Molecular phylogenetics of eimeriid coccidia (Eimeriidae,Eimeriorina, Apicomplexa,Alveolata): A preliminary multi-gene and multi-genome approach

Coccidia possess three distinct genomes: nuclear, mitochondrial, and plastid. Sequences from five genes located on these three genomes were used to reconstruct the phylogenetic relationships of members of the phylum Apicomplexa: 18S rDNA sequences from the nuclear (nu) genome, partial cytochrome c oxidase subunit I sequences from the mitochondrial (mt) genome, and partial 16S and 23S rDNA sequences and RNA polymerase B sequences from plastid (pl) genomes. Maximum parsimony, maximum likelihood, and Bayesian inference were used in conjunction with nuclear substitution models generated from data subsets in the analyses. Major groups within the Apicomplexa were well supported with the mitochondrial, nuclear, and a combination of mitochondrial, nuclear and concatenated plastid gene sequences. However, the genus Eimeria was paraphyletic in phylogenetic trees based on the nuclear gene. Analyses using the individual genes (18S rDNA and cytochrome c oxidase subunit I) resolved the various apicomplexan groups with high Bayesian posterior probabilities. The multi-gene, multi-genome analyses based on concatenated nu 18S rDNA, pl 16S, pl 23S, pl rPoB, pl rPoB1, and mt COI sequences appeared useful in resolving phylogenetic relationships within the phylum Apicomplexa. Genus-level relationships, or higher, appear best supported by 18S rDNA analyses, and species-level analyses are best investigated using mt COI sequences; for parasites for which both loci are available, nuclear 18S rDNA sequences combined with mitochondrial COI sequences provide a compact and informative molecular dataset for inferring the evolutionary relationships taxa in the Apicomplexa.     

Combined mitochondrial 16S and 12S rDNA sequences: an effective genetic marker for inter-species phylogenetic analysis of zoonotic trematodes

The present study studied the genetic variation among Schistosoma japonicum isolates from different endemic regions in mainland China and examined the phylogenetic relationships of zoonotic trematodes using the combined mitochondrial 16S and 12S ribosomal DNA sequences. The fragments of 16S and 12S rDNA were amplified from 22 S. japonicum isolates, and sequenced, and the relevant sequences of other nine trematode species belonging to six genera in four families were downloaded from GenBank, and their phylogenetic relationships were re-constructed by unweighted pair-group method with arithmetic averages analyses using the combined 16S and 12S rDNA sequences, with Trichinella spiralis as outgroup. The results showed that the partial sequences of mitochondrial 16S and 12S rDNA of S. japonicum were 757 and 797 bp, respectively, and they were quite conserved among the S. japonicum isolates. Phylogenetic analysis revealed that the combined 16S and 12S rDNA sequences were not able to distinguish S. japonicum isolates in mountainous areas from those in lake/marshland areas in mainland China. However, the combined sequences could distinguish different species of zoonotic trematodes. Therefore, the combined mitochondrial 16S and 12S rDNA sequences provide an effective molecular marker for the inter-species phylogenetic analysis and differential identification of zoonotic trematodes.     

Phylogenetic relationships in Demodex mites (Acari: Demodicidae) based on mitochondrial 16S rDNA partial sequences

To confirm phylogenetic relationships in Demodex mites based on mitochondrial 16S rDNA partial sequences, mtDNA 16S partial sequences of ten isolates of three Demodex species from China were amplified, recombined, and sequenced and then analyzed with two Demodex folliculorum isolates from Spain. Lastly, genetic distance was computed, and phylogenetic tree was reconstructed. MEGA 4.0 analysis showed high sequence identity among 16S rDNA partial sequences of three Demodex species, which were 95.85?% in D. folliculorum, 98.53?% in Demodex canis, and 99.71?% in Demodex brevis. The divergence, genetic distance, and transition/transversions of the three Demodex species reached interspecies level, whereas there was no significant difference of the divergence (1.1?%), genetic distance (0.011), and transition/transversions (3/1) of the two geographic D. folliculorum isolates (Spain and China). Phylogenetic trees reveal that the three Demodex species formed three separate branches of one clade, where D. folliculorum and D. canis gathered first, and then gathered with D. brevis. The two Spain and five China D. folliculorum isolates did not form sister clades. In conclusion, 16S mtDNA are suitable for phylogenetic relationship analysis in low taxa (genus or species), but not for intraspecies determination of Demodex. The differentiation among the three Demodex species has reached interspecies level.     

