporting worm burdens similar to those recorded in dead sheep on St Kilda. Anthelmintic treatment of a group of animals increased daily survival rates in ewes and male lambs, although treated animals became re-infected as the 'crash' progressed. It is suggested that parasites contribute to mortality in malnourished hosts, exacerbating the effects of food shortage.
The epidemiology of nematode infections of Soay sheep on the island of St Kilda over a period of 2 years (August 1988-August 1990) spanning a host population crash is described. Infective larvae (L3) levels on pasture were high (2422 +/- 365 L3/kg D.M. grass in midsummer 1988) when host population density was high, decreasing after the sheep population declined by 70% in early 1989 (601 +/- 14 L3/kg D.M. in midsummer 1989). The availability of infective larvae to sheep increased during the winter of 1988-1989, probably as a result of concentration of existing larvae on grass as vegetation was destroyed by bad weather and overgrazing. Increased availability of pre-parasitic stages was accompanied by a marked increased in faecal egg counts from sheep of all ages and both sexes. Prevalence and intensity of infection (faecal egg counts) were higher in males than females throughout the 2-year study (chi 2 = 208.3, P < 0.005 and F1, 2000 = 304, P < 0.001 respectively), except during the lambing periods, and decreased with age in both sexes. Changes in prevalence and intensity of strongyle infections were associated with changes in host population density. Prevalence and intensity of Dictyocaulus filaria larvae in faeces increased during the host population crash. Infection intensity decreased with age (F1, 203 = 44.02, P < 0.001) and was higher in males than females (F1, 203 = 13.45, P < 0.001).
We have sequenced one complete rDNA tandem repeat from the nematode C. elegans. By comparative analysis we derive secondary
structures for the 18s, 5.8s, and 26s rRNA molecules, and comment on other important features of the sequence. We also present
the sequence of a Junction between the rDNA and non-ribosomal DNA. Finally, we use our data to quantify the evolutionary relationships
among several organisms currently studied in developmental biology.
We have determined the DNA sequences encoding 18 S ribosomal RNA in man and in the frog, Xenopus borealis. We have also corrected the Xenopus laevis 18 S sequence: an A residue follows G-684 in the sequence. These and other available data provide a number of representative examples of variation in primary structure and secondary modification of 18 S ribosomal RNA between different groups of vertebrates. First, Xenopus laevis and Xenopus borealis 18 S ribosomal genes differ from each other by only two base substitutions, and we have found no evidence of intraspecies heterogeneity within the 18 S ribosomal DNA of Xenopus (in contrast to the Xenopus transcribed spacers). Second, the human 18 S sequence differs from that of Xenopus by approx. 6.5%. About 4% of the differences are single base changes; the remainder comprise insertions in the human sequence and other changes affecting several nucleotides. Most of these more extensive changes are clustered in a relatively short region between nucleotides 190 and 280 in the human sequence. Third, the human 18 S sequence differs from non-primate mammalian sequences by only about 1%. Fourth, nearly all of the 47 methyl groups in mammalian 18 S ribosomal RNA can be located in the sequence. The methyl group distribution corresponds closely to that in Xenopus, but there are several extra methyl groups in mammalian 18 S ribosomal RNA. Finally, minor revisions are made to the estimated numbers of pseudouridines in human and Xenopus 18 S ribosomal RNA.
N. battus is a species which does not belong to the original helminth fauna of sheep. Furthermore it has not been recorded from the native deer species in Europe, except on one occasion from the roe deer in Great Britain. Presumably N. battus was imported into Britain somewhere in the twenties or thirties of this century with some foreign species of deer. The deer situation in Britain, however, gives no support to this hypothesis. One can only speculate about this as there is no concrete evidence of its occurrence in other animals than sheep, cattle, roe deer, wild goats and rabbits in Britain.
The development of polymerase chain reaction-based methods for assessing the genotypes of small individual organisms will promote groundbreaking investigations of the genetic architecture of parasite populations. Both quantitative genetic models and general knowledge of parasite natural history are useful for making general predictions about the distribution of genetic variation over geographic space. However, designing experimental studies to assess relationships between specific life history variables and patterns of genetic structure in natural populations will be challenging. Traditional biochemical-genetic methods have already been used to study a limited number of parasite populations, and inferred patterns of genetic structure are distinctly different between certain species. Some of these differences in genetic architecture may be explained by parasite or host factors that either promote or retard the dissemination of life cycle stages over geographic space. Many additional empirical studies are needed to characterize basic features of parasite populations, including the spatial distribution and group size of random mating populations and levels of gene flow among parasite subpopulations.
Background:
The individual copies of tandemly repeated genes, such as ribosomal DNA (rDNA), evolve coordinately within a species. This phenomenon has been called concerted evolution, and is thought to be caused by sequence-homogenizing mechanisms, such as gene conversion or unequal crossing-over between individual copies of the gene family. As these processes would act between the arrays on homologous and non-homologous chromosomes, the whole family of repeats would be expected to undergo homogenization in a given interbreeding population.
