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SID 5 Research Project Final Report

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NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

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Project identification

1. Defra Project code M0432

2. Project title

PREDATE - Detecting predation of fish eggs and larvae

3. Contractororganisation(s)

CefasPakefield RoadLowestoftSuffolkNR33 0HT

54. Total Defra project costs £ (agreed fixed price)

5. Project: start date................ 01 July 2005

end date................. 28 February 2010

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6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.Most marine finfish spawn huge numbers of eggs and substantive cumulative mortality rates on fish eggs and larvae of up to 99% are typical. Providing that spawning stock biomass remains above a critical threshold these natural levels of mortality will not lead to stock decline. However, the role played by mortality due to predation in the early life-stages of marine fish stocks is currently poorly understood. Field studies of predation have traditionally proved challenging, due for example to the difficulties involved in identifying partially digested fish, eggs or other remains in predator stomachs post-ingestion. Furthermore, many different types of predators may be responsible for egg and larval mortality including fish, crustacea and gelatinous predators, several of which have recently undergone large changes in abundance. Such changes may be linked to recent warming of UK shelf seas but may also have been triggered by fisheries. If the abundance of potential predators on the early stages of commercial species continues to increase, they may have the potential to slow stock rebuilding or even lead to further declines in some commercial species.

The aim of PREDATE was to use existing and novel techniques to study the full range of predators feeding on eggs and larvae of a typical commercial species. We modified existing, and developed new molecular tools to detect the presence of eggs and larvae in the stomachs of predators of cod, haddock, whiting and plaice. Probe reliability was tested both in laboratory and field trials, and the techniques developed were applied in a field study to identify all predators targeting plaice eggs and larvae on a spawning ground in the eastern Irish Sea. A secondary aim was to further develop particle tracking models to simulate the dispersal of eggs and larvae in the sea. The model outputs can be used to help guide the field sampling program, and can also predict locations where sampling needs to be conducted.

The work described is of direct policy relevance to Defra because it addresses a key issue in stock sustainability that is currently not well understood and tends to be ignored in single species fisheries models. The results of our study, listed under 5 main research objectives below, provide the tools required for the effective study of such of predation on fish eggs and larvae, in support of ecosystem-based management and conservation. The results will feed into the provision of policy advice to Defra and the EU on recovery plans and long-term sustainability of stocks under climate change.

Modify existing TaqMan probes for cod, haddock and whiting and demonstrate their specificity against other species.

Existing probes for cod, haddock and whiting DNA were tested against DNA from 27 co-occurring non-teleost marine organisms and a further 28 fish species (in addition to cod, haddock and

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whiting) Probes responses were demonstrated to be highly species-specific.

Develop DNA based probe for plaice and demonstrate its specificity against other species

A real-time Polymerase Chain Reaction (PCR) based assay using a TaqMan probe targeting the cytochrome b gene of plaice (Pleuronectes platessa) was developed

The plaice probe was tested against DNA from 27 co-occurring non-teleost marine organisms and a further 28 fish species and was demonstrated to be highly species-specific.

Determine effects of digestion on reliability of detecting presence of target prey in predator stomachs and intestines

For application to crustacean predator stomach contents, two tissue preservation and two DNA extraction methods were tested. Quality of extracted DNA was comparable for both preservation and extraction techniques, and detectability was similar for both.

Levels of PCR inhibition were significant for shrimp and crab predators, but could be overcome using serial dilution to reduce the incidence of false-negative results.

The plaice probe was capable of detecting 0.001 ng of undigested plaice DNA with high repeatability.

In time-course experiments, detection of prey DNA possible for up to 24 h in crustacean predator and up to 48 h in fish predator stomachs.

Twenty jellyfish fed on cod eggs all tested positive for cod DNA even when no eggs visible in gastric cavity.

The half-life detection rate(T50) for plaice DNA was ~10 h at 14-16ºC in shrimp, ~7 h at 6-10ºC in crab and ~31h for whiting at 6-8 ºC.

The T50 for cod DNA ingested by whiting was ~26 h at 8-10ºC for cod larvae, and ~31 h for cod eggs at 6-10 ºC.

Detection of predator DNA was possible even when no material visibly present in predator gut.

Modify existing coupled physical-biological model of plaice to cover cod and haddock and extend the geographical domain

The Irish Sea life-stage dispersal model was extended to simulate the dispersal of eggs and larvae of 5 fish species with contrasting early life histories (cod, plaice, witch, sprat and pogge).

The original coupled physical-biological model for the Irish Sea was extended to cover the North Sea and improved to provide better predictions of stratified layer thickness and improved transport predictions of fish eggs and larvae in inshore areas.

The model was run for 20 consecutive years for cod and haddock, using the generated distributions of newly spawned eggs from the 2004 international North Sea PLACES field survey, and maps of the densities of settled particles produced.

The displacements observed were much less than have been suggested by other recent studies, indicating that inter-annual variability in wind stress cannot fully explain inter-annual variability in larval displacement derived from these observations.

The possibility and utility of predicting egg-mass dispersal using near real time (NRT) forcing data have been demonstrated in the choice of field survey area and execution of the field work.

Undertake field studies on predation of fish eggs and larvae in the Irish Sea and/or North Sea

Research cruises were undertaken on the CEFAS Endeavour in the eastern Irish Sea on a well described plaice spawning ground located off the North Wales coast in February 2008 and 2009.

The full range of predators consuming plaice eggs (and larvae) and their spatial and temporal distributions were measured through the combined use of vertical plankton sampling, acoustic sampling, trawling and stomach sampling for application of the TaqMan plaice probe, and visual content analysis.

Sprat and herring were the dominant predators of plaice eggs in both the years studied. The TaqMan probe also revealed several other species not previously identified as consumers of

plaice eggs or larvae, including mackerel, whiting, poor cod and squid. Examination of sprat stomach contents revealed that these prey preferentially on a variety of eggs

including plaice, however TaqMan was able to detect plaice DNA even when the prey were substantively digested or regurgitation had occurred.

Possible future work

Field work carried out under PREDATE was limited to just two 10 day surveys, and therefore represents

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“proof of concept”, where we have demonstrated our ability to model egg dispersion in real-time, to sample and map predator communities, and to describe the predator-prey dynamics of the system. Our application of genetic probes has already demonstrated two important features: Firstly, we have shown that solid material need not be present within the predator stomach in order to detect predation. Given the tendency of many fish species to regurgitate their stomach contents on capture, the use of genetic probes potentially provides a means to estimate the level of under-reporting of predation in studies which directly examine stomach contents. Secondly, the successful detection of plaice DNA in non-fish predators (e.g. squid) may prove an effective means of monitoring predation in communities currently being affected by climate change, in which non-fish predators have frequently been observed to be increasing. Given the failure of the western Irish Sea cod stock to recover following 8 years of protection and a recovery plan, a next logical step would be to establish the level of predation experienced by the early-life history stages in Irish Sea cod, so as to either eliminate this or identify it as a significant factor in cod stock recovery.

Furthermore, the molecular approach as described does not yet provide information on the number of eggs or larvae consumed, nor on the developmental stages of the prey. Unless eggs and larvae of a target prey are spatially well separated e.g. due to advection, it will not be possible to conclude which stage is being consumed. As well as the more general quantification issue, future research will be required to quantify the fraction of early life stage mortality accounted for by clupeoid predation and to examine whether the status of the sprat and herring stocks can be linked to subsequent plaice year-class strength (Daan 1976). Further progress in the design of effective conservation measures for species or communities will need an integrated approach taking account of key aspects of early life history and behaviour.

Finally, by using coupled physical-biological models, we have been able to make increasingly sophisticated, multi-decadal predictions of the early life-history dispersal trajectories of marine fish. This is potentially valuable in the North Sea, where the settlement patterns of juvenile gadoids are not well understood. However, although we can predict the settlement patterns of these fish, and are currently using the model to test a hypothesis raised in the literature that recent shifts in wind patterns have altered the dispersal of cod eggs and larvae in the North Sea, there are currently few data available with which to validate our observations, suggesting a requirement for a supporting field survey.

Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with

details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

Contents:

1. General Introduction2. Scientific Objectives3. Summary of research results in relation to Scientific Objectives4. Modification of existing taqman probes for cod, haddock and whiting 5. Development of a DNA-based probe for plaice6. Determination of the effects of digestion on the reliability of gene-probes7. Modification and extension of coupled physical-biological model8. Field studies on predation of fish eggs and larvae9. Future Work

1. GENERAL INTRODUCTION

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It is generally accepted that most of the variance in adult fish stock abundance is generated during the egg and larval stages (Platt et al. 2007), when marine temperate fish typically experience mortality rates of between 5-20% per day. Since the duration of the egg larval and juvenile phases typically extends over several months this leads to cumulative mortalities of 98-99%. Under normal circumstances, most marine teleosts spawn huge numbers of eggs and such high mortality can be absorbed and viable populations sustained, providing that spawning stock biomass remains above a critical threshold. A consequence of these high mortality rates of pre-recruit stages is that small shifts in daily mortality rate accumulate to generate large changes in the numbers of survivors, and hence the stock size. In addition to the generation of exceptional year-classes which can dominate commercially harvested stocks for several years, shifts in the average recruits per spawner affect longer term sustainability (Clark et al. 2003, deYoung et al. 2008, Payne et al. 2009). This is why recruitment studies are considered of key importance in fisheries science.

Recruitment studies often focus on the egg and larval stages but strictly cover all life-history stages up to recruitment of the fish to the fishery. Recruitment studies can be broadly divided into three main groups: firstly the physical processes influencing egg and larval dispersal; secondly the feeding success of larvae which implicates reduced growth (and possibly starvation) as a principal factor controlling survival; thirdly mortality due to predation. Disease may also play a role. Of course none of these factors operate independently but this division forms a useful framework in which to develop a comprehensive research approach. Under a previous project, M0423 we examined the physical dispersal of plaice (Pleuronectes platessa) eggs and larvae in the Irish Sea (Fox et al. 2006), demonstrating that the interaction of larval behaviour with tidal flows is the main determinant in the distribution of larval settlement in the Irish Sea (Fox et al. 2009). M0431 has looked at historical records of zooplankton distribution and abundance, resulting in the development of models predicting the effects of physical parameters on larval growth and survival of fish species. PREDATE, by contrast, addresses the third and much less well understood area of recruitment studies, mortality due to predation.

Field studies of predation have proved challenging due to the difficulties involved in identifying partially digested fish eggs and larvae in predator stomachs (Bailey and Houde, 1989; Heath, 1992). Prey are often digested rapidly and become unidentifiable after only an hour or so following ingestion(Hunter & Kimbrell 1980) e.g. Schooley et al. 2008). Many other predators, particularly crustaceans, macerate their prey which can make visual identification of stomach contents practically impossible (e.g. Garrod and Harding, 1981; Taylor, 2004). In some studies, identification of remaining hard-parts such as otoliths and eye lenses has been used to assess predation (e.g. Nakaya et al., 2004) but visual identification of remains is always time consuming and requires either skilled analysts, or ground-truth evidence of species occurrence in the study area. Moreover, prey are sometimes abandoned following partial consumption, as has been observed for example in Crangon crangon predating on juvenile plaice (van der Veer and Bergman, 1987; Gibson et al., 1995). Visual analysis of stomach contents following partial consumption will therefore underestimate overall feeding incidence.