Characterization of Baylisascaris schroederi from Qinling subspecies of giant panda in China by the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA

In the present study, a total of 20 nematode isolates, (including 10 male and 10 female worms) representing Baylisascaris schroederi from 5 Qinling subspecies of giant pandas (Ailuropoda melanoleuca) in Shaanxi Province of China, were characterized and grouped genetically by the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA (rDNA). The rDNA fragment spanning 3′ end of 18S rDNA, complete ITS-1 rDNA, and 5′ end of 5.8S rDNA were amplified and sequenced. The sequence variability in ITS-1 rDNA was examined within B. schroederi and among parasites in order Ascaridata available in GenBank™, and their phylogenetic relationships were also reconstructed. The sequences of ITS-1 rDNA for all the B. schroederi isolates were 427 bp in length, with no genetic variation detected among these isolates. Phylogenetic analyses based on the ITS-1 rDNA sequences revealed that all the male and female B. schroederi isolates sequenced in the present study were posited into the clade of genus Baylisascaris, sistered to zoonotic nematodes in genus Ascaris, and the ITS-1 rDNA sequence could distinguish different species in order Ascaridata. These results showed that the ITS-1 rDNA provides a suitable molecular marker for the inter-species phylogenetic analysis and differential identification of nematodes in order Ascaridata.     

Cytochrome oxidase subunit 1 and mitochondrial 16S rDNA sequences of Trichuris skrjabini (Tricocephalida: Trichuridae)

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.     

Muscular sarcocystosis in two arctic foxes (Vulpes lagopus) due to Sarcocystis arctica n. sp.: sarcocyst morphology,molecular characteristics and phylogeny

The arctic fox (Vulpes lagopus) is a critically endangered species in Norway, and therefore, the small population is closely monitored, and most foxes found dead are subjected to necropsy. In two deceased foxes, thin-walled muscular sarcocysts were first detected in histological sections, and numerous sarcocysts were later found in frozen and thawed muscle samples from Fox 1. These sarcocysts measured 1–12?×?0.1–0.25 mm and had closely spaced, short, knob-like protrusions, giving the cysts a serrated outline. Genomic DNA was extracted from eight isolated sarcocysts (Fox 1) and two muscle samples (Fox 2) and subjected to polymerase chain reaction amplification at four loci: the nuclear 18S and 28S ribosomal RNA genes and internal transcribed spacer 1 region and the mitochondrial cytochrome c oxidase subunit 1 gene (cox1). Both foxes were infected by the same Sarcocystis sp., which displayed little or no genetic variation at the three nuclear loci (99.9–100 % identity) and slightly more variation at cox1 (99.4–100 % identity). Sequence comparisons and phylogenetic analyses revealed that this species was distinct from other named Sarcocystis spp. but was closely related to various species using avian intermediate hosts and possibly identical to an unnamed species reported from two American dogs. The species described from the two arctic foxes was named Sarcocystis arctica n. sp.     

Molecular characterization of Sarcocystis species from Polish roe deer based on ssu rRNA and cox1 sequence analysis

Sarcocysts from four Polish roe deer were collected and examined by light microscopy, small subunit ribosomal RNA (ssu rRNA), and the subunit I of cytochrome oxidase (cox1) sequence analysis. This resulted in identification of Sarcocystis gracilis, Sarcocystis oviformis, and Sarcocystis silva. However, we were unable to detect Sarcocystis capreolicanis, the fourth Sarcocystis species found previously in Norwegian roe deer. Polish sarcocysts isolated from various tissues differed in terms of their shape and size and were larger than the respective Norwegian isolates. Analysis of ssu rRNA gene revealed the lack of differences between Sarcocystis isolates belonging to one species and a very low degree of genetic diversity between Polish and Norwegian sarcocysts, ranging from 0.1 % for Sarcocystis gracilis and Sarcocystis oviformis to 0.44 % for Sarcocystis silva. Contrary to the results of the ssu rRNA analysis, small intraspecies differences in cox1 sequences were found among Polish Sarcocystis gracilis and Sarcocystis silva isolates. The comparison of Polish and Norwegian cox1 sequences representing the same Sarcocystis species revealed similar degree of sequence identity, namely 99.72 % for Sarcocystis gracilis, 98.76 % for Sarcocystis silva, and 99.85 % for Sarcocystis oviformis. Phylogenetic reconstruction and genetic population analyses showed an unexpected high degree of identity between Polish and Norwegian isolates. Moreover, cox1 gene sequences turned out to be more accurate than ssu rRNA when used to reveal phylogenetic relationships among closely related species. The results of our study revealed that the same Sarcocystis species isolated from the same hosts living in different geographic regions show a very high level of genetic similarity.     