Results:
In order to study the homogenization process, we have examined polymorphisms within the internal transcribed spacer (ITS) of the rDNA in populations of Drosophila melanogaster at the sequence level, by DNA sequencing and temperature-gradient gel electrophoresis. Among 84 ITS clones sequenced from five different wild-type strains, we found three polymorphic sites that are apparently in the process of homogenization. However, these three sites, as well as combinations of them, occurred at different frequencies in the different strains. Moreover, temperature-gradient gel electrophoresis analysis of an ITS fragment including these three sites shows that single chromosomes from locally interbreeding populations can harbor rDNA arrays that are largely homogenized for different sequence variants.
Conclusions:
The presence of chromosomal arrays that are homogeneous for different variants in interbreeding populations of Drosophila melanogaster indicates that there is little recombination between the chromosomes while new mutations are being homogenized along the individual arrays. The most likely explanation for this finding is that intrachromosomal recombination events occur at much higher rates than recombination between homologous chromosomes. Thus, the first step of the homogenization process would occur mainly within chromosomal lines. Such behavior of tandem repeat arrays suggests a simple explanation of how selection can act on a multigene family, namely by acting on whole chromosomally confined repeat arrays rather than on individual repeat units.
The small subunit ribosomal RNA gene (SSU rRNA) from the nematode parasite, Nematodirus battus, was cloned from amplified genomic DNA using polymerase chain reaction primers complementary to the 5' and 3' ends of Haemonchus contortus small subunit ribosomal DNA (SSU rDNA). The 1,758 base pair cloned fragment was sequenced in both directions using internally generated primers and aligned with those from the genus Haemonchus. Results indicated a 1.9% sequence difference between the genera with a 14.2% difference between N. battus and Caenorhabditis elegans. Data presented here in conjunction with previously published work comparing rDNA sequences within the genus Haemonchus demonstrate limited divergence in this group of genes between these subfamilies and suggest further that this molecule alone may be inappropriate for assessing phylogenetic relatedness within the superfamily Trichostrongyloidea.
The entire 1766 bases of the 18S rRNA gene of Strongyloides stercoralis have been sequenced. The gene has a 38% G+C content. Although it is similar in length to the 18S rRNA gene of Caenorhabditis elegans, the only other completely sequenced nematode 18S rRNA gene, it is only 69% identical. Closely related helminths will need to be sequenced in order to delineate sequences specific for the diagnosis of strongyloidiasis.
To determine whether nuclear rDNA sequences provide a useful means for assessing the structure of populations of Ixodes ticks, we compared variability among copies of an internal transcribed spacer (ITS-2) sequence within individual ticks to the variability between ticks. At least 4% of the nucleotides comprising this sequence vary among the copies present within individual ticks. ITS-2 diversity in each of two ticks is nearly half as great as that reported between ticks from geographically disparate populations. Because individual ticks retain ancestral polymorphism, ITS-2 variation does not accurately reflect descent relationships among these ticks. Sequencing single copies of PCR-amplified ITS-2 therefore does not permit assessment of the phylogenetic relationships among the I. ricinus-like ticks in eastern North America. We recommend caution in future analyses, and emphasize the importance of procedures designed to ensure that the many paralogous copies of the rDNA cistron have been sufficiently homogenized by concerted evolutionary processes. Such precautionary measures will make certain that phylogenetic trees based on these gene sequences reflect the phyletic relatedness of the biological species.
Isoelectric focusing was performed on extracts from Nematodirus spathiger, Nematodirus filicollis, Nematodirus helvetianus, and 3 geographic isolates of Nematodirus battus. Gender-specific differences were noted within species; however, the overall protein profile of each species and isolate was distinct and reproducible and allowed unequivocal differentiation. A coefficient of similarity (Sm) for males of each species and isolate was calculated, and a dendrogram, based on evaluation of Sm by the unweighted pair-group method with arithmetic means, was produced. Although cluster analysis of the 3 isolates of N. battus indicates the North American and Weybridge isolates are similar, interpretation of the relationships and thus the history of introduction based on these data is equivocal. Isoelectric focusing is a robust method for establishing identity and has great utility in diagnostics. However, in the absence of selective histochemical staining, interpretation of identity and homology for specific bands and banding patterns is problematic, thus limiting the utility of this method for phylogenetic inference.