By contrast, molecular methods now offer a rapid and unambiguous alternative for identifying species present in gut contents (Symondson, 2002; King et al., 2008). Some studies have applied polyclonal or monoclonal antibodies (e.g.Taylor, 2004), but DNA-based methods are now more prevalent. They are easier and cheaper to develop, and allow rapid screening against the full range of prey species likely to be encountered in the field in any particular ecosystem (e.g. Agustí et al., 2003; Juen and Traugott, 2007). Although more widely applied in terrestrial systems, marine examples of DNA-based identification have included fishes (Rosel and Kocher, 2002; Smith et al., 2005), copepods (Nejstgaard et al., 2003) and appendicularians (Troedsson et al., 2007). Rapid progress has also been made in developing detection methods (e.g. Asahida et al., 1997), and more recently, real-time PCR has been applied in several predation studies. This technique has proven faster, more sensitive and offers improved specificity over conventional PCR approaches (e.g. McBeath et al., 2006). The application of DNA probes takes advantage of unique, species-specific conserved sequences of nucleotides that are present in mitochondrial or nuclear DNA, which enables the development of highly specific and sensitive assays. Several examples of successful applications of DNA-based probes include the identification and estimation of toxic algal abundance (Galluzzi et al., 2004), fish parasites and crab larval abundance in plankton samples (McBeath et al., 2006) and the identification and quantification of algae in copepod stomachs (Durbin et al., 2008; Nejstgaard et al., 2008).

A large range of potential predators are potentially responsible for fish egg and larval mortality including other fish (e.g. herring and sprat), crustacea (e.g. mysids) and gelatinous predators (e.g. the moon-jellyfish - Aurelia). Several of these predators have undergone recent large changes in abundance in UK waters. For example, since the 1980s the herring stock in the North Sea has undergone a significant recovery (e.g. Schmidt et al. 2009) and several studies suggest that the abundance of gelatinous predators may also be increasing (Attrill et al. 2007). Such changes may be linked to recent warming of the North Sea and changes in its zooplankton composition (Phillipart 2007). These changes have the potential to exert increased mortality on early life history stages of commercial fish such as cod and plaice and damage stock viability or inhibit stock recovery.

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Under M0432, we have both modified existing, and developed new molecular tools for detecting the presence of eggs and larvae of several commercial species in the stomachs of predators. The reliability of these probes was tested in the laboratory, and the technique applied in a field study aimed at identifying the full suite of predators preying on plaice eggs and larvae in the eastern Irish Sea. The work described is of direct policy relevance to Defra because it addresses a key issue in stock sustainability that is currently poorly understood and tends to be ignored in fisheries models. Our results provide new tools which can be used to study these predator-prey inter-actions. Such research supports the development of ecosystem-based management and conservation, and the results will ultimately feed into the provision of policy advice to Defra and the EU on recovery plans and long-term sustainability of stocks.

2. SCIENTIFIC OBJECTIVES

1. Modify existing Taqman probes for cod, haddock and whiting and demonstrate their specificity against other species.

2. Develop DNA based probe for plaice and demonstrate its specificity against other species. 3. Determine effects of digestion on reliability of detecting presence of target prey in predator stomachs and

intestines. 4. Modify existing coupled physical-biological model of plaice to cover cod and haddock and extend the

geographical domain. 5. Undertake field studies on predation of fish eggs and larvae in the Irish Sea. 6. Undertake field studies on predation of fish eggs and larvae in the North Sea.

3. SUMMARY OF RESEARCH RESULTS IN RELATION TO SCIENTIFIC OBJECTIVES

Modify existing Taqman probes for cod, haddock and whiting and demonstrate their specificity against other species.

Existing probes for cod haddock and whiting tested against DNA from 27 co-occurring non-teleost marine organisms and a further 28 fish species (in addition to cod, haddock and whiting)

Probes responses demonstrated to be highly species-specific.

Develop DNA based probe for plaice and demonstrate its specificity against other species

A real-time Polymerase Chain Reaction (PCR) based assay using a TaqMan probe targeting the cytochrome b gene of plaice (Pleuronectes platessa) was developed

Specificity of the plaice probe against a wide range of potential targets was confirmed.

Determine effects of digestion on reliability of detecting presence of target prey in predator stomachs and intestines

For application to crustacean predator stomach contents, two tissue preservation and two DNA extraction methods were tested. Quality of extracted DNA was comparable for both preservation and extraction techniques, and detectability was similar for both.

Levels of PCR inhibition were significant for shrimp and crab predators, but could be overcome using serial dilution to reduce the incidence of false-negative results.

The plaice probe was capable of detecting 0.001 ng of undigested plaice DNA with high repeatability. Detection of prey DNA possible for up to 24 h in crustacean predator and up to 48 h in fish predator

experiments. Twenty jellyfish fed on cod eggs all tested positive for cod DNA even when no eggs visible in gastric

cavity. The half-life detection rate(T50) for plaice DNA was ~10 h at 14-16ºC in shrimp, ~7 h at 6-10ºC in

crab and ~31h for whiting at 6-8 ºC. The T50 for cod DNA ingested by whiting was ~26 h at 8-10ºC for cod larvae, and ~31 h for cod

eggs at 6-10 ºC. Positive detection of predator DNA possible even when no material obviously present in predator gut.

Modify existing coupled physical-biological model of plaice to cover cod and haddock and extend the geographical domain

The Irish Sea life-stage dispersal model was extended to simulate the possible dispersal of eggs and larvae of 5 fish species with contrasting early life histories (cod, plaice, witch, sprat and pogge).

The original coupled physical-biological model for the Irish Sea was extended to cover the North Sea and improved to provide better predictions of stratified layer thickness and improved transport predictions in inshore areas.

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The model was run for 20 consecutive years for cod and haddock, using the generated distributions of newly spawned eggs from the 2004 international North Sea PLACES field survey, and maps of the densities of settled particles produced.

The displacements observed were much less than have been suggested by other recent studies, suggesting that inter-annual variability in wind stress cannot fully explain inter-annual variability in larval displacement derived from these observations.

The possibility and utility of predicting egg-mass dispersal using near real time (NRT) forcing data have been demonstrated in the choice of field survey area and execution of the field work.

Undertake field studies on predation of fish eggs and larvae in the Irish Sea and/or North Sea

Research cruises were undertaken on the CEFAS Endeavor in the eastern Irish Sea on a well described plaice spawning ground located off the North Wales coast in February 2008 and 2009.

The full range of predators consuming plaice eggs (and larvae) and their spatial and temporal distributions were measured through the combined use of vertical plankton sampling, acoustic sampling, trawling and stomach sampling for application of the TaqMan plaice probe.

Sprat and herring were the dominant predators of plaice eggs in both the years studied. The TaqMan probe also revealed several other species not previously identified as consumers of

plaice eggs or larvae, including mackerel, whiting, poor cod and squid. Examination of sprat stomach contents revealed that these predate preferentially on a variety of eggs

including plaice, however TaqMan was able to detect plaice DNA even when the prey were substantively digested or regurgitation had occurred.

4. MODIFICATION OF EXISTING TAQMAN PROBES FOR COD, HADDOCK AND WHITING

4.1 Introduction

Under MF0156, real-time Tacqman Polymerase chain reaction (PCR) probes were successfully applied for species identification of eggs in plankton samples from the Irish Sea, and more recently from the North Sea. The probes were able to identify visually indistinguishable early stage gadoid eggs of the cod Gadus morhua L., the haddock Melanogrammus aeglefinus L. and the whiting Merlangius merlangus L., with greater than 98% accuracy (Taylor et al., 2002; Fox et al., 2005 and 2008). These tools have subsequently been used in catch independent stock assessments (Irish Sea cod monitoring program), and in the mapping of spawning sites (Fox et al. 2008). However, although probe specificity has been proven in egg samples known to originate from gadoids, for application in more general studies of predators of fish eggs and larvae, it was first necessary to test probe specificity against a much broader range of organisms.

4.2 Materials and methods

Tissue samples were collected from 27 non-teleost marine organisms covering a broad range of phyla, along with a further 28 fish species (in addition to cod, haddock and whiting). DNA was extracted from these samples and was then tested using the cod, haddock and whiting probes (see Appendix 1).

4.3 Results

The 3 existing probes demonstrated no positive reactions against any of the other fish or non-fish DNA samples tested (for results, see Appendix 1).

4.4 Discussion

The ecological application of molecular tools relies both on the specificity and sensitivity of the PCR assay (Harwood et al., 2007). Our results demonstrate that the TaqMan probes developed for cod, haddock and whiting are highly species-specific (Appendix 1). The risk of cross-hybridisation between the TaqMan assay and DNA from other species is therefore minimal. Whereas in previous trials these probes were applied only to samples containing eggs known to be gadoid, our results here demonstrate that the existing probes are suitable for wider use in predation studies and stomach contents analysis.

5. DEVELOPMENT OF A DNA-BASED PROBE FOR PLAICE

5.1 Introduction

Previously published studies on predation of recently settled plaice juveniles have relied on visual identification of gut contents. Although plaice commence spawning earlier in the year than many other species, several species with similar eggs and larvae occur simultaneously on the same spawning and nursery grounds, especially later in the season. Discrimination of the early developmental stages of plaice from closely related species, such as dab

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and flounder, requires counts of anal and fin rays (Nichols, 1971), however this is often only possible in un-damaged specimens. Maceration by crustacean predators further complicates the identification of plaice eggs, larvae and juveniles in crustacean stomachs, although retained otoliths (ear-stones) have occasionally been used as an index (van der Veer and Bergman, 1987; Wennhage and Pihl, 2001; Nakaya et al., 2004). However, the accuracy of such an approach can be brought into question when whole fish are not generally consumed (Gibson et al., 1995). In the present study, the TaqMan approach, already successfully employed to develop the highly species specific cod, herring and whiting probes (described in section 4, above), was extended to develop a probe specific for plaice.


5.2.1 Taqman assay

The TaqMan assay is an hydrolysis probe-based method that employs real-time PCR to allow species-specific DNA identification and quantification by computing the fluorescence emission during the different cycles of the PCR. The TaqMan technique measures the emission of fluorescence during the exponential stage of the PCR (40 PCR cycles). In brief, the species-specific TaqMan probe combines with the complementary target sequence during PCR. The probe carries a fluorescent dye (reporter) and a fluorescence emission inhibitor (quencher). During each PCR replication cycle the fluorescent dye and the inhibitor are separated, allowing the emission of fluorescence that is then quantified by a detector. Fluorescence intensities in the 40 PCR cycles are used to create amplification plots of fluorescence (Rn) versus cycle number (see Figure 1), allowing calculation of the Ct (threshold cycle), the number of PCR cycles at which a significant exponential increase in fluorescence (measured as ΔRn ) is detected. The latter is directly correlated with the number of copies of target DNA present in the reaction, thereby yielding quantification of target DNA presence in the predator’s stomach.

5.2.2 Tissue samples and DNA extraction

Tissue samples from adult plaice (Pleuronectes platessa), dab (Limanda limanda) and flounder (Platichthys flesus) were obtained from locations covering close to the full geographical range of these species (Table 1). DNA extraction followed a modified salt extraction protocol (Aljanabi and Martinez, 1997). Extractions were made from 77 plaice from 4 geographic regions, 36 dab from 8 localities, and 24 flounder from 5 localities. Four hundred bp of the mitochondrial cytochrome-b (Cyt-b) gene was then PCR amplified using the universal mitochondrial primers GLU-(L)-TGACTTGAAGAACCAYCGTTG-3’ (Palumbi, 1996) and CB2-(H) AAACTGCAGCCCCTCAGAATGATATTTGTCCTCA-3’ (Kocher et al., 1989), prior to PCR purification and sequencing.