Phylogenetic position of Linguatula arctica and Linguatula serrata (Pentastomida) as inferred from the nuclear 18S rRNA gene and the mitochondrial cytochrome c oxidase subunit I gene

Genomic DNA was isolated from a Linguatula serrata female expelled from a dog imported to Norway from Romania and from four Linguatula arctica females collected from semi-domesticated reindeer from northern Norway and subjected to PCR amplification of the complete nuclear 18S rRNA gene and a 1,045-bp portion of the mitochondrial cytochrome c oxidase subunit I gene (cox1). The two species differed at two of 1,830 nucleotide positions (99.9 % identity) of the complete 18S rRNA gene sequences and at 102 of 1,045 nucleotide positions (90.2 % identity) of the partial cox1 sequences. The four isolates of L. arctica showed no genetic variation in either gene. The new cox1 primers may facilitate the diagnosis of various developmental stages of L. arctica and L. serrata in their hosts. In separate phylogenetic analyses using the maximum likelihood method on sequence data from either gene, L. arctica and L. serrata clustered with members of the order Cephalobaenida rather than with members of the order Porocephalida, in which the genus Linguatula is currently placed based on morphological characters. The phylogenetic relationship of L. arctica, L. serrata and other pentastomids to other metazoan groups could not be clearly resolved, but the pentastomids did not seem to have a sister relationship to crustaceans of the subclass Branchiura as found in other studies. A more extensive taxon sampling, including molecular characterisation of more pentastomid taxa across different genera, seems to be necessary in order to estimate the true relationship of the Pentastomida to other metazoan groups.     

Genetic diversity in the trypanorhynch cestode <Emphasis Type="Italic">Tentacularia coryphaenae</Emphasis> Bosc, 1797: evidence for a cosmopolitan distribution and low host specificity in the teleost intermediate host

Partial large subunit (28S) rRNA gene (LSU) sequences were studied from Tentacularia coryphaenae (Cestoda: Trypanorhyncha) plerocercoids from the southern Java coast, Indonesia, collected from two different localities and five different host species. The teleost hosts belonged to four fish families with an overlapping depth range of 0–885 m. The LSU sequences were identical, demonstrating that all specimens belonged to the same species. They also corresponded to a sequence of T. coryphaenae from the Blue shark Prionace glauca in the North Atlantic, giving genetic evidence for the cosmopolitan distribution of the species. A 1,851 bp region of mitochondrial (mt) DNA (coding for partial cytochrome c oxidase subunit I (cox1), complete trnT and partial 16S ribosomal RNA) showed a very low level of intra-specific variation of 1%. Pairwise comparisons of published sequences for partial LSU rDNA and the same region of mtDNA demonstrated that the same regions varied by 8% in the mtDNA for two genotypes (G1 and G4) of Echinococcus granulosus (order Cyclophyllidea), at 16% in newly sequenced Kotorella pronosoma from the same trypanorhynch family and at 23% in Grillotia pristiophori from a different superfamily. The high genetic homogeneity in T. coryphaenae is explained by a constant gene flow between different regions and hosts along the Indonesian coast caused by extensive migrations of the second intermediate/paratenic and also the final hosts. Implications for the zoogeographical distribution, host specificity of the species and future research are discussed.     

