Molecular phylogeny provides new insights on the taxonomy and composition of Lyperosomum Looss, 1899 (Digenea, Dicrocoeliidae) and related genera

Lyperosomum Looss, 1899 is one of the largest genera of the Dicrocoeliidae and is one of the best examples of the systematic complexity and taxonomic instability within this family. We present the molecular analyses based on novel sequences of nuclear and mitochondrial genes obtained from 56 isolates of adult flukes and larval stages of dicrocoeliids belonging to Lyperosomum, Skrjabinus, Zonorchis as well as previously available sequence data. According to obtained results we propose to return Zonorchis clathratus and Z. petiolatus into Lyperosomum, and to recognize L. alagesi as a synonym of L. petiolatum. Our study shows that L. petiolatum commonly occurs in Europe in corvids as well as in several species of migratory songbirds, e.g. Sylvia atricapilla. At the same time, the Turdidae appear to host a distinct species of Lyperosomum. The phylogenetic analysis has clearly demonstrated the paraphylepic nature of Lyperosomum and indicated the need of its thorough revision preferably using specimens from type hosts and type territories of nominal species. In addition, inclusion of numerous not yet sequenced dicrocoeliid genera into future phylogenetic studies is necessary to clarify the interrelationships of taxa within the family and stabilize its system

Leptus kattikus Haitlinger 2009

Leptus kattikus Haitlinger, 2009 Diagnosis. Larva. One seta on palp femur and palp genu. Scutum in the shape of irregular hexagon, with sharpened antero-lateral corners. Anterior and posterior margin of scutum concave. Four intercoxalae at the level of coxae III. Anterior intercoxalae III (3 a 1) 2.0– 2.9 times shorter than the posterior ones (3 a 2). Tibia I (283–328) with two solenidia (φ). ISD = 78–105. Ti III/AW = 3.2–4.5. DS max = 73–97. L/W = 0.8–1.1. Ti III = 362–440. Description. Larva (Figs 1 –14). For morphometric data see Table 1. TABLE 1. Morphometric data on larvae of Leptus kattikus. Leptus kattikus Haitlinger, 2009 Sample size Mean Range Data for holotype (original description (Haitlinger 2009)/present studies) Character GNATHOSOMA ...... continued on the next page Leptus kattikus Haitlinger, 2009 Sample size Mean Range Data for holotype (original description (Haitlinger 2009)/present studies) Character PW/AW 20 1.3 1.1–1.3 1.1 / 1.2 DS max. 20 87 73–97 88 / 90 VS 20 78 75–85 no data/ 78 3 a 1 20 33 27–39 25 / 25 3 a 2 20 81 67–95 65 / 70 3 a 2 / 3 a 1 20 2.5 2.0– 2.9 2.6 / 2.8 LEGS Cx I (L) 20 102 75–120 106 / 100 Tr I (L) 20 64 45–83 60 / 73 bFe I (L) 20 169 157–183 160 / 175 tFe I (L) 20 151 138–162 162 / 155 Ge I (L) 20 213 188–229 236 / 225 Ti I (L) 20 304 283–320 328 / 328 Ta I (L) 20 216 197–234 208 / 230 Ta I (W) 20 32 20–62 19 / 20 LEG I ( Σ ) 20 1219 1145–1267 1260 / 1286 Cx II (L) 20 121 92–133 112 / 108 Tr II (L) 20 67 54–78 70 / 70 bFe II (L) 20 138 120–154 146 / 145 tFe II (L) 20 132 113–142 152 / 138 Ge II (L) 20 174 161–194 180 / 180 Ti II (L) 20 276 253–301 290 / 298 Ta II (L) 20 203 177–234 200 / 203 Ta II (W) 20 28 24–33 19 / 25 LEG II ( Σ ) 20 1110 1039–1161 1150 / 1142 Cx III (L) 20 124 100–144 116 / 118 Tr III (L) 20 71 58–86 70 / 85 bFe III (L) 20 164 151–180 190 / 183 tFe III (L) 20 171 151–187 186 / 185 Ge III (L) 20 199 184–210 212 / 213 Ti III (L) 20 383 362–406 420 / 440 Ta III (L) 20 207 192–224 – Ta III (L) 20 25 20–28 – LEG III ( Σ ) 20 1319 1250–1386 – IP 20 3649 3434–3810 – Ti I/AW 20 2.8 2.4 –3.0 3.2 / 3.3 Ti I/Ge I 20 1.4 1.3–1.5 1.4 / 1.5 Ti II/PW 20 2.0 1.9–2.1 2.6 / 2.6 Ti II/Ge II 20 1.6 1.5–1.7 1.6 / 1.7 Ti III/AW 20 3.5 3.2–3.7 4.0/ 4.5 Ti III/Ge III 20 1.9 1.8–2.1 2.0/ 2.1 Ti III/Ti I 20 1.3 1.2–1.3 1.3 / 1.3 1) distorted (underestimate), due to the fold in the posterior part of the shield, 2) broken, 3) one with broken tip, one missing, 4)distorted (underestimate), due to the broken tip of AL. Gnathosoma. Chelicera (Figs 1, 12) composed of basal segment and movable claw. Mouth (Figs 4, 6, 12) surrounded with lamellar, narrowing apically fimbriae. At mouth—c. 4–5 pairs of digit-shaped and terminally diverged processes. Dorsally, a pair of setiform adoral setae (cs) (c. 15) placed anteriorly and a pair of club-shaped supracoxal setae (elcp) (c. 2) located postero-laterally, at gnathosoma base (Fig. 4). Ventrally (Fig. 6) a pair of very short (c. 3) spine-like setae (as) and a pair of longer (c. 35) subcapitular setae (bs), covered with few short barbs, arising at medial part of the stem. Pedipalp formula: 0-B-B-BBB-NNBBBωζ. Palp femur and palp genu (Fig. 4) with one setulose seta. Palp tibia (Figs 2, 