FISHING FOR A FEEDING FRENZY

Transcription

1 UNIVERSITY OF GHENT- FACULTY OF SCIENCES RESEARCH GROUP MARINE BIOLOGY ACADEMIC YEAR FISHING FOR A FEEDING FRENZY Effect of Shrimp Beam Trawling on the diet of Dab and Plaice in Lanice conchilega habitats Submitted by MARIA INÊS COELHO MEIRELES RIBEIRO PROMOTER: Prof. Dr. Ann Vanreusel SUPERVISORS: Jochen Depestele and Jozefien Derweduwen Master thesis submitted for the partial fulfilment of the title of MASTER OF SCIENCE IN MARINE BIODIVERSITY AND CONSERVATION Within the International Master of Science in Marine Biodiversity and Conservation EMBC+

2 No data can be taken out of this work without prior approval of the thesis promoter Prof. Dr. Ann Vanreusel and supervisors Jochen Depestele and Jozefien Derweduwen

3 TABLE OF CONTENS LIST OF FIGURES... 3 LIST OF TABLES... 4 EXECUTIVE SUMMARY...5 ABSTRACT INTRODUCTION MATERIALS AND METHODS EXPERIMENTAL DESIGN STUDY AREA AND FISHING GEARS FISH CONTENT ANALYSIS DATA ANALYSIS: EFFECT OF TRAWLING ON THE DIET OF DAB AND PLAICE RESULTS DAB UNIVARIATE DIETARY INDICES MULTIVARIATE ANALYSIS Biomass Abudance PLAICE UNIVARIATE DIETARY INDICES UNIVARIATE ANALYSIS Biomass Abudance COMPARISON OF DAB AND PLAICE s DIET ACROSS TIME UNIVARIATE DIETARY INDICES UNIIVARIATE ANALYSIS Biomass

4 Abudance DISCUSSION UNACCOUNTED VARIABILITY OF FEEDING ACTIVITY DAB DIET AFTER FISHING PLAICE DIET AFTER FISHING COMPARISON BETWEEN DAB AND PLAICE s DIET AFTER FISHING CONCLUSION ACKOWLEDGMENTS REFERENCES APPENDIX

5 LIST OF FIGURES Fig 1. A schematic overview of all the hauls performed over time, the vessel type, the vessel name and the type of gear....9 Fig 2. Location of the study area (blue), Box Fig 3. Map of the study area bathymetry (black polygon). Green as shallow mud; Brown as shallow coarse and mixed sediment; Yellow as shallow sands 11 Fig 4. Total number of complete stomachs sampled by time step, time of sampling and name of the vessel...12 Fig 5.Stomach Fullness Index (SFI) (±SE) and Stomach Vacuity Index (%V) for Dab before (T0) and after fishing (T1, T2 and T3)...17 Fig 6. Mean number of prey species per stomach (S) and Shannon-Wiener Index (H ) for Dab before (T0) and after fishing (T1,T2 and T3). 18 Fig 7. Frequency of occurrence (%F) of higher taxon on the diet of Dab in each time step...19 Fig 8. Percentage Index of Relative Importance (%IRI) of higher taxon on the diet of Dab in each time step. 19 Fig 9. Mean prey biomass by higher taxon and by time (T0- before fishing; T1, T2 and T3 after fishing) for Dab. 21 Fig 10. Mean prey abundance by higher taxon and by time (T0- before fishing; T1, T2 and T3 after fishing) for Dab. 22 Fig 11. Stomach fullness (SFI) (±SE) and Stomach Vacuity (%V) indices for Plaice before (T0) and after fishing (T1, T2 and T3)..23 Fig 12. Mean number of prey species per stomach (S) (±SE) and Shannon-Wiener Index (H ) (±SE) for Dab before (T0) and after fishing (T1, T2 and T3). 24 Fig 13. Frequency of occurrence of higher taxon on the diet of Plaice to all time steps.25 Fig 14. Percentage Index of Relative Importance (%IRI) by higher taxon on the diet of Plaice to all time steps..25 Fig 15. Mean prey biomass by higher taxon and by time (T0 before fishing; T1,T2,T3 after fishing) for Plaice. 27 Fig 16. Mean prey abundance by higher taxon and by time (T0 before fishing; T1,T2,T3 after fishing) for Plaice. 28 Fig 17. Stomach fullness (SFI) (±SE) for Dab (Green) and Plaice (Blue) before (T0) and after fishing (T1, T2 and T3). 29 Fig 18. Frequency of occurrence (%F) of higher taxon before (T0) and after fishing (T1, T2 and T3) for Dab and Plaice..30 Fig 19. Percentage Index of Relative Importance (%IRI) of higher taxon before (T0) and after fishing (T1, T2 and T3) for Dab and Plaice..30 Fig 20. MDS-plot for prey biomass by time (T0,T1, T2 and T3 Different symbols) and by species (Dab Green and Plaice Blue). 32 3

6 Fig 21. MDS-plot for prey abundance by time (T0,T1, T2 and T3 Different symbols) and by species (Dab Green and Plaice Blue). 33 LIST OF TABLES Table 1. Study area coordinates (Box9)..11 Table 2. ANOSIM-test with the p-values for the differences between Time steps for prey biomass for Dab. (Global R = 0.037)...20 Table 3. ANOSIM-test with the p-values for the differences between Time steps for prey abundance for Dab. (Global R = ).21 Table 4. ANOSIM-test with the p-values for the differences between Time steps for prey biomass for Plaice. (Global R = 0.135) 26 Table 5. ANOSIM-test with the p-values for the differences between Time steps for prey abundance for Plaice. (Global R = 0.087) 28 Table 6. Average similarities, from SIMPER, between Dab and Plaice s diet by time and for the prey species biomass and abundance..31 4