Molecular phylogeny and evolutionary relationships among mosquitoes (Diptera: Culicidae) from the northeastern United States based on small subunit ribosomal DNA (18S rDNA) sequences

The phylogenetic relationships of Culicidae native to the northeastern United States were investigated by analyzing small subunit ribosomal DNA (18S rDNA) sequences obtained from 39 species representing nine genera. Molecular phylogenies were consistent with traditional classifications based on morphological characters except for the placements of Psorophora Robineau-Desvoidy and Uranotaenia Lynch Arribalzaga. In our analyses, 1) Anopheles Meigen was strongly supported as the sister taxon to the remaining Culicidae; 2) Toxorhynchites Theobald was represented as a distinct monophyletic sister group to the Culicinae; 3) Psorophora formed a basal clade to Culiseta Felt, Coquillettidia Dyar, and Culex L. but also was shown as a sister taxon to Aedes Meigen and Ochlerotatus Lynch Arribalzaga; 4) Coquillettidia perturbans (Walker) seems to be a sister group to Culiseta; 5) placement of Uranotaenia was inconclusive and seemed to be either a sister group to the Aedes and Ochlerotatus or a basal taxon to all other culicines; and 6) Aedes and Ochlerotatus formed two separate and distinct clades, providing phylogenetic data consistent with the recent elevation of Ochlerotatus to the generic level as proposed by Reinert (2000). The utility of 18S rDNA for evaluating phylogenetic and evolutionary relationships among mosquito taxa was demonstrated at the genus and species levels. To our knowledge, this study represents the first molecular-based phylogenetic study of mosquito species occurring within this geographic region of North America and contains the largest number of species that have been examined among the genera Aedes and Ochlerotatus.     

Sarcocystis dehongensis n. sp. (Apicomplexa: Sarcocystidae) from water buffalo (Bubalus bubalis) in China

Water buffalo (Bubalus bubalis) is the intermediate host for at least four species of Sarcocystis: S. fusiformis, S. buffalonis, S. levinei, and S. sinensis/S. dubeyi. Here, a new species, Sarcocystis dehongensis, is reported in 51 of 756 (6.7%) water buffaloes in China. By light microscopy, the cysts of S. dehongensis were macroscopic, up to 18.5 mm long and 95 μm in diameter; 4.9–11.9 μm villous protrusions extended beyond the sarcocyst wall. Using transmission electron microscopy, the sarcocyst wall had lancet- or leaf-like protrusions in longitudinal section, but the cross section showed that the protrusions appeared as mushroom-like in shape with a core of tightly packed microtubules, similar to “type 24.” BLAST searches revealed that S. dehongensis shared the most similarities with the 18S rDNA sequence of S. hardangeri (92.4%) and mitochondrial cox1 gene sequence of S. ovalis (81.0%), whereas no sequences in GenBank were found to be significantly similar to the ITS-1 region of S. dehongensis. A phylogenetic analysis based on 18S rDNA and mitochondrial cox1 gene sequences suggested that S. dehongensis was closely related to Sarcocystis species from cervids that employ corvids as definitive hosts.

     

Characterisation of full-length mitochondrial copies and partial nuclear copies (numts) of the cytochrome b and cytochrome c oxidase subunit I genes of Toxoplasma gondii, Neospora caninum, Hammondia heydorni and Hammondia triffittae (Apicomplexa: Sarcocystidae)

