Appendix A
Abstracts of presentations on the various project methods
WESTHER First Co-ordination Meeting, Aberdeen 18-20 January, 2003.
Body-morphometry: Soenke Jansen, BFA-Fi.
The aim of this method is to provide metric data of the external body dimensions. The central tendencies and variances for the samples of the reference collection (spawning adults) are to be compared of those of the samples from aggregations of juveniles and non-spawning adults. The measurements are to be made on digital images of the fish taken in a standardised way during sampling. Evaluation will include the use of truss networks delivering data for multivariate statistics of body proportions. The pre-requisite for this WP is a photograph of each fish of an appropriate quality, as described in the photography set-up and procedures leaflet (Appendix C).
Otolith shape analysis: Jurgen Schlickeisen, BFA-Fi.
The goal of this workpackage is to collect metric data from the shape of herring otoliths. For this purpose the otoliths are scanned using an image analysis system consisting of a stereo microscope equipped with an analogue CCD camera and connected via a frame-grabber to a PC. After scanning the images are used to extract morphometric data as well as the outline which is described as a set of xy-co-ordinates. These co-ordinates are the basis for the elliptic fourier analysis. This analysis provides the normalised data which are further analysed by means of multivariate analysis. The major requirements for the otoliths used for this package is that they are unbroken, clean and not crystallised (vaterite).
Pyloric caeca counts: Ken MacKenzie, UNIABDN.
Pyloric caeca are finger-like projections of the gut in fish. Previous studies on herring have shown that numbers of caeca range from 13 to 34, with most herring having from 20 to 23. The mean number of caeca per sample tends to increase from south to north and appears to be related to the temperature at which the herring larvae develop. In the WESTHER project the numbers of pyloric caeca in each herring examined will be noted. Two forms of caeca abnormality, bifurcate and looped, will also be recorded.
Parasites as biological tags: Ken MacKenzie, UNIABDN.
Based on previous experience and published information on the parasite fauna of herring in the WESTHER study area, we expect to find about 14 species of parasite in our samples of herring viscera. For biological tag purposes two categories of parasite are recognized: permanent and temporary. Permanent parasites have life spans of several years in herring and include larval helminths, a myxosporean and two protozoans. Temporary parasites have life spans in herring of less than one year and include adult digeneans and larval tapeworms in the lumen of the alimentary tract. The most useful tag parasites for stock identification are likely to be larval digeneans and cestodes, while the temporary parasites may prove useful for following seasonal migrations of herring.
Parasite genetics: Carey Cunningham, FRS.
Anisakid nematodes and Cercarian digeneans were selected as examples of the major groups of parasites that will be examined during this project. The amount of relevant information that is available for the two groups is variable. Investigation of DNA (particularly ribosomal RNA genes, rDNA) of parasites has provided alternative methods of species identification. This has shown that some anisakid nematodes exist as cryptic or sibling species. The rDNA of anisakids and digenea will be examined to determine if other herring parasites exist as cryptic species and if so, if the distribution of each type varies and can add to previous information on the use of these species as biological tags. The molecular biology of herring digeneans has not been studied, but methods for other digeneans will be readily transferable. Again, the rDNA will be examined, and also mitochondrial DNA, which provides a better chance of yielding sub-species variation. The development of microsatellite DNA markers for parasites is in its' infancy. This project will include development of such markers, but time constraints are likely to limit their application.
Genetic characterisation using microsatellites: Tom Cross, UCC
The aims and methods of this workpackage were presented. Microsatellite DNA analysis is the method of choice since these markers are usually highly variable and have been used successfully in population discrimination in many other commercial fish species (e.g. cod, hake, whiting, blue whiting, poor cod, halibut, turbot). Ten loci are in routine use by the HERGEN project and these will initially be tested in both Cork and Liverpool. Some of these will be utilised by WESTHER and other loci will be developed as required. We intend to first analyse spawning (reference) samples, to search for genetic structure. A potential bottleneck will be the availability of these samples. However fish from at least five locations should be available by the end of March, with the others being collected in the subsequent autumn and winter. Between these periods the analysis of the first years nursery and putative mixed stock samples will be carried out. The requirement is a part or whole pectoral fin from each fish (at least 0.5 cm2) stored as soon as possible in absolute ethanol (10 times solid tissue volume) in a 2ml screw cap rubber O-ring Eppendorf tube (or similar).