7 EXECUTIVE SUMMARY Beam-trawl flatfish fisheries are one of the most important fishery in the North Sea. Nevertheless, it is known that beam-trawling has a significant impact on the seabed, on physical properties and on the benthic fauna inhabiting the substrate. The benthic animals may be killed directly by the passage of the trawl or they may be caught by the gear and discarded afterwards (dead or alive). This study investigated the changes in the diet of two flatfish, Dab (Limanda limanda) and Plaice (Pleuronectes platessa), due to fishing disturbance by a shrimp beam trawl in Lanice conchilega habitats. Lanice conchilega is a tube-worm, known as a bio-engineer. This tube-polychaete provides an important habitat for benthic communities, mainly by creating a three-dimensional structure in the seabed. Two hauls of 20 minutes were carried out by an 11-m commercial shrimp beam trawler with a fishing intensity of 150% (based on swept area). The gear was hauled after the first passage and the stomachs were collected, i.e. without prior fishing disturbance (T0). Consecutive hauls were carried out on-board of a research vessel with a sampling beam trawl (2 m long, 3 m wide) and fish stomachs were sampled at three different times following commercial fishing, namely after about 5 h (T1), 10 h (T2) and 20 h (T3). Afterwards, in the lab, the stomach contents were extracted, identified, weighted, dried and muffled. In order to investigate the diet composition, several dietary indices were calculated. Statistical univariate and multivariate analysis were carried out for all indices and for the biomass and abundance estimates of prey species found in the stomachs of Dab and Plaice. The statistical analysis were first carried out for each fish species separately, followed by a comparative analysis between both. Results showed a higher stomach fullness and prey species biomass at T1 for Dab and Plaice. For Dab, no significant differences in stomach fullness were found for the different time steps. For Plaice however, the stomach fullness at T1 and T3 was significantly different from each other and from the other time steps. Additionally, an increase of the biomass of prey species was observed in Dab and Plaice. Both Dab and Plaice fed upon annelids, but more polychaete species were found in a higher abundance in Plaice stomachs then in Dab. Moreover, bivalves were more present in Plaice stomachs while crustaceans were observed in higher abundances and biomass in Dab. The diets of Dab and Plaice were more similar at T1 and T2, although 5

8 the diet composition of Dab and Plaice stomachs differed significantly at each time step. We conclude that shrimp beam trawl fishery has an acute effect on both fish species diet but the effect was rapidly diluted over time. ABSTRACT This study investigated the effect of shrimp beam trawl disturbance on the diet of two abundant flatfish species Dab (Limanda limanda) and Plaice (Pleuronectes platessa) in a Lanice conchilega habitat in the Belgium part of the southern North Sea. Lanice conchilega is a tubeworm known as a bio-engineer, because it structures its habitat and enhances the biodiversity of inhabiting benthic communities. Stomachs contents of Dab and Plaice were collected at four different time steps in relation to fishing disturbance: T0 (before fishing), T1, T2 and T3 (within 24 h after fishing). Before fishing, the diet of Plaice and Dab was mainly composed of annelids including L. conchilega, and complemented with crustaceans for Dab and bivalves for Plaice. Directly after fishing, the diets of Dab and Plaice were more similar with increasing contributions of annelids. In addition, prey biomass in the stomachs of both fish species and the stomach fullness was significantly higher immediately after fishing (T1). The increased contribution of annelids in the diet of both fish species was a clear effect of trawling disturbance but was rapidly (at T3) reduced to pre-fishing diet compositions. 6

9 1. INTRODUCTION Beam-trawling is one of most widely distributed fisheries in the southern North Sea (Anon, 1997). It is well known that bottom fishing is one of the most important agents of sea bottom habitat disturbances, not only altering the structure of the habitat but also leaving behind disturbed, damaged and dead benthic fauna (Groenewold and Fonds, 2000; Auster and Langton, 1999; Kaiser et al., 2002; Ryer et al., 2004). In the southern part of the North Sea, flatfishes are an important element of the fish assemblage (Daan et al., 1990; Rijnsdorp and Millner, 1996; Piet et al., 1998) and they may be severely affected by the benthic habitat alterations (Gibson, 1994; McConnaughey and Smith, 2000) as they are especially adapted to a benthic life style (Gibson, 1994). Dab (Limanda limanda) is one of the highly abundant flatfish species in the North Sea (Dann et al., 1990). Despite some potential disturbing factors, the population levels of this species remained relatively stable in the past (early 1980s) or in some parts an increase was observed in comparison with other flatfish species as Plaice (Pleuronectes platessa) (Hessen and Daan, 1996). However, more recently it was reported that Plaice biomass increased strongly from 2009 onwards (ICES, 2014: Borges et al., 2014). Furthermore, in the southern part of the North Sea, where Plaice is also highly abundant, the beam-trawl flatfish fishery on Sole (Solea solea) and Plaice is one of the most important fishery (ICES, 2014). Dab and Plaice are visual benthic feeders, having well-developed eyes, and feeding upon prey organisms that are found along and in the seabed (Wychie and Shackley, 1986). Several studies (Rijnsdorp and Vingerhoed, 2001; Braber and De Groot, 1973; Schückel et al., 2012) revealed that stomach contents of Plaice and Dab mainly comprised polychaetes. Bivalves supplemented plaice s diet, while Dab s diet is supplemented by decapods and amphipods (Schückel et al., 2012). In addition, earlier studies characterized Dab as a general crustacean and polychaete feeder (Tood, 1914; Richie, 1938; De Groot, 1964; Braber and de Groot, 1973; Ntiba and Harding, 1993; Hinz et al., 2005) and Plaice as a common polychaete and mollusc feeder (Lande,1976; Basimi & Grove, 1985; Carter et al., 1991; Ntiba & Harding, 1993; Shucksmith et al., 2006). Beam trawls are extensively studied, in particular beam and otter trawls as this two fishing gears are the most widely used in the North Sea (Anon, 1997). It is known that these gears reduces the overall abundance and biomass of available prey for Dab and Plaice and indeed 7