Genomic DNA was extracted from three oocyst isolates of Hammondia triffittae from foxes and two oocyst isolates of Hammondia heydorni from dogs, as well as from cell culture-derived tachyzoites of Toxoplasma gondii (RH strain) and Neospora caninum (NC-Liverpool strain), and examined by PCR with primers targeting the cytochrome b (cytb) and the cytochrome c oxidase subunit I (cox1) genes in order to characterise both genes and, if possible, the remainder of the mitochondrial genome of these species. Several primers were designed and used in various combinations to amplify regions within and between both genes and to determine gene order. When certain forward primers targeting cytb were used in combination with certain reverse primers targeting cox1, two overlapping sequences were obtained for each species and isolate studied, which showed that a full-length copy of cytb was followed 36–37 bp downstream by a full-length copy of cox1, and these sequences are believed to represent the true mitochondrial genes and the gene order in the mitochondrial genome of the four species examined. The cytb of T. gondii, N. caninum, H. heydorni and H. triffittae comprised a total of 1,080 bp (359 amino acids) and used ATG and TAA as start and stop codon, respectively. The cox1 of these species also used TAA as stop codon, whereas the most likely start codon was ATG, resulting in a gene comprising 1,491 bp (496 amino acids). Pair-wise sequence comparisons based on either cytb or cox1 clearly separated T. gondii from N. caninum and both of these species from the two Hammondia species, whereas the latter two species were 100 % identical at cytb and shared 99.3 % identity at cox1. Phylogenetic analyses using the maximum-likelihood method confirmed these findings and placed T. gondii in a clade separate from the three other species and all four Toxoplasmatinae in a sister clade to Eimeria spp. PCR with other primers and/or primer pairs than those used to obtain the full-length mitochondrial genes yielded several types of about 1–1.5 kb long sequences, which comprised stretches of the primer-targeted genes at both ends and an intervening non-coding sequence of various length and composition. Thus, portions of cytb could be found both upstream and downstream from portions of cox1 and portions of the same gene could be found adjacent to each other (cytb→cox1; cox1→cytb; cytb→cytb; cox1→cox1). Sequence comparisons revealed that some of these gene fragments were truncated genes, whereas others included the putative start or stop codon of the full-length mitochondrial genes. From the nature of the gene fragments and/or their flanking sequences, they are assumed to be located on the chromosomes of the nuclear genome and to represent nuclear mitochondrial DNA segments (numts) or pseudogenes. In the four species examined, there were no nucleotide differences between the full-length mitochondrial copies of cytb and cox1 and their various incomplete nuclear counterparts. With a few exceptions, identical numt types and closely similar flanking sequences were obtained for all four species, which would indicate that the original transfer of these mitochondrial genes to the nuclear genome and/or the majority of any subsequent rearrangements of these gene fragments within the nuclear genome happened before the four species diverged. Yet, there were species-specific differences in the nucleotide composition of the nuclear gene fragments, identical to the differences in the mitochondrial genes, which would indicate that the incomplete nuclear copies of cytb and cox1 have been continuously updated during evolution to conform to their mitochondrial parent genes. The PCR-based findings of numts were further supported by Basic Local Alignment Search Tool (BLAST) searches against genome sequences of T. gondii and N. caninum using the concatenated mitochondrial cytb/cox1 sequences as queries. These searches revealed the presence of numerous numts of eighth distinct types in both species, with each one having a fixed starting and end point with respect to the nucleotide positions in the full-length mitochondrial genes. Four numt types were completely homologous between both species, whereas four other types differed with respect to their end point and/or the absence/presence of a 96-bp deletion. Each starting and end point was associated with a unique 100–200-bp long flanking sequence, which further revealed the presence of numts. For both species, the numt types and their various arrangements with respect to each other were identical or similar to those obtained by PCR in all four species examined. None of the identified numts covered a full-length gene, but together, the various numts covered the entire mitochondrial cytb and cox1 genes in an overlapping manner. In addition, they were fairly closely spaced on the chromosomes, and these features may explain why the nuclear copies were preferentially amplified to the exclusion of the true mitochondrial genes with most primers and primer pairs used in the present study. The possibility of a similar high prevalence of numts occurring in the nuclear genome of dinoflagellates is discussed.     

Molecular identification of Diphyllobothrium latum and a brief review of diphyllobothriosis in China

Two tapeworm specimens collected in northeast China in 2009 and 2011 were identified as Diphyllobothrium latum based on morphological criteria. Molecular methods were used to confirm their identity and analyze genetic variations compared with published data for this species. Species identity was confirmed by molecular characterization of the 18S rDNA partial sequence, complete sequences of internal transcribed spacers (ITSs) and 5.8S rDNA, and partial sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) and mitochondrial NADH dehydrogenase subunit 5 (nad5). PCR amplification and sequence analysis of 18S rDNA (1472 bp), ITS regions (1218 bp), cox1 (885 bp), and nad5 (1028 bp) revealed that these four sequences showed more than 99% identity to reference sequences for D. latum, confirming that this species is D. latum. To date, a total of 12 diphyllobothriosis cases have been documented in China. This study represents the first molecular characterization of D. latum in China, providing molecular evidence of human diphyllobothriosis in China.     