The sequences from plaice, dab and flounder, and other flatfish species retrieved from GenBank (Table 1) were aligned using Bioedit (Hall, 1999). Primers, PLA-F and PLA-R, were designed to amplify a 72 bp region of the Cyt-b gene (Figure 1) using the Primer Express 3.0 software package (Applied Biosystems). The TaqMan assay was then tested against a panel of plaice, dab and flounder adult tissue (Figure 2) and a panel of DNA extracts from a taxonomically diverse range of species (appendix 1). TaqMan assays were run on an Applied Biosystems 7900 real-time sequence detection system. Post-PCR, the results were analysed using the Sequence Detection Software version 2.3 (Applied Biosystems). The ΔRn values for each cycle and dye layer were then exported to MS Excel and processed further manually. Samples which had ΔRn values larger than the value of z*M were deemed to have a fluorescence significantly greater than the NTCs, and were therefore considered to be positive reactions.

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Table 1. Flatfish tissue samples used for the development of the plaice probe.

Figure 1 Conserved region of the mitochondrial Cytochrome b gene (120bp) in plaice from 4 different regions, compared with the same region in other flatfish species.

5.3 Results

Primers and probe were tested against plaice samples from their full geographic range (Table 1), and also against dab and flounder. There was no geographical intra-specific variation in plaice in the region of the cytochrome b gene tested. Also shown in Figure 1 are sequences from the same region from other flatfish species. There are 12 nucleotide differences between plaice and dab, and 7 between plaice and flounder. Thus ample inter-specific variation in the cytochrome b region was present to allow the design of a species-specific plaice probe.

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The TaqMan probe amplified 100% of plaice samples from different locations (Figure 2) and tested negative against all dab and flounder DNA, whereas it was positive in all plaice tests. No positive reactions were found when using the plaice probe against a panel consisting of a taxonomically wide range of samples (Appendix 1).

Figure 2. Detection of plaice DNA and non amplification of flounder and dab DNA using Taqman primers and probes. Note that lower Ct values are representative of higher quantities of target DNA.

5.4 Discussion

Our results demonstrate that the TaqMan plaice probe is highly specific, reacting positively with plaice samples from the North Sea, Irish Sea, Scottish west coast and English Channel, but failing to react to DNA from many other species from different phyla (Appendix 1). The risk of cross-hybridisation between the TaqMan assay and DNA from other species, and the risk of sequential polymorphism between populations from different localities, is therefore minimised.

6. DETERMINATION OF THE EFFECTS OF DIGESTION ON THE RELIABILITY OF GENE- PROBES

Even when eggs and larvae can be visually identified in the stomachs of predators, the period during which they remain identifiable is related to both the rate and means of digestion. Hunter and Kimbrell (1980) reported that although anchovy(Engraulis mordax) egg chorions remained identifiable for up to 8 h in anchovy stomachs, ingested larvae were unidentifiable after only 30 min. Folkvord (1993) reported that three-day old cod (Gadus morhua) larvae could only be identified in the stomachs of cannibalistic juvenile cod for 15-90 min post-ingestion. Schooley et al. (2008) reported similarly rapid digestion times for larvae of native fish species ingested by non-native fish predators (only 50% being identifiable after 30 min post consumption, reducing to 3% after 60 min).

Before application to field studies, any DNA-based probe must be tested against a range of potential prey for specificity (see section 5 above). Furthermore, experiments are needed to assess the extent to which digestion of the prey DNA reduces the effectiveness of the probe or sequencing method. In order to assess the effects of digestion on the reliability of the gene-probes on a range of potential predators with contrasting digestive systems, fish, crustacean and gelatinous predators were chosen as test subjects.


6.2.1 Whiting feeding experiments

Whiting were obtained locally and transported to the Cefas laboratory, Lowestoft, where they were transferred to tanks containing local seawater at ambient temperature, and fed ad libitum on sandeels. Initial experiments tested the voluntary uptake of fish eggs and larvae. Cod eggs were placed either inside intact portions of sandeel flesh or were incorporated into macerated sandeel flesh pellets, set with an agarose gelling agent. Feeding success between individuals was variable however, and regurgitation rates were high in fish that consumed food items, making quantification of eggs or larvae ingested difficult to track, and subsequent molecular analysis of the stomach contents produced no positive reactions. An alternative method was therefore developed whereby prey items were set in an agarose gel ‘pellet’, then introduced directly via the mouth into the stomach under mild anaesthesia (experiments conducted under Home Office licence). Stray eggs or larvae observed in the mouth, or

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Plaice

Flounder & Dab

retained in the syringe, were noted. After each procedure the fish were placed in individual tanks containing well-aerated water, to recover before being returned to their original observation tanks. Initially, two whiting were sacrificed after 2 hours, then a further 3-5 individuals per time point at varying intervals up to 48 hours post-ingestion. The stomach and intestines were removed and immediately frozen (-80°C) for later analysis using the Taqman method (Taylor et al. 2002).

6.2.2. Shrimp and crab feeding experiments

Previous laboratory and field studies have suggested that both the common shrimp Crangon crangon and the common shore crab Carcinus maenas are important predators on recently settled juvenile plaice (e.g. van der Veer and Bergman, 1987; Pihl, 1990; Burrows et al., 2001). Common shrimp and common shore crab were collected from Red Wharf Bay (Anglesey, United Kingdom) in spring 2008 and acclimatised to aquarium conditions. Shrimp were fed on ground mussel (Mytilus edulis) and crabs on whiting tissue for a minimum of 2 weeks after collection. They were then starved for 48h before feeding trials commenced (Asahida et al., 1997). Experimental animals were fed adult plaice tissue ad libitum. After 2 hours, approximately 10 individuals were preserved and the remaining animals were transferred into aquaria with clean water and no plaice tissue. Around 10 individuals were sacrificed at 6-8 time points between 0 and 24h after the end of feeding; animals were preserved either frozen (-80ºC) or in 50 ml of 80% ethanol [preservation in 100% ethanol results in tissue brittleness, making subsequent stomach dissection difficult (Passmore et al., 2006)]. The 80% ethanol was changed twice within 24 h and samples were subsequently stored at -20º C. Digestion trials were run at several temperatures for C. maenas but only one temperature for C. crangon (Table 4).

6.2.3 Jellyfish feeding experiments

Captive young Aurelia aurita (6 to 9 cm in diameter) were obtained from a public aquarium (Weymouth Sealife Centre). The jellyfish were transferred to a pseudokreisel tank (Raskoff et al, 2003) and fed with nauplii of the brine shrimp Artemia. Survival over periods longer than one week was limited, compromising the range of experimentation possible. Individual jellyfish were removed from the main holding tank and placed in individual glass vessels in approximately 1.5 l of seawater. The vessels were transferred to a constant temperature room and the temperature was gradually lowered from 13ºC to the experimental temperature of 10ºC over 24-48 h. Attempts to initiate the voluntary uptake of prey items by introducing cod eggs into the water or onto the oral arms were unsuccessful. Eggs were therefore carefully pipetted directly into the stomach of each jellyfish via the mouth. In many instances a proportion of the eggs were expelled. Jellyfish that had regurgitated nearly all of the eggs were not sampled. Four jellyfish were removed at varying time intervals up to 12 hours post-feeding and the bell diameter recorded. An area of tissue incorporating the stomach and gastric pouches was removed and viewed under a microscope. Undigested or partly digested eggs were counted and noted. The tissue was frozen at -80°C and submitted for geneprobe analysis (described above).

6.2.4 Effect of preservation and extraction method on DNA yield and purity (shrimp and crab only)

DNA yield (ng/μl) and purity indexes for the extractions were determined using a NanoDrop ND-1000 Spectrophotometer and, compared among both preservation (rapid freezing -80ºC or 80% ethanol) and extraction methods (Salt and Phenol-chloroform methods) where both salt and phenol-chloroform extractions were applied to the same stomach’s contents.

6.2.5 Investigation of PCR inhibition (shrimp and crab only)

PCR inhibitors in crab tissue extracts have been identified in previous studies (Pan et al., 2008) which can lead to a high proportion of false-negative results. In order to determine the extent of inhibition, a series of 10-fold dilutions was performed, from 1/10 to 1/100 for shrimp and to 1/1000 for crab, to the extracted DNA from those experiments. Only animals from 4 different digestion times, all of them preserved in 80% ethanol were used for the dilution test.

6.2.6 Determination of detection limit (shrimp and crab only)

Four standard dilutions were undertaken (for each preservation and DNA extraction method) to test the efficiency of the TaqMan assay to detect low quantities of plaice DNA. Plaice DNA was extracted from raw adult tissue (where PCR inhibition is not a problem). Concentration was determined using a NanoDrop ND-1000 Spectrophotometer, then serially diluted in 10-fold increments (until 1/10000) before applying the Taqman assay.

6.2.7 Determination of prey detectability over predator digestion time

Experimental data were analysed to determine how long plaice DNA remained detectable in predator stomachs. The decline in DNA detection success was calculated using the half-life detection rate (T50) which is the time after which only half of the individuals test positive for prey DNA (Greenstone and Hunt, 1993). Based on PCR

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inhibition results, for all samples testing negative for plaice, we repeated the assay using a 1/10 dilution of the DNA extract for shrimp while both 1/10 and 1/100 dilutions were assayed for crab.

6.3 Results

6.3.1 Effect of preservation and extraction method on DNA yield and purity

A comparison of salt and phenol-chloroform extraction techniques for shrimp and crab revealed that DNA yields were significantly higher using phenol-chloroform extraction compared with salt. Furthermore DNA quality was high for both preservation and extraction methods for shrimp (>1.8 in all cases) but lower in crab extracts. No differences in these patterns were noted if considering all the different digestion times together or separately

6.3.2 Investigation of PCR inhibition

Results dealing with the inhibition effects on the TaqMan assay were limited to 80% ethanol preserved animals using the digestion times where both extraction methods were applied (dilutions test). In total, 38 shrimp and 41 crab stomach extracts were subjected to 10-fold dilutions. The extent of the inhibition on plaice detectability is expressed in terms of percentage of false-negatives; a false-negative corresponds to a stomach where plaice DNA was positively detected in some or all of the dilutions but not in the undiluted (1/1) extract. Overall 7% and 25% of the shrimp stomachs presented a false-negative result for, respectively, salt and phenol-chloroform extractions, while these values increased up to 86% and 88% for crab; no apparent differences in the inhibition extent were found if considering all the different digestion times together or separately. For both species extracts it was also noted that higher DNA yields needed a higher dilution to overcome inhibition indicating that inhibition is caused by some factor extracted from the stomachs along with the DNA. Consequently in the digestion experiments, all shrimp testing negative with the TaqMan probe were diluted to 1/10 and re-tested while crab testing negative were diluted to both 1/10 and 1/100 and re-tested with the TaqMan probe.

6.3.3 Determination of minimum quantity of tissue (detection limit)

The Taqman assay was able to detect 0.001ng of plaice DNA (corresponding to a 1/10000 dilution of salt extracted DNA from frozen preserved raw plaice tissue).

6.3.4 Determination of detectability over digestion time

6.3.4.1 Whiting

In total, a series of eight experiments using a fish predator were carried out. The effect of digestion on the detectability of prey DNA using the Taqman probe was investigated at 6°C, 8°C and 10°C for both cod eggs and larvae, and at 6°C and 8°C for the eggs of plaice. Availability of plaice spawn and unsuccessful efforts to hatch plaice larvae from the eggs limited the number of experiments possible using plaice DNA. Unfortunately, it was not possible to test the cod larvae/6°C samples.

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Figure 3. Detection in whiting stomachs of a) cod eggs at 6, 8 and 10ºC, b) plaice eggs at 6 and 8 ºC and c) cod larvae at 8 and 10ºC, using TaqMan probes.