10 changes in the diet of Plaice and Dab were observed along a trawling gradient in the northeastern Irish sea (Johnson et al., 2015). Impact of trawling fisheries on benthic ecosystems and on fish diet has been reported to vary depending both on the type of gear used and on the type of substrate (e.g. Kaiser et al., 2006; Brylinski et al., 1994). Kaiser et al. (2002) mentioned that unconsolidated sediment habitats are less affected than biogenically structured habitats. This study was developed in a Lanice conchilega habitat. This tube worms, known as bioengineers (Jones et al., 1994; Rabaut et al., 2007), influence the habitat structure creating 3-dimensional structures that affect several environmental elements: (1) altered sediment properties; (2) modified the hydrodynamic regime; (3) improved availability of attachment surface; (4) increased refuge from predators (Callaway, 2006). Studies in this type of habitat are important as they have a positive influence in the benthic communities, enhancing biodiversity (Dittmann, 1999; Ager, 2002). Studies have been made on the impact of beam trawling in Lanice conchilega habitats (Rabaut et al., 2008), however no studies have been made revealing the effect of Shrimp Beam trawl on the diet of flatfishes. Moreover, the changes on food habits of Dab and Plaice following trawling were not studied in this type of habitat. In this study the diet of both species was investigated along transect in the North Sea following trawling with shrimp beam trawl. It was hypothesized that Dab, as an opportunistic feeder, is more readily adapted to changes in the prey species composition after Shrimp Beam trawling disturbance, while Plaice s diet would be directly more affected due to its lower niche breadth. The differences in the diet are expected to be especially visible during daylight hours as both species are visual predators. 8

11 2. MATERIAL AND METHODS The effect of shrimp beam trawling on the diet of two different species of flatfish was investigated from a Before-After impact study in a pre-selected site. The stomachs were collected and the size of the fishes recorded. The stomach content was identified, measured and analysed in other to identify the dietary changes in Dab and Plaice following Shrimp Beam Trawling. 2.1 EXPERIMENTAL DESIGN Two hauls were carried out by a commercial fishing vessel on 11 June 2015 in a twohour time frame resulting in a fishing intensity of 150% (by swept area). This two hauls were carried out between 12h28 and 14h30 (Fig 1). Moreover, nine hauls were conducted by a Research vessel Simon Stevin at consecutive time steps following fishing disturbance. This nine hauls, were divided in three different times, occurring three hauls of 20 minutes at each time step. In detail, the hauls were executed between the evening of the 11 June 2015 (T1 19h16,20h19 and 20h42) and the afternoon of 12 June 2015 (T2 04h04, 04h53 and 05h12; T3 13h02, 13h26 and 13h56). The gear used on board of RV Simon Stevin was a lighter gear. Given that, we assumed that the short deployment and the smaller dimension (including weight) of the gear did not affect the benthic communities. 9

12 Fig 2. A schematic overview of all the hauls performed over time, the vessel type, the vessel name and the type of gear. Assumed negligible impact of the sampling gear. 10

13 2.2 STUDY AREA AND FISHING GEAR This experiment was carried out in a Laniche conchilega habitat in Belgian waters in the South part of the North Sea (Fig 2 and 3; Table 1). The area is characterized by a wide range of sediments types, going from muddy to sandy muddy subtract (Fig 3). All hauls were carried out during day light hours (Fig. 1) within the experimental site. First, the whole area was fished with a commercial shrimp beam trawl with electric pulses deployed by a commercial fishing vessel ( O82 Nautilus ). The beam width was an 11 m and the trawl had 12 rubber bobbins rigged in a perpendicular direction of fishing. Stomach that were sampled during these hauls were assumed to originate from fish that did not scavenge after fishing disturbance (T0). Consecutive hauls were carried out with a sampling beam trawl (2m long, 3m wide, 9 x 9 mm mesh size) on board of the research vessel Simon Stevin. Fig 2. Location of the study area (blue), Box 9. Fig 3. Map of the study area bathymetry (black polygon). Green as shallow mud; Brown as shallow coarse and mixed sediment; Yellow as shallow sands. Table 1. Study area coordinates (Box9) BOX 9 Shrimp Pulse Beam Trawl 150% (12 bobbins) Longitude Latitude

14 Number of complete stomachs 2.3 FISH STOMACH CONTENT ANALYSIS A total of 264 complete stomachs were sampled (Fig 4). For Dab 130 stomachs were sampled and 134 for Plaice. All were extracted and stored in 8% formaldehyde solution with seawater for both species of flatfish: Dab (Limanda limanda) and European Plaice (Pleuronectes platessa). Besides, the total length of each one of the individuals were measured to the nearest cm below. Each one of the stomach was removed from the formaldehyde solution in the laboratory, rinsed with deionized water and cut open along the longitudinal axis. All the preys items found in the stomach were counted and identified under a LEICA stereo microscope with a magnification up to 16x. All the preys were identified, by preference, on species level or, as result of digestion to a higher taxonomic level. All unidentifiable components were classified as digested debris or fish scales (if they were fish digested debris) h28-15h00 19h16-21h00 04h00-05h30 13h02-14h15 T0 T1 T2 T3 O82 SiSt Dab Plaice Fig 4. Total number of complete stomachs sampled by time step. Time of sampling and name of the vessel. 12