The complete DNA sequence of the mitochondrial genome of the dermatophyte fungus Epidermophyton floccosum

We report here the complete nucleotide sequence of the 30.9-kb mitochondrial genome of the dermatophyte fungus Epidermophyton floccosum. All genes are encoded on the same DNA strand and include seven subunits of the reduced nicotinamide adenine dinucleotide ubiquinone oxireductase (nad1, nad2, nad3, nad4, nad4L, nad5, and nad6), three subunits of cytochrome oxidase (cox1, cox2, and cox3), apocytochrome b (cob), three subunits of ATP synthase (atp6, atp8, and atp9), the small and large ribosomal RNAs (rns and rnl), and 25 tRNAs. A ribosomal protein gene (rps5) is present as an intronic ORF in the large ribosomal subunit. The genes coding for cob and cox1 carry one intron and nad5 carries two introns with ORFs. The mtDNA of E. floccosum has the same gene order as Trichophyton rubrum mtDNA, with the exception of some tRNA genes. Maximum likelihood phylogenetic analysis confirms T. rubrum as a close relative of E. floccosum. This is the first complete mitochondrial sequence of a species of the order Onygenales. This sequence is available under GenBank accession number AY916130.     

Toxocara canis and Toxocara vitulorum: molecular characterization, discrimination, and phylogenetic analysis based on mitochondrial (ATP synthase subunit 6 and 12S) and nuclear ribosomal (ITS-2 and 28S) genes

Toxocara canis and Toxocara vitulorum are two important parasites of dogs and buffaloes with public health concern. The objectives of the present study are to identify molecular markers to discriminate these closely related parasites and to determine their phylogenetic position and genetic diversity within the genus Toxocara. Thus, two mitochondrial genes (complete ATPase 6 and partial small subunit ribosomal RNA (12S rDNA)), two nuclear ribosomal genes (second internal transcribed spacer region (ITS-2)), and part of the large subunit 28S region were analyzed. Nucleotide sequence (597 bp) and predicted amino acid sequences of the complete ATPase 6 gene (199 amino acids) of both species (T. canis and T. vitulorum) are similar in size with the Toxocara cati and Toxocara malaysiensis. There was 88% nucleotide similarity between T. canis and T. vitulorum and many transversions present in the 12S gene. Analyses of the ITS-2 and 28S regions revealed that the 28S region was more conserved (95% nucleotide similarity between T. canis and T. vitulorum) than the ITS-2 region (85%). This study has provided useful molecular markers for the molecular epidemiological investigation of Toxocara species. Further, phylogenetic analyses of the ITS-2 and 28S genes have indicated that the members of the genus Toxocara form a distinct group with reference to their definitive hosts.     

The first determination of Trichuris sp. from roe deer by amplification and sequenation of the ITS1-5.8S-ITS2 segment of ribosomal DNA

Trichuris nematodes were isolated from roe deer (Capreolus capreolus). At first, nematodes were determined using morphological and biometrical methods. Subsequently genomic DNA was isolated and the ITS1-5.8S-ITS2 segment from ribosomal DNA (RNA) was amplified and sequenced using PCR techniques. With u sing morphological and biometrical methods, female nematodes were identified as Trichuris globulosa, and the only male was identified as Trichuris ovis. The females were classified into four morphotypes. However, analysis of the internal transcribed spacers (ITS1-5.8S-ITS2) of specimens did not confirm this classification. Moreover, the female individuals morphologically determined as T. globulosa were molecularly identified as Trichuris discolor. In the case of the only male molecular analysis match the result of the molecular identification. Furthermore, a comparative phylogenetic study was carried out with the ITS1 and ITS2 sequences of the Trichuris species from various hosts. A comparison of biometric information from T. discolor individuals from this study was also conducted.     

Multiplex PCR identification of <Emphasis Type="Italic">Taenia</Emphasis> spp. in rodents and carnivores

The genus Taenia includes several species of veterinary and public health importance, but diagnosis of the etiological agent in definitive and intermediate hosts often relies on labor intensive and few specific morphometric criteria, especially in immature worms and underdeveloped metacestodes. In the present study, a multiplex PCR, based on five primers targeting the 18S rDNA and ITS2 sequences, produced a species-specific banding patterns for a range of Taenia spp. Species typing by the multiplex PCR was compared to morphological identification and sequencing of cox1 and/or 12S rDNA genes. As compared to sequencing, the multiplex PCR identified 31 of 32 Taenia metacestodes from rodents, whereas only 14 cysts were specifically identified by morphology. Likewise, the multiplex PCR identified 108 of 130 adult worms, while only 57 were identified to species by morphology. The tested multiplex PCR system may potentially be used for studies of Taenia spp. transmitted between rodents and carnivores.     