Analysis of deviance found the proportion of positive detections declined gradually with time after ingestion (P<0.001) and there is some evidence of differences in detections between the two development stages of prey (P<0.05). No significant differences between the digestibility of cod and plaice eggs were apparent, and both had an estimated half life detection rate (T50) of approximately 31 h, compared with 26 h for cod larvae. Time after ingestion accounted for significantly more variation than other factors (Table 3), with higher levels of detection being observed over longer time periods at the lower temperatures. Fish egg DNA was detectable in the majority of the whiting stomachs for up to 24 hours after feeding, and sometimes after the maximum interval of 48 hours

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(Figure 3). However, for both cod and plaice eggs, reactions varied considerably between replicates. The plots in figure 3 show that this difference was strongly dependent on one experiment where ingested larvae were still being detected 48 hours after ingestion for most individuals.

In contrast, the strength of the reactions between temperatures did not reveal a clear pattern. This may be due to differences in the DNA content between the egg batches. For cod larvae there were slightly more positive detections at 10ºC than 8ºC but the difference was neither sufficiently marked (in effect 4 fish), or over a long enough time-scale to rule out random variation being the root cause.

Visual assessment of stomach contents during the course of the digestion experiments showed that stomach fullness reduced progressively with increasing digestion times in both species from total fullness in the first hours to nearly or totally empty at the end of the experiment (Table 2). Out of 39 crab stomachs visually identified as empty (index 0, see methods), four tested positive for plaice DNA.

6.3.4.2 Shrimp and Crab

Table 2. Detectability of plaice DNA during digestion by shrimp C. crangon and crab C. maenas. Results show no. TaqMan testing +ve by treatment following application of the dilution protocol to overcome PCR inhibition.

Half-life detection rates of the target DNA (T50 values) and maximum detection times were estimated for both predator species in the different experimental setups using the dilutions described above to overcome inhibition. Plaice DNA could be amplified from shrimp and crab stomachs up to a maximum of 24 h after feeding. For shrimp extracts the T50 was ~10 h at water temperatures of 14-16º C. The effects of a wider range of temperatures were tested for crab where the half-life detection rates (T50) were ~7 h at 6-10ºC, ~ 6 h at 14-16ºC but only 2 h at 19-20ºC. The extraction method had little effect on T50s for either species.

6.3.4.3 Jellyfish

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Species Temp. (oC) Preservation Time (h) n Mean size Mean fullness Positives Positives (std. dev.) (std. dev.) Salt Phenol-

C. crangon 14-16 -80oC 0 10 4.95 (0.27) 2.8 (0.32) 10 101 10 5.1 (0.6) N/A 10 102 10 4.93 (0.59) N/A 10 N/A4 10 5.1 (0.77) 1.8 (0.64) 9 N/A6 10 4.85 (0.57) N/A 8 89 10 5.33 (0.69) 1.2 (0.32) 6 N/A18 10 4.78 (0.63) N/A 0 024 10 5.08 (0.28) 1 (0) 1 N/A

C. crangon 14-16 80% EtOH 0 10 5.25 (0.55) 2.9 (0.18) 10 101 10 5.25 (0.7) N/A 10 102 10 5.75 (0.5) N/A 10 N/A4 8 5.31 (0.69) 1.88 (0.22) 8 N/A6 10 5.35 (0.62) N/A 8 99 10 5.58 (0.84) 1.3 (0.42) 5 N/A18 8 5.31 (0.48) N/A 3 324 10 5.63 (0.55) 1 (0) 1 N/A

C. maenas 6-10 -80oC 2 7 49.57 (4.94) N/A N/A 64 6 54.83 (7.83) N/A N/A 66 7 54.14 (7.27) N/A N/A 58 7 58.86 (4.41) N/A N/A 312 6 58.67 (3.44) N/A N/A 118 6 54.33 (6.44) N/A N/A 124 6 57.17 (7.5) N/A N/A 0

C. maenas 14-16 -80oC 0 8 55.69 (6.31) 3 (0) 8 83 8 55.75 (5.56) 2.5 (0.5) 7 86 8 53.25 (8.69) 0.75 (0.56) 6 29 8 53.25 (9.5) 0.5 (0.63) 2 324 8 49 (6.38) 0.13 (0.22) 0 1

C. maenas 19-20 80% EtOH 0 10 54.25 (9.3) 2.6 (0.48) 9 91 8 56.88 (7.25) 2.88 (0.22) 7 82 10 58.95 (9.36) 2.5 (0.7) 5 53 10 55.85 (8.78) 1.6 (0.72) 4 36 10 55.4 (8.12) 0.4 (0.48) 4 412 10 55.25 (10.55) 0.5 (0.5) 1 218 10 54.4 (9.1) 0.4 (0.48) 0 024 10 53.75 (8.6) 0.7 (0.42) 1 0

From the 18 jellyfish sampled, visual inspection of the dissected material revealed at least 1 egg to be present (maximum 5) in 10 individuals, however the TaqMan analysis detected cod DNA in all 18 samples.

6.4 Discussion

A key factor for successful application of PCR is the use of appropriate tissue preservation and DNA extraction methods (e.g. Passmore et al., 2006; Lopera-Barrero et al., 2008). Although the effect of different preservation and extraction methods on the subsequent success of PCR is fairly well understood for high quality tissue, there is a paucity of reported research on protocols for degraded material, including stomach contents. Passmore et al. (2006 concluded that 80% ethanol was superior to freezing for maintaining the integrity of prey krill stomach contents, as DNA degradation by nucleases may accelerate in stomachs if not rapidly deactivated. Our results showed little difference in detectability using the TaqMan plaice assay on DNA extracted from predator stomachs which had either been rapidly frozen (-80°C) or preserved in 80% ethanol. For field sampling either preservation method appears suitable. Preservation of crustacea in 80% ethanol has the advantage of partially dehydrating the specimens making subsequent dissection easier but clearly ethanol is flammable. In addition there are ethical concerns about sampling of decapod crustaceans and the recommended procedures for working with these animals in Europe at least are currently being reviewed. Rapid freezing may be a more humane sampling method and for field-work dry-shippers which are charged with liquid nitrogen are both safe and effective.

To our knowledge, this was the first time salt extraction methods have been applied to invertebrate stomach contents and compared with phenol-chloroform extraction. For all the samples where salt and phenol-chloroform extractions were compared we obtained comparable results for DNA purity, T50 detection values and maximum TaqMan probe detection times. Because of its low toxicity, cost and speed and the comparable TaqMan results salt extraction may be a better technique to extract DNA from crustacean stomachs.

PCR inhibition has been reported in extracts from insect stomach contents (Juen and Traugott, 2006) whilst Pan et al. (2008) stated that inhibition was a significant factor in application of molecular methods for species identification of crab larvae. In our study PCR inhibition was apparent in extracts from both shrimp and crab, but was much stronger in extracts from the latter (approx 87 % of false-negatives for undiluted template). The relative size of the two types of crustacean predators (Table 2) used in our experiments may have had an effect.

Our results further demonstrate that the TaqMan probe is capable of detecting as little as 0.001 ng of raw (undigested) plaice DNA with high repeatability. This compares well with detection limits of e.g. 0.002 ng in a real time PCR assay for the identification of Anguilla japonica eggs and larvae (Minegishi et al., 2009), and 0.006 ng for a PCR based assay for stomach contents analysis in the insect genus Homalodisca (de Leon et al., 2006). Because of the high sensitivity of PCR, field sampling and subsequent laboratory analysis should include rigorous blank procedures designed to detect any cross-contamination (King et al., 2008).

A key consideration in applying either visual or molecular stomach content analysis is the impact of digestion on the prey detectability. Molecular probes for predation studies tend to target short-sequences of DNA in order to improve their effectiveness with degraded material (e.g. Hoogendoorn and Heimpel, 2001; King et al., 2008; Troedsson et al., 2009). We were able to detect plaice DNA in whiting stomachs up to 48 h after ingestion. In several incidences a positive result was even obtained with stomachs which appeared visually empty. This shows that it is possible to detect traces of target DNA after almost complete digestion or gut clearance.

Water temperature is another factor which must be taken into account as it often has a strong effect on digestion rates. Previous studies have reported prey detection up to 5 h after ingestion for stone flounder, Kareius bicoloratus fed to shrimp Crangon affinis at ~9ºC, (Asahida et al., 1997) and up to 12 h for cod (Gadus morhua) fed to mackerel (Scomber scombrus) at 10ºC (Rosel and Kocher, 2002). Unsurprisingly, TaqMan detection with time decreased with increasing water temperature as previously demonstrated in insect predator guts assayed with immunoassays (Hagler and Naranjo, 1997) and PCR (Hoogendoorn and Heimpel, 2001).

In future studies, it is worth considering that digestion rates may be further affected by meal size (Dos-Santos and Jobling, 1991), mixed diets (Andersen and Beyer, 2005) and starvation. Pre-experimental starvation is often used in laboratory studies to ensure that the predators feed when exposed to the prey (Asahida et al., 1997) but reports on the effects of pre-starvation on digestion rates and the subsequent detectability by molecular methods are conflicting (Symondson and Liddell, 1995). Furthermore, predators in the wild usually ingest a range of prey and this may reduce the amount of target tissue ingested. However, the high sensitivity of the TaqMan assay should enable detection even where only small quantities of plaice tissue have been ingested.

7. MODIFICATION AND EXTENSION OF COUPLED PHYSICAL-BIOLOGICAL MODEL

7.1 Introduction

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Many marine species produce pelagic propagules which, because of their life-history characteristics and the local hydrodynamics, can disperse considerable distances from the point of release. Distances travelled are affected by factors such as: release time and location, egg and larval stage duration, local environmental conditions and active swimming and settlement behaviours. Understanding such dispersal patterns is important for the design of effective ecosystem-conservation strategies.

Under a previous project (M0423), a coupled physical-biological model was further developed to examine the dispersion patterns of plaice eggs and larvae in the Irish Sea. Such models can provide valuable guidance in the design and execution of field programs that rely on tracking patches of eggs and larvae, whose dynamics are frequently incompletely understood. Under PREDATE, we have aimed to extend the species range of our existing model to include cod and haddock by modifying the growth, mortality and behaviour parameters in the existing model by applying supplementary life-history data from the existing literature. Furthermore, the model has been extended to cover the North Sea area, and re-written to upgrade from the underlying physical model from the Princeton Ocean Model (POM) to the General Estuarine Transport Model (GETM). Drawing on existing maps of spawning areas for cod and haddock in the Irish and North Seas (from M0151 and PLACES), the upgraded models have been applied in the examination of the transport pathways for early life history stages of cod, haddock and plaice in the Irish and North Seas. The results from this work have been applied not only in the prediction of the settlement trajectories of commercially valuable species, but also by applying the same particle-tracking techniques in real-time, we have been able to demonstrate the utility of this approach in the targeting of suitable study locations for sampling eggs and larvae, and their predators.


For the Irish Sea extensive egg distribution data based on surveys from 1995, 2000 and more recent years for the eastern Irish Sea, were available. For the North Sea, we were able to utilise egg distribution data from the international co-ordinated PLACES survey in 2004.

7.2.1 Extension of the Irish Sea life-stage dispersal model

A regional scale, coupled physical-biological model for the Irish Sea, originally developed under M0423, was used to simulate the possible dispersal of eggs and larvae of five species of fish with contrasting early life histories (cod Gadus morhua, plaice Pleuronectes platessa, witch Glyptocephalus cynoglossus, sprat Sprattus sprattus and pogge Agonus cataphractus). The hydrodynamic model was forced with meteorological data for 1995, a year when extensive plankton surveys were conducted in the Irish Sea. A particle tracking method featuring particle release (spawning) and species-dependent particle development and behaviour was then run based on flow and temperature fields from the hydrodynamical model.

