Supplementary Material Online Resource 1 pcr amplification and parasite species identification




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Supplementary Material

Online Resource 1

PCR amplification and parasite species identification

Genomic DNA was isolated and purified from parasite individuals using a genomic DNA extraction kit (Peqlab Biotechnology GmbH, Germany) according to the instructions of the manufacturer. Two different genetic markers were analysed (rDNA: Acanthocephala, Nematoda; COI: Acanthocephala): The rDNA region comprising the ITS-1, 5.8S, ITS-2 was amplified using primers BD1 (5’-GTC-GTA-ACA-AGG-TTT-CCG-TA-3’) and BD2 (5’-TAT-GCT-TAA-ATT-CAG-CGG-GT-3’) (Král’ová-Hromadová et al. 2003). A part of the cytochromeoxidase subunit 1 (COI) was amplified using primer HCO2198 (5´-TAA-ACT-TCA-GGG-TGA-CCA-AAA-AAT-CA-3´) and LCO1490 (5´- GGT-CAA-CAA-ATC-ATA-AAG-ATA-TTG-G-3´) (Folmer et al. 1994). PCR-reactions (50μl) included 25μl Master-Mix (Peqlab Biotechnology GmbH, Erlangen, Germany) containing dNTP, MgCl2, Buffer and Taq-Polymerase, 3μl of each primer (10 pmol µl-1), 14 ddH2O and 5μl genomic DNA. Each PCR reaction was performed in a thermocycler (Eppendorf, Germany) under the following conditions: BD1/BD2: an initial denaturation step at 94ºC for 120 sec, 40 cycles of 94ºC for 20 sec (denaturation), 51ºC for 20 sec (annealing), 72ºC for 50 sec (extension), followed by a final extension at 72ºC for 5 min. HCO2198/LCO1490: an initial denaturation step at 94ºC for 180 sec, 40 cycles of 94ºC for 45 sec (denaturation), 37ºC for 45 sec (annealing), 72ºC for 60 sec (extension), followed by a final extension at 72ºC for 10 min. Samples without DNA were included in each PCR run (control). PCR products were examined on a 1% agarose gel. A 100bp ladder marker (peqGOLD, Erlangen, Germany) was used to estimate the size of the PCR products. To prepare the samples for sequencing, PCR products were purified with Cycle-Pure Kit (Peqlab Biotechnology GmbH, Germany). Afterwards the PCR product were sequenced by Seqlab (Goettingen GmbH, Germany) using primer BD1 (5’-GTC-GTA-ACA-AGG-TTT-CCG-TA-3’) and LCO1490 (5´-GGT-CAA-CAA-ATC-ATA-AAG-ATA-TTG-G-3´). For species identification, the sequences were compared with Genbank data using the BLASTn algorithm (Altschul et al. 1990).


Results

A subsample of n = 6 nematode specimens from the gut of Amatitlania were identified to species level using light microscopy and molecular analyses. Based on the morphology of the digestive tract and cephalic end (Stumpp 1975; Menezes et al. 2006; Moravec and Justine 2006), all nematodes were determined as adult Camallanus cotti. Alignment of the sequences with Genbank data confirmed the preliminary morphological identification and revealed 99-100% identity (e-value: 0.00) with a sequence of Camallanus cotti from Saurogobio dabryi (Genbank-Acc.: EU598901.1) suggesting that the individuals in this study belong to the same species (Genbank-Acc.: KR136205-KR136210). Species identification for the acanthocephalan subsample was difficult. The general morphology of the proboscis (hook configuration, quantity), specimen size, and embryophore morphology resembled that of Acanthocephalus anguillae (Golvan 1969). However, molecular analysis (n = 22: ngut = 9, nbodycavity = 6, nA. aquaticus = 7) did not reveal clear-cut results. Sequence data for ITS-1/5.8S/ITS-2 (n = 18: ngut = 8, nbodycavity = 4, nA. aquaticus = 6) showed low similarity values (identity: max 84%; e-value: 5.0 x 10-138-4.0 x 10-89) with a sequence of Acanthocephalus sp. JDC-2005 (Genbank-Acc.: DQ768187.1) and alignment of cytochromeoxidase subunit I (COI) data (n = 18: ngut = 7, nbodycavity = 6, nA. aquaticus = 5) with Genbank data revealed an average 93% identity (e-value: 6.0 x 10-156-0.0) with a sequence of Acanthocephalus dirus (DQ089718.1) from Asellus aquaticus but not with A. anguillae as could have been expected from morphological analysis. In this context, Benesh et al. (2006) reported the occurrence of pseudogenes (Numts) in acanthocephalans of the same genus, such “abnormalities” (i.e. indels and/or stop codons) in sequence data obtained by using universal primers (HCO2198/LCO1490) might have hindered proper species identification in the present study. Due to the clear morphological match with A. anguillae, the morphological mismatch with A. dirus (following Golvan (1969)) as well as the fact that A. anguillae is a widespread parasite in Germany (Taraschewski 1988), we describe this parasite as A. anguillae.


References

Altschul SF, Gish W, Miller W, et al (1990) Basic local alignment search tool. J Mol Biol 215:403–410. doi: 10.1016/S0022-2836(05)80360-2


Benesh DP, Hasu T, Suomalainen L-R, et al (2006) Reliability of mitochondrial DNA in an acanthocephalan: The problem of pseudogenes. Int J Parasitol 36:247–254. doi: 10.1016/j.ijpara.2005.09.008
Folmer O, Black M, Hoeh W, et al (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol 3:294–299
Golvan YJ (1969) Systématique des Acanthocéphales (Acanthocephala, Rudolphi 1801). Muséum, Parais
Král’ová-Hromadová I, Tietz DF, Shinn AP, Špakulová M (2003) ITS rDNA sequences of Pomphorhynchus laevis (Zoega in Müller, 1776) and P. lucyi Williams & Rogers, 1984 (Acanthocephala: Palaeacanthocephala). Syst Parasitol 56:141–145. doi: 10.1023/A:1026127219358
Menezes RC, Tortelly R, Tortelly-Neto R, et al (2006) Camallanus cotti Fujita, 1927 (Nematoda, Camallanoidea) in ornamental aquarium fishes: pathology and morphology. Mem Inst Oswaldo Cruz 101:683–687
Moravec F, Justine J (2006) Camallanus cotti (Nematoda: Camallanidae), an introduced parasite of fishes in New Caledonia. Folia Parasitol (Praha) 53:287
Stumpp M (1975) Untersuchungen zur Morphologie und Biologie von Camallanus cotti (Fujita, 1927). Parasitol Res 46:277–290. doi: 10.1007/BF00418521
Taraschewski H (1988) Host-parasite interface of fish acanthocephalans. I: Acanthocephalus anguillae (Palaeacanthocephala) in naturally infected fishes: LM and TEM investigations. Dis Aquat Organ 4:109–119


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