Testing different theories of concerted evolution experimentally has been hampered mainly due to the lack of appropriate model systems and technical limitations. In this study, we employed a denaturing gradient gel electrophoresis (DGGE) approach for the display and definition of nucleotide variations in the second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA) of the parasitic nematode, Haemonchus contortus. The ITS-2 was amplified from individual adult nematodes by PCR and subjected to DGGE. Of the 94 individuals (representing nine different populations) analysed, 13 different DGGE profiles were displayed. Eighteen bands representing those profiles were excised and sequenced. Sequencing defined 13 different types of ITS-2 with 12 nucleotide variations (4 transitions, 5 transversions, 1 insertion and 2 deletions) which could be related to particular positions of the predicted secondary structure for the ITS-2 pre-rRNA. The results showed that individuals of interbreeding populations of H. contortus can have rDNA arrays that are partially or fully homogenised for different sequence variants (despite interindividual variation), suggesting that the homogenisation process is driven mainly by intrachromosomal exchange. The findings also demonstrated the capacity of the DGGE-sequencing strategy to quantify the frequency of ITS-2 sequence types within individual nematodes from different populations without the need for cloning or Southern blot procedures. This has important implications for studying the mechanisms of sequence homogenisation in rDNA and pre-rRNA processing as well as for elucidating speciation events and population differentiation at the molecular level.
Among root knot nematodes of the genus Meloidogyne, the polyploid obligate mitotic parthenogens M. arenaria, M. javanica, and M. incognita are widespread and common agricultural pests. Although these named forms are distinguishable by closely related mitochondrial DNA (mtDNA) haplotypes, detailed sequence analyses of internal transcribed spacers (ITSs) of nuclear ribosomal genes reveal extremely high diversity, even within individual nematodes. This ITS diversity is broadly structured into two very different groups that are 12%-18% divergent: one with low diversity (< 1.0%) and one with high diversity (6%-7%). In both of these groups, identical sequences can be found within individual nematodes of different mtDNA haplotypes (i.e., among species). Analysis of genetic variance indicates that more than 90% of ITS diversity can be found within an individual nematode, with small but statistically significant (5%-10%; P < 0.05) variance distributed among mtDNA lineages. The evolutionarily distinct parthenogen M. hapla shows a similar pattern of ITS diversity, with two divergent groups of ITSs within each individual. In contrast, two diploid amphimictic species have only one lineage of ITSs with low diversity (< 0.2%). The presence of divergent lineages of rDNA in the apomictic taxa is unlikely to be due to differences among pseudogenes. Instead, we suggest that the diversity of ITSs in M. arenaria, M. javanica, and M. incognita is due to hybrid origins from closely related females (as inferred from mtDNA) and combinations of more diverse paternal lineages.
Genetic differences in the nucleotide sequence of the second internal transcribed spacers (ITS-2) among Trichostrongylus axei, Trichostrongylus colubriformis, Ostertagia ostertagi, Cooperia oncophora, Cooperia punctata, Nematodirus helvetianus, Nematodirus filicollis, and Haemonchus contortus are described. The ITS-2 sequences of the 8 species ranged between 230 and 241 base pairs in length. Sequence similarities between the different genera varied between 60% and 80%. Identities between the different species within a genus varied between 99% for C. oncophora and C. punctata, 95% for T. axei and T. colubriformis, and 89% for N. helvetianus and N. filicollis. The ITS-2 sequences proved to be useful for species differentiation. Except for the species of Cooperia (2.07% intraspecific variations for C. oncophora and 0.83% for C. punctata) the degree of intraspecific variations (N. filicollis 0.85%, T. colubriformis 1.26%, T. axei 1.27%, H. contortus 2.60%, O. ostertagi, and N. helvetianus no variation) was markedly lower than the interspecific variations allowing a reliable differentiation within the ITS-2 region between single species.
A brief description of Nematodirus battus has been given elsewhere (Crofton and Thomas, 1951), but this included only sufficient details for specific diagnosis. A more detailed account is given below.
The worms are long, slender and more or less straight. The cuticle is smooth except in the cephalic region where it is inflated and is lightly striated. The cephalic inflation extends 0.12 mm. from the anterior end and is 0.06 mm. in diameter (see Fig. 1). In some specimens the cephalic inflation may be reduced and relatively inconspicuous. Although this variation may be due to fixation, it occurs in members of a batch which have been fixed simultaneously. The head is 0.03 mm. in diameter and bears six papillae round the mouth. The “mouth capsule” is small and bears a tooth on the dorsal side. This tooth is not easily visible in Fig. 1 because of contraction of the oesophagus. (The term mouth capsule is retained because it has been used by other workers (see Rajewskaja, 1918, Travassos, 1987) but if the definitions of Baylis and Daubney (1926) are adopted it would be more correct to refer to the tooth as lying in an oesophageal funnel). The oesophagus is relatively short, 0.35–0.54 mm. in length.
OUTBREAKS of parasitic disease in lambs have occurred in the north of England during June and July 1951. Deaths have been associated with the presence of large numbers of nematodes of the genus Nematodirus. We are grateful to Mr. W. Lyle Stewart, who sent us the contents of the intestines of five lambs which had been sent to him for examination. About two million worms were received, and the collection was remarkable not only because of the large numbers but also because more than 90 per cent of the worms belonged to the genus Nematodirus. Examination showed that there were no N. spathiger, a large number of N. filicollis and a large number of a third species of Nematodirus. This last species cannot be referred to any known member of the genus, and it is proposed to name it Nematodirus battus. The description is as follows.
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