7.2.2 Extension of coupled physical-biological model to cover North Sea

The original model for the Irish Sea (which used the POM, was re-written in M0432 to use GETM input, with the aim of providing better predictions of stratified layer thickness and improved transport predictions in inshore areas. For this upgrade, the biological elements of the model were largely rewritten, to facilitate increased flexibility in entering species characteristics into the model. Furthermore, the particle tracking model was parallelised to ensure optimal use of the high performance parallel computing cluster at Cefas.

A routine to automatically generate spatial and temporal distributions of newly spawned eggs from the 2004 international North Sea PLACES field survey (ICES Cooperative Research Report, No. 285, 2007) was made. Available survey data were too infrequent in time to determine temporal distributions of spawning effort, so a number of methods to distribute egg release around the mean date of observed egg presence were designed.

The model was applied to the North Sea for cod and haddock, using the generated distributions of newly spawned eggs. Maps of the densities of settled particles were produced. Furthermore, to investigate the hypothesis that cod eggs and larvae are displaced to the north in wet, warm and windy years (Rindorf & Lewy, 2006), the North Sea model was run for 20 consecutive years, using the same initial egg distributions, and the displacement of the 'centre of mass' of the larval distributions was calculated to reproduce the field-observations based results of Rindorf & Lewy (2006).

7.2.3 Near real-time prediction of egg and larval dispersal

To support the planning and conduct of the first PREDATE field survey (see section 8, below), a coupled physical-biological model was used to predict egg and larval dispersal in the survey area. The GETM model was run in forecasting mode, set up as follows: The hydrodynamics in the model were brought into a realistic state by running coarse grid meteorological reanalysis data from the European Centre for Medium-Range Weather Forecasts (ECMWF) as forcing for the years 2001-2007. For the years 2004 and onwards, river runoff data, which are also required as model forcing, were not available, so an extrapolation was made based on previous years.

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For January and February 2008 (up to several days preceding the start of the cruise), the hydrodynamical model was run using high resolution NRT meteorological forecast data received daily from ECMWF (through the British Atmospheric Data Council, BADC). From four days before the cruise to four days before the end of the cruise, the GETM model was run daily in combination with the particle tracking model to make an eight day forecast of the dispersal of eggs spawned in both the survey areas on day 1 of the cruise.

7.3 Results

7.3.1 Extension of the Irish Sea life-stage dispersal model

Modelled larval distributions and settlement areas corresponded favourably with observations from field sampling. The settlement destinations (or onset of shoaling for sprat) were affected both by their initial spawning location and by the species-specific development rates and behaviours coded into the model. Eggs and larvae typically remained within 160 km of their spawning origin, although a minority travelled up to 300 km. Even in a relatively enclosed sea such as the Irish Sea, fish eggs and larvae can be dispersed over 100s of km. The application of different vertical swimming patterns had a very strong influence on the dispersal pathways and final settlement destinations of the larvae. By contrast, rates of horizontal swimming did not appear to be effective in influencing their dispersal (for full results, see Van der Molen et al. 2007).

Figure 4. Density of particles for cod (above) and haddock (below): spawning positions (left) and settling positions (right) for 2004.

7.3.2 Extension of coupled physical-biological model to cover North Sea

The reconstructed spawning density based on the PLACES survey, and the modelled settling positions are given in Figure 4. The results show considerable transport of the eggs and larvae, and distinctly higher concentrations of settled particles in certain areas.

The positions of the 'centre of mass' of modelled settled cod larvae for the years 1983-2003 is shown in Figure 5. The modelled displacements are much less than the displacements reported by Rindorf & Lewy (2006), suggesting that interannual variability in wind stress is not fully responsible for displacements derived from the observations.

7.3.3 Near real-time prediction of egg and larval dispersal

The possibility and utility of predicting egg mass dispersal using NRT forcing data have been demonstrated at several levels. Firstly, model predictions of daily egg-dispersal were applied initially to aid choice of survey area (Dundalk Bay in the NW Irish Sea or Liverpool Bay in the SE Irish Sea). Secondly, model predictions were used to aid cruise planning before and during the cruise. The predicted daily egg distributions were e-mailed to the research vessel as compressed, low resolution black and white maps. The graphical quality was deliberately reduced to a minimum due to the limited band width available to communicate with the research vessel.

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Figure 5. Locations of modelled centre of mass of settled cod particles for the years 1983-2003 in current study compared with those from Rindorf & Lewy (2006)..7.4 Discussion

The capability to model the transport of eggs and larvae in the seas around the UK at Cefas has been greatly enhanced by the work carried out within the PREDATE project. The modelling tools have been developed from a method with fixed species characteristics associated with an older type hydrodynamic model running on single processor PC's to a much more flexible method linked to a state of the art hydrodynamic model and running on a high performance parallel computing system. The ability to use a parallel computer has made model runs with several tens of thousands of particles, and spanning several decades possible. The model is already being used successfully within other projects (A1148: van der Molen et al., in prep.; C3576), while additional projects are at proposal, planning or initial stages. The model is beginning to elucidate the various ways in which the eggs and larvae of pelagic and benthic marine specific species interact with the marine hydrodynamics to achieve unique transport pathways from spawning areas to nursery grounds.

A significant conclusion from this work is that even for well studied species such as plaice and cod there is a lack of substantial data on the behaviour of early life stages. To generate a functioning model we have had to use parameter estimates and behavioural observations from disparate systems e.g. Georges Bank. The extent to which observations for the same species can legitimately be transferred across systems is unclear as local conditions and stock differences could be significant. This provides a major challenge for the design of effective spatial management strategies if it is necessary to protect a species across its life-history stages.

8. FIELD STUDIES ON PREDATION OF FISH EGGS AND LARVAE

8.1 Introduction

Following the successful development of TaqMan probes specific for cod, haddock, whiting (see section 4) and plaice (see section 5) DNA, and the testing of these for the effects of digestion on detection efficiency by crustacean, teleost and gelatinous predators (see section 6), the final objective of the project was to prove the efficacy of these techniques under field conditions. Although visual detection of predation on plaice eggs by fish is possible (Ellis & Nash, 1997), we were interested to compare the overall rates of detection by visual and molecular methods, as regurgitation of gut contents in trawl hauls is a common problem in predation studies. For PREDATE we chose to measure predation on a temporally stable and well studied patch of plaice eggs and larvae in the eastern Irish Sea. Over two surveys on the RV Cefas Endeavour in February 2008 and 2009, each of 10 days duration (see Appendices 2 and 3), we aimed to address the following questions:1. To identify the full range of predators consuming plaice eggs and larvae from the egg patch.2. To compare the percentages of predators (at least for the main fish predators) estimated to be consuming

plaice eggs by molecular and visual approaches. 3. To examine the temporal feeding patterns of the main predators.4. To examine how the predator and prey distributions overlapped.


8.2.1 Field survey

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Two research cruises were undertaken on the CEFAS Endeavour in the eastern Irish Sea focussing on a well described plaice spawning ground located off the North Wales coast (Fox et al., 2000). The first cruise took place between 25th Feb and 2nd March 2008. Poor weather curtailed some of the planned sampling so results from this cruise were used to develop the sampling protocols and to conduct an initial evaluation of the range of predators present. The second cruise was conducted from 19th – 28th Feb 2009 and included plankton sampling to locate the egg patch, semi-pelagic trawling and dedicated acoustic grids. In addition, a limited amount of sampling using a BIONESS multi-net (Ocean Seas Instrumentation Inc. Nova Scotia, Canada) was undertaken.

On both cruises, a mini-grid of up to 12 plankton stations was sampled to locate an area with high abundance of plaice eggs within the region known to be a plaice spawning ground from previous surveys (Fox et al., 2000). In 2008 one grid was surveyed at the start of the cruise, in 2009 two grids were completed, one at the start and one towards the end of the cruise. Samples were sorted on-board for plaice eggs to gain a rapid estimation of the patch location but were subsequently fully re-sorted in the laboratory for all fish eggs and larvae (Russell, 1976). Egg and larval numbers were expressed as nos m-2 of sea surface.

8.2.2 Vertical plankton sampling

In 2009, despite technical problems, a single deployment of a BIONESS, multi-net system was made. The BIONESS comprises a frame with a 1 m square opening containing up to 10 opening/closing nets. Because of the relatively shallow water depth (33 m), only four, 300 μm mesh nets were used in this deployment. The system was deployed around 15:00 on the 27th Feb. Depth strata sampled were from 26.4 to 20.6 m, 20.6 to 13.7 m, 13.7 to 9.2 m and 9.2 m to surface. On recovery, samples were pre-sorted on-board and representative examples of organisms present frozen in well plates. The remainder of the samples were fixed in 4% sodium acetate buffered formalin and subsequently sorted and organisms identified to broad taxonomic categories in the laboratory.

8.2.3 Acoustic sampling

Data on the behaviour, distribution and abundance of sprat and herring were collected during the 2009 survey using a Simrad EK60 splitbeam echosounder operating at 3 frequencies (38, 120 and 200kHz). Acoustic data were collected continuously (day and night). Three high resolution dedicated acoustic mini grids were also surveyed at the beginning, middle and end of the survey, covering all but the eastern and western most plankton stations. The acoustic data from the three mini surveys combined was used to estimate biomass of herring and sprat. Directly after the first plankton grid was completed, a small area (7 x 10 nmi) of high pelagic fish abundance, positioned in the plankton grid was sampled continuously for four days to investigate the diurnal behaviour of clupeoids. Regular trawls were made during this period, using a sandeel trawl to investigate the composition and length frequency of the fish observed.

Acoustic data were analysed using Myriax’ Echoview post-processing software (version 4.6). Using the virtual echogram module a filter was created to remove all weaker scatterings leaving only acoustic marks associated with fish with swimbladders. A dilation filter was also applied to account for reduced beam overlap between the transducers at shallow depths. A final erosion filter aimed to maintain the original school shape and size. These techniques were adequately selective to filter clupeoid marks, including most of the loose scatterings observed at night, yet excluding weaker scatterings from other organisms. Morphology, vertical position and energetic values of schools and aggregations of sprat and herring collected during the fishing experiment were plotted against time of day to explore diurnal patterns. Vertically binned, filtered acoustic density was also plotted against time of day.

8.2.4 Trawl sampling

Fishing in 2008 used a Portuguese high-headline trawl (PHHT) and in 2009 this was supplemented with a sand-eel trawl. Tow lengths with the PHHT varied between 15 and 30 mins but were up to 50 mins with the sandeel trawl. Once landed the catch was sorted into species and catch, weights and lengths recorded. Heavy catches were sub-sampled by weight ensuring that the sub-sample was randomised and yielded at least 30 individual fish length measurements. Catch data were recorded to an ACCESS database using the Cefas electronic fisheries data capture system. Catches weights were expressed standardised to a 30 min tow. Additional individual weight and length data were collected for sprat in 2009.

8.2.5 Predator sampling for TaqMan analysis and visual analysis of gut contents

A key consideration for stomach sampling was avoidance of cross-contamination between samples. Plaice were immediately removed from the catch on landing, and were transferred to a dedicated measuring work-station which was not used to process any other species. On each haul a number of negative controls (a piece of vinyl disposable glove inserted into another glove) were mixed in with the catch in the hopper, and were processed as for real samples. After the catch had been weighed, sorted and measured, 20 to 30 individuals of each fish

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species (or up to the maximum caught for lesser abundant species) were selected across the size range present. In 2008 both the stomach and hind-guts were dissected and placed in individual plastic sample bags and immediately frozen to -20ºC. In 2009, only the stomachs were preserved. For organisms such as crabs and squid, the whole animal was frozen. Sampling was discontinued after 60 mins from the completion of catch sorting due to concerns about post-mortem degradation. Total time taken for sorting catches ranged from 30 mins to two hours depending on the catch size. Each dissection was performed with clean gloves and instruments, batches of instruments were decontaminated using Microsol detergent (Anachem, Luton, Bedfordshire, UK) between trawl hauls. In addition to the negative controls inserted into the fish hopper, swabs were taken of instruments and processing stations after cleaning and processed for TaqMan analysis.