The occurrence of mtDNA heteroplasmy in multiple cetacean species

In population genetics and phylogenetic studies, mitochondrial DNA (mtDNA) is commonly used for examining differences both between and within groups of individuals. For these studies, correct interpretation of every nucleotide position is crucial but can be complicated by the presence of ambiguous bases resulting from heteroplasmy. Particularly for non-model taxa, the presence of heteroplasmy in mtDNA is rarely reported, therefore, it is unclear how commonly it occurs and how it can affect phylogenetic relationships among taxa and the overall understanding of evolutionary processes. We examined the occurrence of both site and length heteroplasmy within the mtDNA of ten marine mammal species, for most of which mtDNA heteroplasmy has never been reported. After sequencing a portion of the mtDNA control region for 5,062 individuals, we found heteroplasmy in at least 2% of individuals from seven species, including Stenella frontalis where 58.9% were heteroplasmic. We verified the presence of true heteroplasmy, ruling out artifacts from amplification and sequencing methods and the presence of nuclear copies of mitochondrial genes. We found no evidence that mtDNA heteroplasmy influenced phylogenetic relationships, however, its occurrence does have the potential to increase the genetic diversity for all species in which it is found. This study stresses the importance of both detecting and reporting the occurrence of heteroplasmy in wild populations in order to enhance the knowledge of both the introduction and the persistence of mutant mitochondrial haplotypes in the evolutionary process.     

Mitochondrial DNA sequences of triatomines (Hemiptera: Reduviidae): phylogenetic relationships

The phylogenetic relationships among 18 species of Triatominae were inferred based on mitochondrial DNA (mtDNA) sequences. The species of Triatoma included 11 belonging to the infestans complex [T. infestans (Klug), T. guasayana Wygodzinsky & Abalos, T. sordida (St?l), T. platensis Neiva, T. brasiliensis Neiva, T. rubrovaria (Blanchard), T. vitticeps (St?l), T. delpontei Roma?a & Abalos, T. maculata (Erichson), T. patagonica Del Ponte, and T. matogrossensis Leite & Barbosa] and four others of the same genus but of different complexes [T. circummaculata (St?l), T. protracta (Uhler), T. dimidiata (Latreille), and T. mazzottii Usinger]. As possible outgroups we used Mepraia spinolai Mazza, Panstrongylus megistus (Burmeister), and Rhodnius prolixus St?l. We analyzed mtDNA fragments of the 12S and 16S ribosomal RNA genes from each of the 18 species, as well as of the cytochrome oxidase I (COI) gene from nine. The 12S, 16S, and COI gene sequences were analyzed individually and combined. All of the phylogenetic analyses unambiguously supported two clusters: one including T. infestans, T. platensis, and T. delpontei, and the other T. sordida and T. mutagrossensis. Inclusion of T. circummaculata into the infestans complex was confirmed, although this is in disagreement with the morphological classification. On the other hand, our analyses showed that T. dimidiata is closely related to a phylosoma complex species, T. mazzottii. This is consistent with the tentative classification previously made based on morphological characters. The issue of the monophyly of the genus Triatoma remains unresolved.     

Genetic comparison of liver flukes, Clonorchis sinensis and Opisthorchis viverrini, based on rDNA and mtDNA gene sequences

To clarify the genetic relationships between Clonorchis sinensis and Opisthorchis viverrini, patterns of inter-/intraspecific polymorphism were compared for four markers with nuclear ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) in liver flukes C. sinensis from Korea (Kimhae) and China (Shenyang and Nanning) and O. viverrini from Laos (Savannakhet). Intra- and interspecific variations in the 18S, ITS2, and 28S rDNA and mitochondrial cytochrome c oxidase subunit I (mtCOI) of mtDNA gene sequences were low and nearly identical. Three isolates of C. sinensis showed a high similarity (99–100%). No variation was detectable in the ITS2 sequence for the C. sinensis from Korea and China. ITS2 region sequences of O. viverrini vs C. sinensis showed 95% identity and differed at 28 nucleotide positions. Pairwise sequence divergence with three C. sinensis isolates and O. viverrini ranged from 0 to 3.94% in mtCOI gene. The mtCOI sequences were more highly conserved relative to the ITS2 sequences. These genetic data from different geographical areas showed that the liver flukes are not variable and are virtually identical almost despite belonging to entirely different genera.