15 Furthermore, each prey item was weighted and the stomach content was placed into a ceramic cup. All the cups were dried at 110ᵒC for a period of 48 hours, and weighted to obtain the dry weight (DW). Ultimately, the stomach contents were muffled at 550ᵒC for 2 hours and the ashes weighted (AW) to determine the Ash Free Dry Weight (AFDW = DW AW). 2.4 DATA ANALYSIS: EFFECT OF TRAWLING ON THE DIET OF PLAICE AND DAB The stomach content of each fish species was analysed by species to evaluate the change in Plaice and Dab diet following fishing. The diets of both species were then compared to evaluate the niche overlap between Plaice and Dab and the potential change following fishing. UNIVARIATE DIETARY INDICES Several indices were calculated and used to quantify the differences in the diets of each one of the species along time, and between them. Mean values of Species richness [1], number of individuals, stomach fullness index [2] and the Shannon-Wiener index [3] were estimated in order to observe more clearly the differences on the diets in the different times and comparing values obtain between Dab and Plaice. Stomach vacuity index [4] was also estimated, to assess the percentage of empty stomachs. [1] Species Richness (Margalef) d = (S 1) log e N Where S is the total number of species and N is the total number of individuals. 13

16 [2] Stomach Fullness Index (%SFI) SFI = AFDW s x 100 AFDW f Where, AFDW s is equal to the ash-free dry weight of the stomach contents. AFDW f is the ash-free dry weight of the whole fish, which was calculated by converting the total wet weight of the fish WW to AFDW f by using the following formula AFDW f 20% x WW (Edgar and Shaw, 1995). WW was estimated by using the exponential length-weight relationship,ww = a x L b where the coefficients a and b were different for each species and were taken from Depestele et al (Plaice) and Fishbase.org (Dab). [3] Shannon-Wiener index (H ) H = Σ S i=1 p i Log p i Where p i is the percentage importance (Peet, 1974). [4] Stomach Vacuity Index (%V) %V = ( V ) x 100 N Where V is the number of empty stomachs, whereas N is the total number of stomachs that have been examined. This index was assessed to express the percentage of empty stomachs and it was based on Rabaut et al (2013). Moreover, several dietary indices were calculated to obtain a more clear view on the differences of Dab and Plaice s prey species composition, likewise along the different times. Frequency of occurrence (%F) [5], gravimetric index (%W) [6], numerical abundance (%N) [7], and Percentage index of relative importance (%IRI) [8] were the indices estimated. 14

17 [5] Frequency of occurrence (%F) %F = ( F i ) x 100 N Where F i is the number of stomachs containing the prey type i of a specific taxon whereas N is the total number of non-empty stomachs. This index reflects the relative importance of a prey item over all the non-empty stomachs. [6] Gravimetric Index (%W) %W = ( W i ) x 100 W Where W i is the total weight of prey type i of a specific taxon, while W represents the total weight of all preys in the stomachs. In this index is considered the mass of a prey over the total mass of all the preys found in the stomachs. [7] Numerical abundance (%N) %N = ( N i ) x 100 N Where N i represents the total number of prey i of a specific taxon and N is the total number of all preys identified. In this index is it provided the percentage of the number of individuals of a specific prey type over the total number of individuals of all preys. [8] Percentage Index of Relative Importance (%IRI) [8.1] IRI = (%N i + %W i )x %F i [8.2] %IRI = ( IRI ) x 100 ΣIRI The IRI is calculated based on three dietary indices calculated before (%N, %W and %F), and it was calculated by the equations [8.1] and [8.2], based on Cortés et al (1997). All the dietary indices were analysed using a multivariate statistics in PRIMER e-package (v.6) software + PREMANOVA + add-on (Anderson et al., 2007). A multivariate analysis, with PERMANOVA extension was performed, using Euclidean distance matrix, in other to observe if there were significance differences between all the time steps. 15

18 MULTIVARIATE DIETARY ANALYSIS In the first place, the dataset went through a preliminary analyses, where all the prey items were combined into Genus level in order to obtain a cleaner dataset, which would simplify the future analyses. Moreover, the fish scales were joint with the digested debris, as only a few were found at T0 for Plaice. The diet of each fish species was compared based on both abundance and biomass of the prey items. Both parameters were used in the analyses as the abundances gives is information about the number of prey species in the stomach. At the same time, it is good to complement it with biomass of prey species, as biomass is the parameter giving information about the prey energy given to the predator. Since, bigger preys provide more energy to the fish species. Differences in the diet composition along time, for both Dab and Plaice, were analysed using a multivariate statistics in PRIMER e-package (v.6) software + PREMANOVA + addon (Anderson et al., 2007). Firstly, a Multidimensional Scaling (MDS) test was carried out for a better visualization of the differences. Afterwards, multivariate analysis, with PERMANOVA extension (permutational ANOVA/MANOVA), were performed in other to see if there were significant differences in the prey species composition (both prey biomass and abundance) of both fish species. For the PERMANONA analysis, the same design was used. One fixed factor was taken into account, time (T0,T1, T2 and T3). All data was previously squared-root transformed, to down-weigh the contribution of dominant species and to approximate normality and homogenize variances (Johnson et al., 2005). To compare the prey species composition of both fish species, the same analyses were performed again. MDS-plots were primarily done for a better visualization of the data. Afterwards, PERMANOVA analyses were performed with using a different design, as this time two fixed factors were taken into account: time (T0,T1,T2 and T3) and fish species (Dab and Plaice). SIMPER analysis indicated the contribution of the prey species to the differences in diet between foraging fish species as well as the effect of fishing and/or time. 16