DNA Pooling: Phill Watts, LIV (Liverpool).
DNA pooling is a process where samples are genotyped using a pool of tens to hundreds of different DNA samples rather than individual DNA samples. The main use of this technique has been in disease association studies where differences in allele frequency between two samples have been identified and correlated with individual genotyping data. The benefit of this technique is that it increases the speed that a sample may be characterised genetically (or allows a much larger sample size) whilst reducing the consumable cost. The main drawbacks of DNA pooling are (i) that genetic information is lost (because the resulting data give whole sample allele frequencies rather than individual genotypes) and (ii) some error in quantifying allele frequencies is introduced. We aim to identify whether DNA pooling can identify biologically relevant differences in allele frequencies between herring samples.
Otolith microstructure: Soenke Jansen, BFA-Fi.
The aim of this WP is to provide information about the hatch season of the individual fish. Apart from testing the hypothesis of the identity of spawning season for the fish of the reference collection, the relative distribution of hatch seasons in the aggregations of juvenile fish as well as in the non-spawning adults will be elucidated. To this end the right otoliths of the fish will be ground down to a plane through the nucleus, etched and observed under a scanning electron microscope to determine the relative distance between the nucleus and the first winter ring and the ring-width of the micro-increments of the first year.
Otolith core microchemistry: Audrey Geffen, LIV
The study of otolith composition (microchemistry) is based on observations that fish from different areas can be distinguished by the relative concentrations of various elements in the otoliths. Otoliths are primarily calcium, carbon, and oxygen (CaCO3), but also contain smaller quantities of minor elements such as Sr, Na, K, Cl. Trace concentrations of many other elements are also found in the otoliths and these form a ‘chemical fingerprint’ for fish from different areas. The composition of the otoliths will be measured by Inductively Coupled Plasma Mass Spectrometry (ICP-MS), which allows multi-element detection at the sensitivity required for otolith work. Previous studies have shown that relative concentrations (primarily Sr, K) can be used to separate herring larvae sampled in different areas of the North Sea.
Sampling considerations – lessons learned from REDFISH & HOMSIR: Chris Zimmermann, BFA-Fi
A short presentation on the purpose and structure of two current EU projects, HOMSIR and REDFISH, was given. HOMSIR deals with Horse Mackerel in the North East Atlantic, North Sea and the Mediterranean, started in 2000 and is about to end. The project structure is very similar to WESTHER, with 2 yrs sampling, fixed sampling sites and each method to be conducted on the same individual fish. Outliers (fish known to belong to a separate stock) were not processed. Some of the WESTHER, -partners are also involved there. REDFISH deals with Redfish in the North Atlantic, started in 1999 and will end in late 2003; 3 yrs sampling, no fixed sampling sites (depending on WP), but use of outliers (from the Pacific).
Problems in these projects were: highly varying quality of samples, only a fraction of samples have been used for all methods (HOMSIR); delay in processing samples, especially Genetics WP’s (both); database still not functional - no integrated analysis possible (HOMSIR); no outliers - uncertainty about distances or differences needed to define “stocks“ (HOMSIR). Interim results show very little differences among sampled areas, especially in the genetics WP’s, but rather promising results from the parasitology WP’s.
Lessons learned from these projects’ experiences: a strict and detailed sampling protocol should be available before sampling starts, and any partner involved in sampling should be aware that the whole project success depends on their work; a database should be defined and set up as soon as possible, and the co-ordinator should only accept data as “being delivered” if it is entered in the database; group should discuss the usefulness of “outliers” for the final statistical evaluation.