At each trawl station 30-50 sprat (and herring) were randomly chosen from the catch and frozen. The stomachs from these specimens were subsequently thawed and the stomachs dissected. Sprat stomach contents were washed into sorting trays and examined under a low-power micrcoscope. Any identifiable fish eggs were enumerated and their diameters measured.

8.3 Results

8.3.1 Plankton surveys

Figure 6. Sea temperature, plaice egg abundance and location of trawl stations in 2009.

In 2008, the water column in the study area was well mixed with depth integrated temperatures ranging from 6.7 – 8.4ºC. Salinity ranged from 32.0 to 34.1. Plaice eggs were concentrated to the north-eastern corner of the sampling grid and the spatial distribution of later stage eggs was similar to that of the early stages. Plaice larvae were only recorded at two plankton stations with a maximum abundance of 1.6 larvae m-2. In 2008, nine trawl tows were completed using the PHHT, most of the trawl stations were located close to the area of high egg abundance but exploratory tows were also conducted to the west of the patch. Trawl hauls were based on known clear tows but some areas had to be avoided due to the presence of underwater obstructions or other shipping.

In 2009 (Fig. 6), the spatial pattern in water temperatures was similar to 2008 but up to 1.7ºC colder. The water column was thermally mixed but surface to bottom salinity differences of up to -1 were noted at several stations. Compared with 2008, plaice eggs were more evenly spread over the study area. Egg spatial distribution changed slightly during the cruise, becoming slightly more concentrated to the NW corner of the study area, but overall, the maximum abundances did not change greatly. Plaice larval abundances were low at the start of the cruise (maximum 2.3 larvae m-2) but increased slightly reaching a maximum of 7.87 larvae m-2 by the end of the cruise. The highest larval abundances were concentrated in the north-western area of the sampling grid.

Eggs of species other than plaice were abundant in both years. The majority could not be identified to species, being less than 1.1 mm in diameter and lacking oil globules or other distinguishing features. Although Russell (1976) suggests spawning typically occurs further offshore, many of these eggs may have been dab (Limanda limanda) as concentrations of their larvae are found off the North Wales coast towards the end of March (Bunn & Fox 2004, Bunn et al. 2004). Other identifiable eggs recorded included late stage cod and those of dragonets (Callionymidae), rocklings, sole (Solea solea) and sprat but all these were present at relatively low abundance.

8.3.2 Vertical distribution of plankton as determined by the BIONESSThe vertical distribution of plankton is shown in Fig. 7. Fish eggs and larvae were dominant being concentrated towards the surface. Eggs identifiable to species included plaice and sprat, which were concentrated towards the surface. Callionymus eggs and a single sandeel egg were caught in the deepest depth strata. The vast majority of the eggs were < 1.1 mm in diameter and lacked oil globules or other distinguishing visual characteristics. Fish larvae identified included Clupea harengus, Ammodytidae, Myxocephalus scorpius, Limanda limanda, Platichthys

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flesus, Gadus morhua, Merlangius merlangus, Liparis montagui, and Pleuronectes platessa. All fish larvae tended to be concentrated towards the surface. Other larger planktonic organisms recorded included amphipods, chaetognaths and ctenophores but at very low densities (< 0.5 m-3).

Figure 7. Vertical distribution of plankton from BIONESS deployment.

8.3.3 Trawling

Trawl catches are summarised in Appendix 4. Herring, sprat and whiting were recorded in nearly all the trawl hauls and dominated the catches in terms of weight. Other species caught frequently included dab, various species of squid, grey gurnard, plaice, poor cod, dragonets, mackerel, thornback ray, flounder and lesser spotted dogfish. The range of species caught in 2008 was greater than in 2009 but excluding the major species listed above, catch quantities were low. Catches with the sandeel trawl were much lower than for the PHHT which was therefore used for most of the sampling. Stomach samples were collected from the full range of species encountered. For 2009, the trawl data were combined with the acoustics data to describe the spatial distribution and make an estimate of clupeoid biomass in the study area.

8.3.3 Acoustic analysis of clupeoid density and behaviour

The sandeel trawl catches revealed that the fish community in the water-column consisted of mixed aggregations of herring, sprat and small numbers of whiting. No clear diurnal patterns were observed in the catch composition ratio at the trawling site, so no further attempt was made to extract species specific data. The average catches obtained from all sandeel trawls combined was therefore used to partition the plankton grid acoustic backscatter into relative species contributions. Herring and sprat biomass in the 370 nmi2 plankton area were estimated at 3873 (~58.7 million individuals) and 707 tonnes (105.2 million individuals) respectively. Biomass based on the high resolution mini-grids data were approximately 50% larger with 6123 (92.8 m individuals) and 1117 tonnes (166.3 m individuals) estimated for herring and sprat respectively. The distribution of clupeoids as recorded during the plankton survey showed higher densities in the west and north of the survey area. The high resolution grid confirmed the more northerly distribution, although schools were found throughout the area (Fig. 8).

Clupeoids diurnal behaviour monitored over 4 consecutive days revealed clear patterns in school shape and acoustic density (Fig.9 a-d). During daylight hours clupeoids tended to form tall, narrow schools, of high density. At dusk these schools tended to disperse into less dense aggregations thinly spread over large areas, continuing until dawn when the process reversed. Patterns in vertical water column occupancy showed that although clupeoids tended to be associated with the seabed throughout most of the cycle, several reductions in median depths were observed, most notably around midday, dusk and after midnight. This, combined with increased variance particularly around midday (but also at dusk and after midnight), suggests that some of the sprat and herring aggregations made excursions in the water-column during these periods. Diurnal behaviour patterns were generally similar to those in the detected schools, showing that the majority of clupeoids were positioned close to the seabed. Again, around midday some higher densities moved up in the water column. Although very small aggregations of clupeoids were in the water column throughout the daily cycle, slightly larger densities also gradually moved up into mid-water from dusk until midnight, after which these scatterings appear to gradually move back to the seabed. At dawn most clupeoids were again predominantly associated with the seabed.

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Figure 8. Density distribution of clupeoids based on the acoustics recorded during the mini grids (left) and plankton grid 1(right).

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8.3.4 Analysis of stomach samples by TaqMan

Overall summaries of the molecular analysis of fish stomach contents are shown in Table 3. Overall, sprat and herring showed the highest proportion of positive TaqMan responses (45% and 33% respectively in 2008, then 91% and 96% in 2009). Around one third of mackerel also tested positive with the plaice in 2008, compared with only 6% in 2009, while whiting was similar in both years at 13% and 14% respectively. However both of the latter species are much less numerous than sprat and herring.. Other species showing some response included poor cod and squid (13-20% and 9% for squid in 2008 only). Several species expected to be predominantly benthic which also gave positive results were lesser spotted dogfish, lesser weever, grey and red gurnard and a single lumpsucker in 2009.

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2008 2009Species n % POS n % POSSprat 173 44.5 537 91.1Herring 160 33.1 383 96.1Whiting 77 13 293 14.3Dab 55 0 181 0.0Flounder 23 0 63 0.0Mackerel 12 33.3 17 5.9Scaldfish 31 0.0Weaver 13 7.7 22 0.0Goby 15 0.0Common dragonette 13 0 30 0.0Dragonette DTX 2 0.0Butterfly blenny 4 0.0Grey gurnard 26 0 37 2.7Red gurnard 10 20.0Tub gurnard 8 0.0Poor cod 23 13 10 20.0Cod 2 0.0Lumpsucker 1 100.0Common dogfish 1 0.0Lesser spotted dogfish 23 4.3 10 0.0Greater sandeel 1 0.0Sandeel 4 0.0Solenette 14 0.0Thickback sole 7 0.0Lemon sole 7 0.0Dover sole 26 0.0Spotted ray 13 0.0Thornback ray 12 0 11 0.0Pogge 13 0.0

Negative controls 25 0 92 1.1

Table 3. Detection of plaice DNA in fish predator stomachs collected from the eastern Irish Sea in February 2008 and 2009, using a TAQMAN probe.

In 2009, crustaceans and molluscs were also sampled extensively, however none tested positively for plaice DNA (Table 4).

Crustaceans n % POS Molluscs n % POSHermit crab 45 0 Octopus 1 0Swimming crab 18 0 Sea snail 2 0Spider crab 20 0 Sepiola 47 0Corystes 26 0 Squid 1 0L. depurator 9 0L. holsatus 149 0Edible crab 3 0Crangon crangon 59 0Pandalus 2 0P. serratus 1 0Prawn 2 0Pink shrimp 1 0Amphipods 7 0Euphausid 8 0

Table 4. Detection of plaice DNA in crustacean and mollusc samples collected from the eastern Irish Sea in February 2008 and 2009, using a TAQMAN probe.

8.3.5 Visual analysis of sprat stomach samples

Results of stomach content analysis on sprat suggests that these fish may feed preferentially on fish eggs, although the stomachs contained more large eggs than plaice eggs. Peak feeding occurring at around midday with stomachs gradually emptying until most were empty (or contained little) around 10:00am the following day.

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8.4 Discussion

Studies on fish egg predation in a variety of locations including the Irish Sea, the North Sea and the Baltic have implicated sprat and herring as important consumers of fish eggs (e.g. Daan 1976, Köster et al. 2001). Our results support the idea that sprat and herring are very important, if not the dominant, predators of plaice eggs off the North Wales coast in both the years studied. Our results also show that several other species consume plaice eggs or larvae, including mackerel, whiting, poor cod and squid. As far as we are aware these species have not previously been identified as predators on plaice eggs or larvae (although Harding et al. (1978) reported the presence of plaice eggs in the stomachs of grey gurnard, Eurtrigla gurnadus). Except for whiting however, the abundance of these latter species off the North Wales coast was low. Because the majority of the plaice eggs occurred in the upper parts of the water column, they are most vulnerable to clupeoid predators. Fish eggs may be a valuable source of nutrition for sprat and herring at this time of year. Further research on the behaviour of these predators in the vicinity of egg concentrations is needed as changes in predator behaviour in patchy prey environments have been widely observed (e.g. Sims et al. 2008). Significant numbers of jellyfish were absent in the samples collected in 2008/09. Predation by gelatinous predators is likely to become more important for fish spawning later in the year. Overall predation by gelatinous predators may often be significantly underestimated due their fragile nature (Madin et al. 1996).

Our results provide further evidence that analysis of stomach contents using molecular tools is a valuable tool in predation studies. Despite the difficulties of sorting trawl catches, cross-contamination between samples did not occur. However, it remains a concern due to the sensitivity of the technique and great care must be taken to avoid cross-contamination and adequate controls embedded within any field sampling scheme. The TaqMan method was capable of detecting traces of target DNA even when the prey were severely digested or regurgitation had occurred. Alternatively, some of the predators may have consumed plaice larvae which were also present in the study area, albeit at lower abundance compared with the eggs. Plaice larvae are likely to be digested rapidly thus reducing the probability of detecting larval remains visually.