19 Stomach Fullness Index Stomach Vacuity Index 3. RESULTS 3.1. DAB UNIVARIATE DIETARY INDICES The stomach fullness (Fig 5) increases from T0 (0,14±0,006) to T1 (0,20±0,005) and from T2 to T3 (0,15±0,010), but decreases from T1 to T2 (0,12±0,008). None of the changes in stomach fullness were significant (p=0.9115). Empty stomachs were only found at T2 (vacuity index = 12,5%). 0,3 0, ,2 0,15 0,1 0, T0 T1 T2 T3 Before After SFI %V 0 Fig 5.Stomach Fullness Index (SFI) (±SE) and Stomach Vacuity Index (%V) for Dab before (T0) and after fishing (T1, T2 and T3) The mean number of species in the diet of Dab varied between 2.36 (±0.233) and 2.87 (±0.283) (Fig 6), but was not statistically different across the times (p=0.1206). The Shannon-Wiener index (H ) was also not statistically significant across times with mean values between 0.61 (±0.097) and 0.68 (±0.091) (p=0.6057). 17

20 3,50 3,00 2,50 2,00 1,50 1,00 0,50 0,00 T0 T1 T2 T3 Before After S H' Fig 6. Mean number of prey species per stomach (S) and Shannon-Wiener Index (H ) for Dab before (T0) and after fishing (T1,T2 and T3). The frequency of occurrence (Fig 7) highlights that Dab mainly feeds on Annelida and Crustacea with values ranging between 50% and 67%, except for Annelida at T1 which occurred more frequently (80%). Annelida were more frequently observed than Crustacea directly after fishing (~4h), while Crustacea were more frequent than Annelida before fishing (T0) and after a time lapse following fishing (>12h). Over time, the frequency of Annelida increased directly following fishing disturbance (T1: 80%), but declined rapidly (T2: 52.5%; T3: 53.6%) to similar values before fishing (T0: 50%). Conversely, the frequency of Crustacea occurrence declined following fishing disturbance (T1: 52%), but increased at T2 (66,7%) and T3 (64,3%) to similar levels of T0 (61,5%). No significant differences were observed for all the indices between times. A similar pattern is observed from the index of relative importance (%IRI) (Fig 8), which highlights that Dab mainly feeds on Annelida (%IRI range: ), followed by Crustacea (IRI range: ) with minimal %IRI values at T0 for the Annelida (36.9%) and at T1 for the Crustacea (17.5%). 18

21 Percentage Index of Relative Importance Frequency of occurence Annelida Bivalvia Cnidaria Crustacea Echinodermata Nematoda Nemertea T0 T1 T2 T3 Fig 7. Frequency of occurrence (%F) of higher taxon on the diet of Dab in each time step Annelida Bivalvia Cnidaria Crustacea digested_debris Echinodermata Nematoda Nemertea 0 T0 T1 T2 T3 Fig 8. Percentage Index of Relative Importance (%IRI) of higher taxon on the diet of Dab in each time step. 19

22 UNIIVARIATE DIETARY ANALYSIS Biomass Firstly, an analysis on higher taxonomic level was performed, revealing that biomass increases were observed for Annelida from T0 (0,0581) to T1 (0,1802) and to T2 (0,1877) (Fig 9). The same pattern was found for the biomass of the digested debris. The biomass of Bivalvia and Crustacea, in contrast, decreased from T0 to T1 and T2. Table 2. ANOSIM-test with the p-values for the differences between Time steps for prey biomass for Dab. (Global R = 0.037). Groups R Statistic Significance level % T1, T2 0, T1, T3 0, T1, T0 0, T2, T3 0, T2, T0-0, T3, T0 0, The ANOSIM test (Table 2) showed significant differences in species biomass composition between T1 and each other time step: T0 (p=0.018), T2 (p=0.014) and T3 (0.047) although explanatory power was limited (global R = 0.037). Biomass changes between times were induced by a small number of taxa. Simper routine, performed on genus level, indicated that most of the differences between T1 and other time steps were explained by Lanice conchilega (SIMPER: ~30%) followed by Polynoidae (~10%). Both L. conchilega and Polynoidae had higher biomass values at T1 (APPENDIX Fig 25). Simper analysis further indicated that the highest dissimilarity in diet composition was observed between T0 and T1 (65,28%) The changes were induced by an increase of Lanice conchilega biomass to almost double, contributing 27,27% to the dissimilarity between T0 and T1, and a substantial increase in the biomass of digested debris, which contributed up to 24,69% to the differences in diets. 20

23 0,4000 0,3500 0,3000 0,2500 0,2000 0,1500 0,1000 Nemertea Nematoda Echinodermata digested_debris Crustacea Cnidaria Bivalvia Annelida 0,0500 T0 T1 T2 T3 Fig 9. Mean prey biomass by higher taxon and by time (T0- before fishing; T1, T2 and T3 after fishing) for Dab. Abundance The abundance of Annelida increased after fishing (T1) and the next morning (T2), but decreased again to pre-fishing levels, as shown by similar numerical percentages in Fig 10. The same pattern was observed in Crustacea, although the differences in abundance were not as notorious as in the analysis of biomass and there was an increase in abundance from T2 to T3. Table 3. ANOSIM-test with the p-values for the differences between Time steps for prey abundance for Dab. (Global R = ) Groups R Statistic Significance level % T1, T T1, T T1, T T2, T T2, T T3, T

