Field preservation of monogenean parasites for molecular and morphological analyses

Strona, Giovanni; Stefani, Fabrizio; Galli, Paolo

doi:10.1016/j.parint.2008.10.001

Cited by 16 publications

(19 citation statements)

References 15 publications

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“…In their study of free-living nematodes and some parasites of invertebrates, Yoder et al (2006) reported successful PCR amplification of nuclear ribosomal gene regions of up to 1,800 bp in ,80% of the specimens tested (Yoder et al, 2006). DESS has also been tested for preservation of monogeneans and fish gill tissues, and the results confirmed excellent, short-term preservation (up to 8 wk tested experimentally, with longer preservation periods suggested) of parasites and gills, with PCR amplification and DNA sequencing results comparable to 99% ethanol (Strona et al, 2009). In other studies, PCR-based evaluations of 5 solutions used for field preservation of DNA for marine invertebrate tissues (NaCl-CTAB, 70% ethanol, DESS, Queen's lysis buffer, and urea buffer) also indicated that DESS was best for preserving both the physical structure of tissues and the DNA, as assessed by amplification (Dawson et al, 1998), a result previously reported for vertebrate tissues (Seutin et al, 1991).…”

mentioning

confidence: 82%

Structural Restoration of Nematodes and Acanthocephalans Fixed in High Percentage Alcohol Using Dess Solution and Rehydration

Naem

Pagan

Nadler

2010

Journal of Parasitology

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mentioning

confidence: 82%

Structural Restoration of Nematodes and Acanthocephalans Fixed in High Percentage Alcohol Using Dess Solution and Rehydration

Naem

Pagan

Nadler

2010

Journal of Parasitology

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“…The emergence of molecular methods has prompted a renewed interest in methods which can process material for both morphological and molecular methods (Harris et al 1999;Košková et al 2011;Naem et al 2010;Strona et al 2009;Toe et al 1997;Yoder et al 2006).…”

Section: Introductionmentioning

confidence: 99%

A quick and simple method, usable in the field, for collecting parasites in suitable condition for both morphological and molecular studies

2012

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Many methods have been proposed for collecting and fixing parasites, but most were written before the molecular age, and were intended to be practised by experienced parasitologists in well-equipped laboratories. We describe here a very simple method, illustrated by photographs, for collecting helminths from the digestive tract of vertebrates. It only requires a few plastic vials, some ethanol and a means to heat water. Basically, the method consists of: (a) the extraction of all organs from the abdominal cavity; (b) opening the digestive system longitudinally; (c) agitate gut and contents in a saline solution (i.e. ca. 9% NaCl or 1/4 sea water in tap water); (d) decant in saline as many times as needed to clean contents; (e) immediately fix parasites in near-boiling saline; (f) discard saline and keep specimens in 95% ethanol. Additional information is given for collecting parasites from fish gills with a similar process. The method will collect most helminths (digeneans, larval cestodes, nematodes, acanthocephalans) from the digestive tract, and monogeneans and isopod and copepod crustaceans from fish gills. The specimens will be suitable for both morphological study and DNA sequencing. The method is simple, fast, inexpensive and can be used by untrained personnel, even in the field without electricity and without a binocular microscope. It can also be used by trained parasitologists who need to expedite treatment of abundant samples.

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“…As a good fixative for both molecular and morphological studies, apart from alcohol, fixation used DMSO-NaCl solution by Strona et al 2009; however, morphological and molecular analyses were conducted separately on different individual of parasites. Other fixatives commonly used for monogeneans such as formalin and alcohol-formalin-acetic acid act as fine preservatives for morphological investigation of parasites.…”

Section: Discussionmentioning

confidence: 99%

“…To overcome these difficulties, the classical morphological determination has been combined with modern molecular approaches, especially utilizing variability of the internal transcribed spacers and genes of ribosomal DNA (Dzikowksi et al 2003;Huyse et al 2004;Matejusová et al 2001Matejusová et al , 2002Matejusová et al , 2004Shamsi et al 2009;Strona et al 2009). Molecular analyses complementing morphological taxonomy help to assign correct molecular data with an appropriate species to ensure its accurate determination and description (Harris et al 1999).…”

Section: Introductionmentioning

confidence: 99%

Ethanol-fixed material used for both classical and molecular identification purposes: Eudiplozoon nipponicum (Monogenea: Diplozoidae) as a case parasite species

et al. 2010

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This study is focused on the feasibility of two treatments of alcohol-fixed monogenean parasites which are intended to be use for the combined morphological and molecular characterizations. The monogenean parasite, Eudiplozoon nipponicum, was selected as a model parasite species; however it is expected that these techniques will be suitable for other monogeneans and other parasitic families. The haptor of diplozoid parasites is equipped with sclerotized attachment clamps and central hooks which are utilized for morphological identification. As parasite tissue become very tough and rigid when preserved in ethanol, using these structures for species identification without additional treatment is difficult. We investigated two different techniques to digest the surrounding tissues, the first was treatment with 10% sodium dodecyl sulphate (SDS) and the second treatment was proteinase K. Tissue was successfully digested in both treatments and all clamps, central hook and even individual sclerites of the clamps were clearly visible and well defined. After treatment, the digest was used to extract genomic DNA, and the second internal transcribed spacer of the ribosomal DNA genes (rDNA) was amplified. Nucleic acid sequence was obtained from 90% of parasite specimens processed by both treatments. Treatment of haptors with SDS was proven to be more successful with no visible changes or damage observed to sclerites even after a month. This method represents a useful tool for the combined morphological and molecular studies as the correct sequence can be assigned to the same individual worm from which haptoral parts have been obtained.

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Field preservation of monogenean parasites for molecular and morphological analyses

Cited by 16 publications

References 15 publications

Structural Restoration of Nematodes and Acanthocephalans Fixed in High Percentage Alcohol Using Dess Solution and Rehydration

Structural Restoration of Nematodes and Acanthocephalans Fixed in High Percentage Alcohol Using Dess Solution and Rehydration

A quick and simple method, usable in the field, for collecting parasites in suitable condition for both morphological and molecular studies

Ethanol-fixed material used for both classical and molecular identification purposes: Eudiplozoon nipponicum (Monogenea: Diplozoidae) as a case parasite species

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