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Evaluation of fixatives and autofluorescence reduction treatments for marine bivalve larvae

Published online by Cambridge University Press:  04 March 2011

S.A. Heaney ,
A.P. Maloy  and
J.W. Slater
S.A. Heaney*
Affiliation:
Centre of Applied Marine Biotechnology (CAMBio), Department of Science, Letterkenny Institute of Technology, Port Road, Letterkenny, County Donegal, Ireland
A.P. Maloy
Affiliation:
Centre of Applied Marine Biotechnology (CAMBio), Department of Science, Letterkenny Institute of Technology, Port Road, Letterkenny, County Donegal, Ireland
J.W. Slater
Affiliation:
Centre of Applied Marine Biotechnology (CAMBio), Department of Science, Letterkenny Institute of Technology, Port Road, Letterkenny, County Donegal, Ireland
*
Correspondence should be addressed to: S.A. Heaney, Centre of Applied Marine Biotechnology (CAMBio), Department of Science, Letterkenny Institute of TechnologyPort Road, Letterkenny, County Donegal, Irelandemail:[email protected]
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Abstract

Improved understanding of the occurrence and spatio-temporal distribution of bivalve larvae holds significant benefits for ecological studies, shellfisheries management and aquaculture. Morphological methods for identification have proved difficult to develop because of the small size of these larvae and similarities in their shape and colour. Molecular methods based on DNA extraction can confirm the presence of a species in a plankton sample, but without sample sorting and individual larval analysis, provide no estimate of larval abundance and are incapable of providing an estimate of larval growth rate. Fluorescence in situ hybridization (FISH) using species-specific DNA probes has the potential to resolve these issues. However, utilization of this technique is constrained by the strong autofluorescence, common in marine larvae. Here we evaluate the effect of eight different fixatives on the autofluorescence intensity of bivalve larvae using fluorescein isothiocyanate (FITC) and Cy3 filters. In addition, fifteen autofluorescence reduction treatments were evaluated and their compatibility with FISH assessed. Relative to fresh larvae, chemically fixed larvae had significantly higher autofluorescence in both filter sets. Larvae preserved by freezing at –80°C exhibited no significant increase in autofluorescence over a 3-year period. Autofluorescence levels were generally lower with the FITC filter set than the Cy3 filter set. For archived larvae preserved in modified saline ethanol and exhibiting fixative-induced autofluorescence, the autofluorescence intensity could be reduced to 20–30% with saturated Sudan Black B and to 30–40% with Chemicon™. Both of these autofluorescence reduction treatments were compatible with subsequent FISH protocols using a FITC-labelled probe.

Keywords

autofluorescencefixativeautofluorescence reductionbivalve larvaeMytilus
Type
Research Article
Information
Journal of the Marine Biological Association of the United Kingdom , Volume 91 , Issue 7 , November 2011 , pp. 1567 - 1576
Copyright
Copyright © Marine Biological Association of the United Kingdom 2011

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