24 The composition of prey species abundance in the stomachs of Dab was not statistically significant for the different time steps (Table 3), although a similar pattern to the biomass changes was observed. Lanice conchilega was the species contributing the most to these differences between times, increasing slightly from T0 to T1, and decreasing slightly from T1 to T2 to maintain the mean abundance on a similar level at T3. The second species showing a high contribution was Pariambus typicus. In contrast to patterns in Lanice conchilega this species slightly decreased in abundance after fishing and returned back to pre-fishing levels at T3. 10,00 9,00 8,00 7,00 6,00 5,00 4,00 3,00 2,00 Nemertea Nematoda Echinodermata Crustacea Cnidaria Bivalvia Annelida 1,00 T0 T1 T2 T3 Fig 10. Mean prey abundance by higher taxon and by time (T0- before fishing; T1, T2 and T3 after fishing) for Dab. 22

25 Stomach Fullness Index Stomach Vacuity Index 3.2. PLAICE UNIVARIATE DIETARY INDICES The stomach fullness (Fig 11) varied across time and was generally higher at T1 (0,24±0,0077) and T3 (0,24±0,0051), thus after fishing. An increase between T0 (0,11±0,0025) and T1 was observed, followed by a sharp decrease to T2 (0,09±0,0031) and the stomach fullness reached it maximum at T3. Significant differences were observed between T1xT2 (p=0,0001) and T0 (p=0,0002) and between T3xT2 (p=0,0001) and T0 (p=0,0003). As it was observed on Dab, empty stomachs were only found at T2 (Stomach vacuity index = 13,3%). 0,3 14 0,25 * * 12 0,2 0,15 0,1 0, T0 T1 T2 T3 0 SFI %V Fig 11. Stomach fullness (SFI) (±SE) and Stomach Vacuity (%V) indices for Plaice before (T0) and after fishing (T1, T2 and T3). No significant differences were found on Plaice s diet diversity across times, for both Shannon-Wiener index (H ) (p=0.6057) (Fig 12) and mean number of prey species (p=0.1206). No big changes were observed on the values of the Shannon-Wiener index (H ), with mean values between 0.68 (±0.091) and 0.61 (±0.089) 23

26 Furthermore, regarding the species richness (S) (Fig 12), some changes were found even though the differences were not significant (p=0.3139), varied between 2.84 (±0,273) and 1.89 (±0,220). Additionally, an increase in the number of individuals was observed after fishing, having T1 (12,11±1,507) the maximum number of individuals (APPENDIX Fig. 22). 3,50 3,00 2,50 2,00 1,50 1,00 0,50 0,00 T0 T1 T2 T3 Before After S H' Fig 12. Mean number of prey species per stomach (S) (±SE) and Shannon-Wiener Index (H ) (±SE) for Dab before (T0) and after fishing (T1, T2 and T3). The frequency of occurrence (%F) (Fig 13) points that Plaice mainly feeds on Annelida and Bivalvia. Annelida values ranging between 68% and 90%, while Bivalvia values range between 68% and 16%. Annelida was more frequent observed than Bilvalvia across all time steps. Over time, the frequency of Annelida slightly increased directly after fishing, but declined at T3. Conversely, the frequency of Bivalvia occurrence declined following fishing disturbance (T1: 34%), declining more at T2 (16%), increasing at T3 (68%). However, no significant differences were observed. A similar trend is observed from the Index of relative importance (%IRI) (Fig 14), which highlights that Dab mainly feeds on Annelida (%IRI range: 84% - 38%), followed by Bilvalvia (%IRI range: 51% - 6%). Annelida has the biggest values, except for T3 where Bilvalvia has higher value for %IRI (Annelida 24

27 Percentage Index of Relative Importance Frequency of occurence 38% ; Bivalvia 51%). Bivalvia also has a bigger contribution gravimetrically. However, at T2 Annelida (54%) showed higher gravimetrical values comparing with Bilvalvia (33%) (APPENDIX Fig 24) Annelida Bivalvia Cnidaria Crustacea Echinodermata Nematoda Nemertea 0 T0 T1 T2 T3 Fig 13. Frequency of occurrence of higher taxon on the diet of Plaice to all time steps Annelida Bivalvia Cnidaria Crustacea digested_debris Echinodermata Nematoda Nemertea 10 0 T0 T1 T2 T3 Fig 14. Percentage Index of Relative Importance (%IRI) by higher taxon on the diet of Plaice to all time steps. 25