The molecular approach is also a powerful tool when applied to predators which macerate their prey e.g. euphausiids (Theilacker et al. 1993). In the present study euphausiids were not abundant but in the deeper parts of the Irish Sea they are much more common (Mauchline 1984). Predation by euphausiids may therefore be significant in the deep parts of the western Irish Sea which lie adjacent to important fish spawning grounds (Fox et al. 2000). We conclude that because the molecular approach is relatively rapid, highly specific and sensitive it is ideally suited to rapid screening of large numbers of stomachs collected from a range of potential predators in order to identify key target species.

Our results support and extend the conclusions of (Daan 1976) from the southern North Sea and of Ellis and Nash (1997) from the Irish Sea. Overall, sprat and herring appear to be a major source of plaice egg (and possibly larval) mortality in these areas. Both sprat and herring are predominantly plankton feeders but may switch to larger prey, such as fish eggs, if they are abundant or if alternative prey are scarce (Daan 1976). As plaice are one of the earliest species to spawn in the year, predation by sprat and herring may be especially important as the abundance of other potential predators such as jellyfish is generally low at this time. Despite the likely importance of predation by clupeoids, links between their abundance and subsequent year-class strength of other fish species have only been firmly established in the Baltic (Köster et al. 2001). This may reflect the fact that the Baltic has a relatively simple food-web so interactions may be easier to discriminate. However, if the present conclusions are supported by further research, predation by sprat and herring on the early stages of other fish such as cod may need to be taken into account when evaluating stock re-building strategies in the Irish Sea and other areas (Köster & Möllmann 2000, Kelly et al. 2006)

9. FUTURE WORK

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Application of genetic probes to detect predation of plaice eggs and larvae in the Irish Sea has already demonstrated two important features. Firstly, we have shown that solid material need not be present within the predator stomach in order to detect predation – it was possible to detect prey DNA from the mucous lining of the sampled stomachs. This has implications for feeding studies which directly examine stomach contents. Given the tendency of many fish species to regurgitate their stomach contents on capture, the use of genetic probes potentially provides a means to estimate the level of under-reporting of predation in such studies. Secondly, the successful detection of plaice DNA in non-fish predators (e.g. squid) may prove an effective means of monitoring predation in communities currently being affected by climate change, in which non-fish predators have frequently been observed to gain increased presence. The techniques developed under PREDATE are also being applied in a study of predation on settled flatfishes funded by the Basque government, indicating wider outside interest in the uptake of these methods.

Field work carried out under PREDATE was limited to just two 10 day surveys, one of which was negatively impacted by adverse weather conditions. The work undertaken therefore represents “proof of concept”, where we have demonstrated our ability to model egg dispersion in real-time, to sample and map predator communities, and to describe predator-prey dynamics. Given the failure of the western Irish Sea cod stock to recover following 8 years of protection and a recovery plan, a next logical step would be to investigate whether the the same species predate the early-life history stages of Irish Sea (and North Sea) cod, so as to eliminate this or otherwise as a significant factor in cod stock recovery. Since early stage cod eggs cannot be identified visually, the use of the molecular approach pioneered in this study will be essential.

The molecular approach as described does not yet provide information on the number of eggs or larvae, nor on the developmental stages of the prey consumed. Unless eggs and larvae of a target prey are spatially well separated e.g. due to advection, it will is not currently possible to determine which stage is being consumed. However progress with quantifying the amount of ingested prey may be possible as the real-time PCR method is quantitative. For such work, a better understanding of digestion rates and diurnal feeding patterns would be necessary. However if, as has shown to be the case for plaice, the majority of predation on cod eggs and larvae proved to derive from clupeoids, such research could focus on these specific predators. As well as the more general quantification issue, future research will be required to quantify the fraction of early life stage mortality accounted for by clupeoid predation and to examine whether the status of the sprat and herring stocks can be linked to subsequent plaice year-class strength (Daan 1976). Further progress in the design of effective conservation measures for species or communities may need to take account of these predator-prey interactions, especially when the abundance of predators or prey has undergone large spatial and/or temporal changes.

Finally, by using coupled physical-biological models, we have been able to make increasingly sophisticated, multi-decadal predictions of the early life-history dispersal trajectories of marine fish. In addition to supporting future field studies, we are using these models to test hypotheses concerning long-term environmental effects on cod egg and larval dispersal in the North Sea, where the settlement patterns of juvenile gadoids are not well understood. Although we can predict the settlement patterns of these fish, there are currently few data with which to validate our observations, suggesting a requirement for future supporting field surveys.

References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.

10. LIST OF PUBLICATIONS

van der Molen, J, Rogers, SI, Ellis, JR, Fox, CJ, McCloghrie, P, 2007. Dispersal patterns of the eggs and larvae of spring-spawning fish in the Irish Sea, UK. Journal of Sea Research. 58 (4), pp. 313-330.

van der Molen, J, Ellis, JR, Rogers, SI, 2010. Potential inter-connect of potential Marine Protected Areas in the North Sea: a model study. Manuscript in preparation.

Albaina, A, Taylor, M, Fox, CJ, 2010. Predation impact on juvenile Plaice (Pleuronectes platessa) in its nursery ground (Ardmucknish Bay, Scotland; 2008-2009); field application of a real-time PCR based assay. Manuscript in preparation.

Albaina, A, Fox, CJ, Taylor, N, Hunter, E, Maillard, M, Taylor, MI, 2010. A TaqMan real-time PCR based assay to detect predation of plaice (Pleuronectes platessa L.) DNA by the brown shrimp (Crangon crangon L.) and the shore crab (Carcinus maenas L.) – Assay development and validation. Journal of Experimental Marine Biology and Ecology. In revision.

Fox, CJ, Taylor, M, Milligan, SM, van der Kooij, J, Taylor, N, Hunter, E, 2010. The application of molecular probes to investigate the predation of plaice early life stages in the eastern Irish Sea. Marine Ecology Progress Series In prep

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11. ADDITIONAL REFERENCES CITED IN THE REPORT

Agustí, N, Shayler, , Harwood, JD, Vaughan, IP, Sunderland, KD, Symondson, WOC, 2003. Collembola as alternative prey sustaining spiders in arable ecosystems: prey detection within predators using molecular markers. Mol. Ecol. 12, 3467-3475.

Aljanabi, SM, Martinez, I, 1997. Universal and rapid salt-extraction of high quality genomic DNA for PCR based techniques. Nucleic Acids Res. 25, 4692-4693.

Andersen, NG, Beyer, JE, 2005. Gastric evacuation of mixed stomach contents in predatory gadoids: an expanded application of the square root model to estimate food rations. J. Fish Biol. 67, 1413-1433.

Asahida, T, Yamashita, Y, Kobayashi, T, 1997. Identification of consumed stone flounder, Kareius bicoloratus (Basilewsky), from the stomach contents of sand shrimp, Crangon affinis (De Haan) using mitochondrial DNA analysis. J. Exp. Mar. Biol. Ecol. 217, 153-163.

Attrill, MJ, Wright J, Edwards, M, 2007. Climate related increases in jellyfish frequency suggest a more gelatinous future for the North Sea. Limnol. Oceanogr. 52, 480–485.

Bailey, KM, Houde, ED, 1989. Predation on eggs and larvae of marine fishes and the recruitment problem. Adv. Mar. Biol. 25, l-83.

Bunn, N, Fox, CJ, 2004. Spring plankton surveys of the Irish Sea in 2000: Hydrography and the distribution of fish eggs and larvae, Science Series Data Report 41, The Centre for Environment, Fisheries and Aquaculture Science, Lowestoft, p 131.

Bunn, N, Fox, CJ, Nash, RDM, 2004. Spring plankton surveys in the eastern Irish Sea in 2001, 2002 and 2003: Hydrography and the distribution of fish eggs and larvae, Science Series Data Report 42, The Centre for Environment, Fisheries and Aquaculture Science, Lowestoft, p 214.

Burrows, MT, Gontarek, SJ, Nash, RDM., Gibson, RN, 2001. Shrimp predation on 0-group plaice: contrasts between field data and predictions of an individual-based model. J. Sea Res. 45, 243-254.

Clark, RA, Fox ,CJ, Viner, D, Livermore, M, 2003. North Sea cod and climate change - modelling the effects of temperature on population dynamics. Global Change Biol. 9:1669-1680.

Daan, N, 1976. Some preliminary investigations into predation on fish eggs and larvae in the southern North Sea. ICES CM 1976/L:15:12pp.

deYoung, B, Barange, M, Beaugrand, G, Harris, R, Perry, RI, Scheffer, M, Werner, F, 2008. Regime shifts in marine ecosystems: detection, prediction and management. Trends in Ecology and Environment 23:402-409.

Dos-Santos, J, Jobling, M, 1991. Factors affecting gastric evacuation in cod, Gadus morhua L., fed single meals of natural prey. J. Fish Biol. 38, 697-713.

Durbin, EG, Casas, MC, Rynearson, TA, Smith, DC. 2008. Measurement of copepod predation on nauplii using qPCR of the cytochrome oxidase I gene. Mar. Biol. 153, 699–707.

Ellis, T, Nash, RDM 1997. Predation by sprat and herring on pelagic fish eggs in a plaice spawning area in the Irish Sea. J. Fish Biol. 50:1195-1202.

Folkvord, A, 1993. Prey recognition in stomachs of cannibalistic juvenile cod (Gadus morhua L.). Sarsia 78, 97-100.

Fox, C, McCloghrie, P, Young, EF, Nash, RDM, 2006. The importance of individual behaviour for successful settlement in juvenile plaice - a modelling and field study in the eastern Irish Sea. Fish. Oceanogr. 15:301-313.

Fox, CJ, McCloghrie, P, Nash, RDM, 2009. Potential transport of plaice eggs and larvae between two apparently self-contained populations in the Irish Sea. Est. Coast. Shelf. Sci. 81:381-389.

Fox, CJ, O'Brien, CM, Dickey-Collas, M, Nash, RDM, 2000. Patterns in the spawning of cod (Gadus morhua L.) sole (Solea solea L.) and plaice (Pleuronectes platessa L.) in the Irish Sea as determined by generalised additive modelling. Fish. Oceanogr. 9:33-49.

Fox, CJ, Taylor, MI, Pereyra, R, Villasana-Ortiz, MI, Rico, C, 2005. TaqMan DNA technology confirms likely over-estimation of cod (Gadus morhua L.) egg abundance in the Irish Sea: implications for the assessment of the cod stock and mapping of spawning areas using egg based methods. Mol. Ecol.14, 879-884.

Fox, CJ, Taylor, MI, Dickey-Collas, M, Fossum, P, Kraus, G, Rohlf, N, Munk, P, van Damme, CJG, Bolle, LJ, Maxwell, DL, Wright, PJ, 2008. Mapping the spawning grounds of North Sea cod (Gadus morhua) by direct and indirect means. Proc. R. Soc. Lond. B 275, 1543-1548.

Garrod, C, Harding, D, 1981. Predation by fish on the pelagic eggs and larvae of fishes spawning in the west central North Sea in 1976. ICES CM 1981/I: 11, 6pp.

Gibson, RN, Yin, MC, Robb, L, 1995. The behavioural basis of predator-prey size relationships between shrimp (Crangon crangon) and juvenile plaice (Pleuronectes platessa). J. Mar. Biol. Ass. U. K. 75, 337-349.

Greenstone, MH, Hunt, JH, 1993. Determination of prey antigen half-life in Polistes metricus using a monoclonal antibody-based immunodot assay. Entomol. Exp. Appl. 68, 1–7.

Hagler, JR, Naranjo, SE, 1997. Measuring the sensitivity of an indirect predator gut content ELISA: detectability of prey remains in relation to predator species, temperature, time and meal size. Biol. Control 9, 112–119.

Hall, TA, 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids. Symp. Ser. 41, 95-98.