13) with three barbed setae. Odontus simple. Palp tarsus (Figs 2, 13) with six normal setae, of which four are covered with setules and two are smooth, one solenidion (ω) located in proximal part of the segment and one prominent distal eupathidium (ζ). Dorsal side of idiosoma. Scutum (Figs 3 –4, 14) with indistinct perforations on the entire surface. Setae AL shorter than PL. AL almost levelled with ASens, PL placed posteriorly to AL, c. at the widest part of scutum. ASens shorter than PSens, both with setules covering the distal half of the stem. Single eyes on circular sclerites located at the level of posterior margin of scutum. The remaining part of idiosoma covered with cuticle displaying a linear design. Folds (Fig. 15) separated with rows of perforations. Each perforation clogged with cuticular plug. Dorsal setae (Fig. 5) inserted in small cuticular rims and covered with setules distributed along the stem, except for its basal most part. ƒD = 43–58 (n = 19). Ventral side of idiosoma (Fig. 6). ƒCx = 1 - 1 - 1. Club-shaped supracoxal setae elc I present on dorsal side, in terminal part of coxae I. Setae 1 b, 2 b, 3 b located on coxae I, II, III, respectively. Setae 1 a, 2 a, 3 a 1 -a 2 placed between the respective coxal plates. Setae 3 a 1 (Figs 6 –7, 15) distinctly shorter than 3 a 2. ƒV = 22–29 (n = 19) (the total number of setae in ƒD and ƒV formula (NDV) = 72–84, n = 19). Ventral setae (Fig. 8) with setules more sharpened than in dorsal setae. Cuticle displays the similar pattern to one on dorsal side of idiosoma; the rows of perforations more distinct (Fig. 15). Legs (Figs 9 –11, 16– 17). Leg segmentation formula: 7 - 7 - 7. For leg chaetotaxy see Table 2. Cuticle on legs arranged in transverse folds, especially distinct on tarsi. Normal setae on legs covered with husk-like setules. Famulus on tarsus I located distally to solenidion. Eupathidia on tarsi I–III covered with delicate fimbriae. Slightly longer fimbriae present on claw-like claw and on empodium at tarsi termination. Tarsal claws without terminal hooks. Leg segment Chaetotaxy Cx I 1 n, 1 supracoxal seta Tr I 1 n bFe I 2 n tFe I 5 n Ge I 8 n, 1 σ, 1 κ Ti I 14 n, 2 φ, 1 κ Ta I 26 n, 2 ζ, 1 ω, 1 ε Cx II 1 n Tr II 1 n bFe II 2 n tFe II 5 n Ge II 7–8 n, 1 κ Ti II 14–15 n, 2 φ Ta II 21–24 n, 2 ζ, 1 ω Cx III 1 n Tr III 1 n bFe III 1 n tFe III 5 n Ge III 8 n Ti III 15 n, 1 φ Ta III 25–27 n, 1 ζ FIGURES 4–8. Leptus kattikus: 4. Dorsal side of the body (legs omitted beyond trochanters); 5. Dorsal idiosomal seta; 6. Ventral side of the body (legs omitted beyond trochanters); 7. Intercoxala 3 a 1; 8. Ventral idiosomal seta. FIGURES 12–17. Leptus kattikus. SEM micrographs. 12. Antero-ventral part of gnathosoma; 13. Palp tibia and palp tarsus; 14. Cheliceral bases and scutum; 15. Intercoxala 3 a 1; 16. Tibia I; 17. Tarsus III. Distribution. Nepal, Vietnam. Deposition of the material. A total number of 121 larvae were separated from a single host. Voucher specimens are deposited in the senior author’s collection (Institute of Biology, Department of Invertebrate Systematics and Ecology, Wrocław University of Environmental and Life Sciences, Wrocław, Poland). Molecular analysis. We expected to obtain an approximately 650 bp long COI gene product from L. kattikus, however after sequencing we obtained only a reliable 438 bp long contig beginning at the 5 ’ end. Unfortunately most of the 3 ’ end of the sequence was illegible due to the presence of double peaks. The PCR reaction as well as sequencing were repeated and, for each repetition, the obtained results were similar to the initial sequencing. BLAST alignment analysis of the COI barcode region of our sequence showed 88 %– 84 % similarity, in the overlapping regions, with the sequences being those of Phasmatidae. The highest levels of similarity (87.9 % and 86.8 %) were observed for Ramulus irregulariterdentatus (AB 477463) and Phraortes illepidus (AB 477460), respectively. The results show that the amplified product of COI barcode belongs to the host of Leptus kattikus which was determined morphologically as Xenophasmina bedoti. Thus, despite the unsuccessful amplification of barcode COI region of the mite larva, we were able to obtain the sequence of its host, which demonstrates molecular host identification based on DNA content isolated from engorged mite larvae.Published as part of Mąkol, Joanna, Felska, Magdalena, Moniuszko, Hanna & Zaleśny, Grzegorz, 2012, Redescription of Leptus kattikus Haitlinger, 2009 (Actinotrichida, Parasitengona, Erythraeidae) and molecular identification of its host from DNA barcoding, pp. 67-78 in Zootaxa 3569 on pages 69-75, DOI: 10.5281/zenodo.20881