28 UNIIVARIATE DIETARY ANALYSIS Biomass An analysed on higher taxonomic level revealed that a biomass increase was observed for Annelida from T0 (0.0858) to all the other time steps (T1: ; T2:0.1801; T3: ) (Fig 15). The biomass of the digested debris followed the same trend as Annelida, increasing its biomass at T1 (0.1013). In contrast, Bivalvia was the following taxa observed in a high biomass on the stomachs, decreasing slightly its biomass from T0 (0.0799) to T1 (0.0758), followed by a large decrease at T2 (0.0124). At T3 (0.2775) the biomass increased sharply. Bivalvia was slightly more abundant than Annelida at T3. The ANOSIM test, performed on genus level, showed significant differences between all the times (Table 4), with the exception for T2 and T0 (p=0.081). Meaning that the differences on the diet composition before and right after fishing, T0 to T1, were significant (p=0.07) although the explanatory power was quite limited (global R = 0.135). Table 4. ANOSIM-test with the p-values for the differences between Time steps for prey biomass for Plaice. (Global R = 0.135) Groups R Statistic Significance level % T1, T2 0,11 0,1 T1, T3 0,131 0,09 T1, T0 0,137 0,07 T2, T3 0,243 0,01 T2, T0 0, T3, T0 0,146 0,02 The SIMPER-test revealed that Lanice conchilega (~30%) and Abra alba (~30%) are the two species contributing more for the differences between times. The biggest differences between times on the prey composition were observed between T3 and T2 (67,15%) and also between T3 and T0 (62,57%). Abra alba was the species contributing the most for these differences as it suffered a sharp increase at T3 (APPENDIX Fig 26). 26

29 For the differences between T1 and T2 and T1 and T0, Lanice conchilega was the one contributing the most with 37% and 33%, respectively. An increase on the biomass (APPENDIX Fig 26) of this species was observed right after fishing (T1: ) followed by a small decrease for the following times (T2: and T3: ). 0,7000 0,6000 0,5000 0,4000 0,3000 0,2000 Nemertea Nematoda Echinodermata digested_debris Crustacea Cnidaria Bivalvia Annelida 0,1000 T0 T1 T2 T3 Fig 15. Mean prey biomass by higher taxon and by time (T0 before fishing; T1,T2,T3 after fishing) for Plaice. Abundance The abundance of Annelida increased after fishing (T1: 10.79) but decreased again in the next morning (T2: 8.30) and in the next afternoon (T3: 8.11) for pre-fishing levels (T0: 7.80) (Fig 16). The same trend was observed for Bilvalvia, except that the abundance increased sharply at T3 (1.89). The ANOSIM-test revealed some significant differences on the diet composition between times (Table 5). However, the differences before and after fishing were only significant between T0 and T3 (p=0,002). Lanice conchilega (38%) and Abra alba (23%) were the species contributing the most to these differences, mainly due to their changes in abundance. Lanice conchilega was more abundant after fishing (T1, T2 and T3), while Abra alba increased it abundance across time, reaching its maximum at T3. 27

30 Table 5. ANOSIM-test with the p-values for the differences between Time steps for prey abundance for Plaice. (Global R = 0.087) Groups R Statistic Significance level % T1, T2 0, T1, T T1, T0 0, T2, T T2, T T3, T Moreover, significant differences were also observed between T1 and T2 (p=0,015) and T3 (p=0,003). These differences on the diet composition are mainly due to the decrease on the abundance of the Annelida (Lanice conchilega, Phyllodoce mucosa, Eumida sanguinea and Polynoidae) and Abra alba from T1 to T2 (APPENDIX Fig 28). 14,00 12,00 10,00 8,00 6,00 4,00 Nemertea Nematoda Echinodermata Crustacea Cnidaria Bivalvia Annelida 2,00 T0 T1 T2 T3 Fig 16. Mean prey abundance by higher taxon and by time (T0 before fishing; T1,T2,T3 after fishing) for Plaice. 28

31 Stomach Fullness Index 3.3. COMPARISON OF DAB AND PLAICE s DIET ACROSS TIME UNIVARIATE DIETARY INDICES A similar pattern over time was observed for the Stomach Fullness index (Fig 17) of Dab and Plaice, i.e. a slight increase directly after fishing (T1). However, the values revealed that Dab had a higher stomach fullness than Plaice at T0 and T2, while Plaice had higher fullness on T1 and T3. Both, Dab and Plaice only had empty stomachs at T2. 0,3 0,25 0,2 0,15 0,1 0,05 0 T0 T1 T2 T3 Before Dab Plaice After Fig 17. Stomach fullness (SFI) (±SE) for Dab (Green) and Plaice (Blue) before (T0) and after fishing (T1, T2 and T3). The Frequency of occurrence shows clearly that while Plaice mainly feeds on Annelida and Bivalvia (Fig 18), Dab feeds mainly on Annelida and Crustacea. Annelida is always more frequent in Plaice s diet. A similar trend is observed from the index of relative importance (%IRI) (Fig 19), where Annelida has more importance in Plaice s diet, except at T3 where it is more important for Dab. Bivalvia also has a bigger importance in Plaice s diet, especially at T3. 29

32 Annelida Bivalvia Cnidaria Crustacea Echinodermata Nematoda Nemertea 10 0 Dab Plaice Dab Plaice Dab Plaice Dab Plaice T0 T1 T2 T3 Before After Fig 18. Frequency of occurrence (%F) of higher taxon before (T0) and after fishing (T1, T2 and T3) for Dab and Plaice Annelida Bivalvia Cnidaria Crustacea digested_debris Echinodermata Nematoda Nemertea 0 Dab Plaice Dab Plaice Dab Plaice Dab Plaice T0 T1 T2 T3 Before After Fig 19. Percentage Index of Relative Importance (%IRI) of higher taxon before (T0) and after fishing (T1, T2 and T3) for Dab and Plaice. 30