Harding, D, Nichols, JH, Tungate, DS, 1978. The spawning of plaice (Pleuronectes platessa L.) in the southern North Sea and English Channel. Rapp. P-V Réun. Cons. Int. Explor. Mer. 172:102-113.

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Harwood, JD, Desneux, N, Yoo, HJS, Rowley, DL, Greenstone, MH, Obrycki, JJ, O’Neil, RJ, 2007. Tracking the role of alternative prey in soybean aphid predation by Orius insidiosus: a molecular approach. Mol. Ecol. 16, 4390–4400.

Heath, MR, 1992. Field investigations of the early life stages of marine fish. Adv. Mar. Biol. 28, 1-174.

Hoogendoorn, M, Heimpel, GE, 2001. PCR-based gut content analysis of insect predators: using ribosomal ITS-1 fragments from prey to estimate predation frequency. Mol. Ecol. 10, 2059–2067.

Hunter, JR, Kimbrell, CA, 1980. Egg cannibalism in the northern anchovy, Engraulis mordax. Fish. Bull. 78, 811-816.

Juen, A, Traugott, M, 2006. Amplification facilitators and multiplex PCR: tools to overcome PCR-inhibition in DNA-gut-content analysis of soil-living invertebrates. Soil Biol. Biochem. 38, 1872–1879.

Juen, A, Traugott, M, 2007. Revealing species-specific trophic links in soil food webs: molecular identification of scarab predators. Mol. Ecol. 16, 1545–1557.

Kelly, CJ, Codling, EA, 2006. The Irish Sea cod recovery plan: some lessons learned. ICES J. Mar. Sci. 63:600-610.

King, RA, Read, DS, Traugott, M, Symondson, WOC, 2008. Molecular analysis of predation: a review of best practice for DNA-based approaches. Mol. Ecol. 17, 947–963.

Kocher, TD, Thomas, WK, Meyer, A, Edwards, SV, Pääbo, SF, Villablanca, FX, Wilson, AC, 1989. Dynamics of mtDNA evolution in animals: amplification and sequencing with conserved primers. Proc. Natl. Acad. Sci. USA 86, 6196–6200.

Köster, FW, Möllman, C, Neuenfeldt, S, St. John, MA, Plikshs, M, Rüdiger, V, 2001. Developing Baltic cod recruitment models. II. Incorporation of environmental variability and species interaction. Can. J. Fish. Aquat. Sci. 58:1534-1556.

Köster, FW, Möllmann, C, 2000. Trophodynamic control by clupeid predators on recruitment success in Baltic cod? ICES J. Mar. Sci. 57:310-323.

de León, JH, Fournier, V, Hagler, JR, Daane, KM, 2006. Development of molecular diagnostic markers for sharpshooters Homalodisca coagulata and Homalodisca liturata for use in predator gut content examinations. Entomol. Exp. Appl. 119, 109–119.

Lopera-Barrero, NM, Povh, JA, Ribeiro, RP, Gomes, PC, Jacometo, CB, da Silva-Lopes, T, 2008. Comparison of DNA extraction protocols of fish fin and larvae samples: modified salt (NaCl) extraction. Cien. Inv. Agr. 35(1), 65-74.

Madin, LP, Bollens, SM, Horgan, E, Butler, M, Runge, J, Sullivan, BK, Klein-Macphee, G, Durbin, E, Durbin, AG, van Keuren, D, Plourde, S, Bucklin, A, Clarke, EM, 1996. Voracious planktonic hydroids: unexpected predatory impact on a coastal marine ecosystem. Deep-Sea Research II 43:7-8.

Mauchline, J, 1984. Euphausiid, Stomatopod and Leptostracan crustaceans: keys and notes for the identification of the species, 30, Linnean Society of London, Estuarine and Brackish-water Sciences Association.

McBeath, AJA, Penston, MJ, Snow, M, Cook, PF, Bricknell, IR, Cunningham, CO, 2006. Development and application of real-time PCR for specific detection of Lepeophtheirus salmonis and Caligus elongatus larvae in Scottish plankton samples. Dis. Aquat. Org. 73, 141–150.

Minegishi, Y, Yoshinaga, T, Aoyama, J, Tsukamoto, K, 2009. Species identification of Anguilla japonica by real-time PCR based on a sequence detection system: a practical application to eggs and larvae. ICES J. Mar. Sci. 66, 1915–1918.

Nakaya, M, Takatsu, T, Nakagami, M, Joh, M, Takahashi, T, 2004. Spatial distribution and feeding habits of the shrimp Crangon uritai as a predator on larval and juvenile marbled sole Pleuronectes yokohamae. Fish. Res. 70, 445-455.

Nejstgaard, JC, Frischer, ME, Raule, CL, Gruebel, RC, Kohlberg, KE, Verity, PG, 2003. Molecular detection of algal prey in copepod guts and faecal pellets. Limnol. Oceanogr. Methods 1, 29-38.

Nejstgaard, JC, Frischer, ME, Simonelli, P, Troedsson, C, Brakel, M, Adiyaman, F, Sazhin, AF, Artigas, LF, 2008. Quantitative PCR to estimate copepod feeding. Mar. Biol. 153, 565–577.

Nichols, JH, 1971. Pleuronectidae. International Council for the Exploration of the seas, Fiches d'identification des oeufs et larves de poissons, 4-6, 18 pp.

Palumbi, SR, 1996. Nucleic acids II: The polymerase chain reaction. In: Hillis, D.M., Moritz, C., Mable, B.K. (Eds.) Molecular Systematics. Sinauer, Sunderland, Massachusetts, pp. 205–247.

Pan, M, McBeath, AJA, Hay, SJ, Pierce, GJ, Cunningham, CO, 2008. Real-time PCR assay for detection and relative quantification of Liocarcinus depurator larvae from plankton samples. Mar. Biol. 153, 859–870.

Passmore, AJ, Jarman, SN, Swadling, KM, Kawaguchi, S, McMinn, A, Nicol, S, 2006. DNA as a dietary biomarker in Antarctic krill, Euphausia superba. Mar. Biotechnol. 8, 686–696.

Payne, MR, Hatfield, EMC, Dickey-Collas, M, Falkenhaug, T, Gallego, A, Gröger, J, Licandro, P, Llope, M, Munk, P, Röckmann, C, Schmidt, JO, Nash, RDM, 2009. Recruitment in a changing environment: the 2000s North Sea herring recruitment failure. ICES J. Mar. Sci. 66:272-277.

Philippart, CJM (Ed.) 2007. Impacts of climate change on the European marine and coastal environment: ecosystems approach. ESF MarineBoard Position Paper, 9. European Science Foundation, Marine Board: Strasbourg, France. ISBN 2-912049-63-6. 82 pp.

Pihl, L, 1990. Year-class strength regulation in plaice (Pleuronectes platessa L.) on the Swedish west coast. Hydrobiologia 195, 79–88.

Platt, T, Sathyendranath, S, Fuentes-Yacom, C, 2007. Biological oceanography and fisheries management: perspective after 10 years. ICES J. Mar. Res. 64:863-869.

SID 5 (Rev. 3/06) of 28

Pommeranz, T, 1981. Observations on the predation of herring (Clupea harengus L.) and sprat (Sprattus sprattus L.) on fish eggs and larvae in the southern North Sea. Rapp. P-V Réun. Cons. Int. Explor. Mer 178:402-404.

Raskoff, KA, Sommer, FA, Hamner, WM, Cross, KM, 2003. Collection and culture techniques for gelatinous zooplankton. Biol. Bull. 204, 68-80.

Rindorf, A, Lewy, P, 2006. Warm, windy winters drive cod north and homing of spawners keeps them there. J. Appl. Ecol. 43: 445–453.

Rosel, PE, Kocher, TD, 2002. DNA-based identification of larval cod in stomach contents of predatory fishes. J. Exp. Mar. Biol. Ecol. 267, 75-88.

Russell, FS, 1976. The Eggs and Planktonic Stages of British Marine Fishes, Academic Press, London.

Schmidt, JO, Van Damme, CJG, Rockmann, C, Dickey-Collas, M., 2009. Recolonisation of spawning grounds in a recovering fish stock: recent changes in North Sea herring. Scientia Marina 73, 153-157.

Schooley, JD, Karam, AP, Kesner, BR, Marsh, PC, Pacey, CA, Thornbrugh, DJ, 2008. Detection of larval remains after consumption by fishes. Trans. Am. Fish. Soc. 137, 1044-1049.

Sims, DW, Southall, EJ, Humphries, NE, Hays, GC, Bradshaw, CJ, Pitchford, JW, James, A, Ahmed, MZ, Brierley, AS, Hindell, MA, Morritt, D, Musyl, MK, Righton, D, Shepard, EL, Wearmouth, VJ, Wilson, RP, Witt, MJ, Metcalfe, JD, 2008. Scaling laws of marine predator search behaviour. Nature 451:1098-1102.

Symondson, WOC, Liddell, JE, 1995. Decay rates for slug antigens within the carabid predator Pterostichus melanarius monitored with a monoclonal antibody. Entomol. Exp. Appl. 75, 245–250.

Symondson, WOC, 2002. Molecular identification of prey in predator diets. Mol. Ecol. 11, 627-641.

Taylor, MI, Fox, CJ, Rico, I, Rico, C, 2002. Species-specific TaqMan probes for simultaneous identification of cod (Gadus morhua L.), haddock (Melanogrammus aeglefinus L.) and whiting (Merlangius merlangus L.). Mol. Ecol. Notes 2, 599-601.

Taylor, DL, 2004. Immunological detection of winter flounder (Pseudopleuronectes americanus) eggs and juveniles in the stomach contents of crustacean predators. J. Exp. Mar. Biol. Ecol. 301, 55-73.

Theilacker, GH, Lo, NCH, Townsend, AW, 1993. An immunochemical approach to quantifying predation by euphausiids on the early life stages of anchovy. Mar. Ecol. Prog. Ser. 92:35-50.

Troedsson, C, Frischer, ME, Nejstgaard, JC, Thompson, EM, 2007. Molecular quantification of differential ingestion and particle trapping rates by the appendicularian Oikopleura dioica as a function of prey size and shape. Limnol. Oceanogr. 52, 416–427.

Troedsson, C, Simonelli, P, Nägele, V, Nejstgaard, JC, Frischer, ME, 2009. Quantification of copepod gut content by differential length amplification quantitative PCR (dla-qPCR). Mar. Biol. 156, 253–259.

van der Veer, HW, Bergman, MJN, 1987. Predation by crustaceans on a newly settled O-group plaice Pleuronectes platessa population in the western Wadden Sea. Mar. Ecol. Prog. Ser. 35, 203-215.

Wennhage, H, Pihl, L, 2001. Settlement patterns of newly settled plaice (Pleuronectes platessa L.) in a non-tidal Swedish fjord in relation to larval supply and benthic predators. Mar. Biol. 139, 877–889.

12. LIST OF PRESENTATIONS

J. van der Molen, C.J. Fox and P. McCloghrie “Modelling the Dispersal of Eggs and Larvae of Plaice in the Southern North Sea; Workshop on advancements in modelling physical-biological interactions in fish early life history”. Nantes, France, 3 April 2006 – 5 April 2006

Aitor Albaina,Taylor MI, Fox CJ, Taylor N. “First steps in order to quantify predation on plaice ( Pleuronectes platessa) nursery grounds using molecular markers”. Seventh International Flatfish Symposium, Sesimbra, Portugal, November 2008.

Fox, C., Taylor, M., Taylor, N., van der Kooij, J., Milligan S. and Hunter, E. “Describing the dynamics of predation in a changing predator landscape”. Fish and Climate Change, Belfast, July 2010.

SID 5 (Rev. 3/06) of 28

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