Host-dependent morphology of Isthmiophora melis (Schrank, 1788) Luhe, 1909 (Digenea, Echinostomatinae) – morphological variation vs. molecular stability

Abstract Background Echinostomes are cosmopolitan digenean parasites which infect many different warm-blooded hosts. Their classification is extremely confused; the host spectrum is wide, and morphological similarities often result in misidentification. During our long-term studies on the helminth fauna of rodents and carnivores we have collected 27 collar-spined echinostomes which differ in morphology to an extent that suggests the presence of more than one species. Here, we describe this material, and the extent of host-related variation in this parasite. Methods Specimens of Isthmiophora isolated from four host species (badger, American mink, hedgehog, striped field mouse) were subject to morphological and molecular examination; the data were statistically analysed. Results Our results show that genetically all the Isthmiophora specimens obtained from all the examined hosts are conspecific and represent I. melis. On the other hand, the individuals isolated from Apodemus agrarius are morphologically distinct and, based on this criterion alone, should be described as a new species. Conclusions The morphological traits of Isthmiophora melis are much variable and host-dependent; without molecular analysis they would suggest a necessity to describe a new species or even genus. Such a high level of intraspecific variability may be affected by the host’s longevity

The genetic structure of populations of Isthmiophora melis (Schrank, 1788) (Digenea: Echinostomatidae). Does the host’s diet matter?

Abstract Background Here we provide a comparative analysis of the genetic structure of populations (based on nad1 mtDNA) of Isthmiophora melis isolated from the American mink (Neogale vison), an introduced invasive species, commonly occurring in the territory of Poland, and from the striped field mouse (Apodemus agrarius). Methods A total of 133 specimens of I. melis were obtained from naturally infected N. vison collected from six localities in Poland (108 samples) and 25 individuals of I. melis from A. agrarius. All sequences of the nad1 gene obtained during the present study were assembled and aligned. The standard statistics for haplotype composition, i.e., the number of haplotypes, haplotype diversity, nucleotide diversity, and average number of nucleotide differences, were calculated. Haplotype analysis and visualization of haplotype frequency among populations were performed using a median-joining network. Results Based on the samples collected from different localities in Poland, our study revealed that the overall genetic diversity of I. melis isolated from the American mink and of the striped field mouse do not differ significantly. The median-joining network showed that the three main haplotypes are in the centre of a star-like structure, with the remaining haplotypes as the satellites, reflecting the recent expansion of the populations. Conclusions The overall genetic diversity of I. melis isolated from the American mink and striped field mouse reveals a high level of homogeneity. Moreover, regional differences in the food composition of the definitive hosts play an important role in shaping the genetic structure of the trematode populations. Graphical Abstrac

Parasitic nematodes of the genus Syphacia Seurat, 1916 infecting Muridae in the British Isles, and the peculiar case of Syphacia frederici.

Syphacia stroma (von Linstow, 1884) Morgan, 1932 and Syphacia frederici Roman, 1945 are oxyurid nematodes that parasitize two murid rodents, Apodemus sylvaticus and Apodemus flavicollis, on the European mainland. Only S. stroma has been recorded previously in Apodemus spp. from the British Isles. Despite the paucity of earlier reports, we identified S. frederici in four disparate British sites, two in Nottinghamshire, one each in Berkshire and Anglesey, Wales. Identification was based on their site in the host (caecum and not small intestine), on key morphological criteria that differentiate this species from S. stroma (in particular the tail of female worms) and by sequencing two genetic loci (cytochrome C oxidase 1 gene and a section of ribosomal DNA). Sequences derived from both genetic loci of putative British S. frederici isolates formed a tight clade with sequences from continental worms known to be S. frederici, clearly distinguishing these isolates from S. stroma which formed a tight clade of its own, distinct from clades representative of Syphacia obvelata from Mus and S. muris from Rattus. The data in this paper therefore constitute the first record of S. frederici from British wood mice, and confirm the status of this species as distinct from both S. obvelata and S. stroma