33 COMMUNITY STRUCTURE Biomass The MDS-plot (Fig 20) shows a clear difference between the diet of Dab and Plaice despite some overlap at times T1 and T2. Permanova routines of the biomass species composition revealed a significant interaction between the factors Time and Species (Dab and Plaice) (p=0,0001). The differences in the diet composition between Dab and Plaice were significant within each time step. Nevertheless, the diets of both species were more similar at T1 and T2, as indicated by the SIMPER analysis (Table 6) Table 6. Average similarities, from SIMPER, between Dab and Plaice s diet by time and for the prey species biomass and abundance. Time steps Average similarity Biomass Abundance T T T T The SIMPER-analysis revealed that Lanice conchilega and Abra alba were contributing most to the differences in the diets. L. conchilega and A. alba showed a higher biomass in Plaice than in Dab stomachs in each time step. The contributions of Lanice conchilega, Phyllodoce mucosa, POLYNOIDAE, Lagis koreni were higher in Plaice than in Dab stomachs, and Dab consumed less different species. The contribution of Bivalvia, mainly Abra alba, was higher in abundance and in biomass in Plaice than in Dab stomachs. Crustacea, in contrast, were observed more frequently and in higher biomass in Dab stomachs (e.g. Pariambus typicus, Abludomelita obtusata, Microprotopus maculatus). 31

34 Fig 20. MDS-plot for prey biomass by time (T0,T1, T2 and T3 Different symbols) and by species (Dab Green and Plaice Blue). Abundance The MDS-plots (Fig 21) showed clear differences between the diet of Dab and Plaice composition, but also highlighted that the differences between times steps are not clear. The diet of Dab and Plaice was significantly different for the interaction of time and species (p=0,005). The differences in the diet composition between both species were significant within each time step. The diet of both species are more similar at T1 and T2 (Table 6). The SIMPER-test revealed that Lanice conchilega and Abra alba are the species contributing the most for the differences in the diet. L. conchilega was always present in a higher biomass in Plaice than in Dab stomachs, the same was observed for A. alba. 32

35 Fig 21. MDS-plot for prey abundance by time (T0,T1, T2 and T3 Different symbols) and by species (Dab Green and Plaice Blue). 4. DISCUSSION This study investigated the diet composition of Dab and Plaice before and after shrimp beam trawl disturbance by a commercial fishing vessel in an experimental site (Box 9). The impact of this fishing gear on the diet of this flatfish species was assessed from stomach contents and the diet composition at several the time steps after disturbance. The diet of Dab and Plaice did not alter substantially, but showed slight differences in biomass and prey species composition. The consumption of annelids generally increased, while crustaceans and bivalves decreased in the diets of Dab and Plaice respectively. The effect, however, was only detected directly after trawling. UNACCOUNTED VARIABILITY OF FEEDING ACTIVITY The original experimental design entailed a control site and replicates, which could not be established due to bad weather conditions. The lack of a control site implies that we did not control for dietary changes, which may have been imposed by environmental conditions such as tidal cycles or the diurnal feeding pattern of Dab and Plaice. Both species show a diurnal cycle with bottom activity restricted to daylight hours. While stomach contents may have been influenced at time step T2, we anticipate no effect on 33

36 any of the other time steps, because the experiments were conducted during days in June with long daylight duration (Verheijen & De Groot, 1967; De Groot, 1971). Moreover, De Groot (1971) reported while investigating the diurnal feeding periodicity in situ, the stomachs of Dab and Plaice were most of the times empty or nearly empty just before the sunrise. In our study, the stomachs from T2 were sampled around the sunrise (04h00-05h00), and the lowest stomach fullness and total prey biomass were found at this time. Another highly relevant effect may be due to the size of the predators. Whereas the diet of small Plaice (<15cm) for instance, consists nearly exclusively of annelids, larger Plaice also target bivalves and crustaceans (Rijnsdorp & Vingerhoed, 2001). The diet of predating fish species is related to their body size, because mouth gape increases with body size (Johnson et al., 2011). The size of the mouth gape was estimated from the body sizes of each fish based on the equations in Johnson et al. (2014). The size distributions of the mouth gapes largely overlapped in range ( mm for Dab; for Plaice) and had similar mean (SE) mouth gapes ( mm for Dab; mm for Plaice). The overlap implied that the differences in prey selection were due to dietary selection rather than mouth gape constraints. DAB DIET AFTER FISHING Dab s diet composition altered after fishing, even though the differences were not significant for each dietary index. Significant differences were found for prey biomass between T1 (first time step after fishing) and the other time steps. This means that shrimp beam trawling exposes the potential food resources increasingly to Dab predation, as the stomach fullness and the biomass of the prey species found in the stomach was the highest at this time. Nonetheless, the global R was really low (R=0,037). Groenewold and Fonds (2000), as results as their stomach analysis, mention that Dab, among other demersal fish, is one of the main users of the food that becomes available after trawling, which could be the reason why the stomach fullness and the prey biomass increased right after fishing (T1). The analysis of Dab s stomach contents revealed that Lanice conchilega, Polynoidae and Ophiura ophiura were most abundant. The ingestion of ophiuroid species seems to be a 34

37 common occurrence in Dab stomachs as observed in other studies (Hinz et al., 2005). This group contributed the most to the observed differences in the stomach content before and after fishing, and showed the highest biomass right after fishing (T1). At the same time, it was observed that the amphipod Pariambus typicus decreased its biomass, even though its abundance increased. Therefore, smaller individuals of this species were caught at T1. Volbehr and Rachor (1997) found that Pariambus typicus lives clinging in some other individuals. As our area is a Lanice conchilega habitat and P. typicus, is one of the dominant associated species in subtidal areas (Rabaut et al., 2008) the specimens were possibly displaced by a disturbance event (Rabaut et al., 2007), in this study by fishing disturbance. According to Johnson et al. (2014) Dab remains largely unaffected by trawling due to its quickly diet adaptation without reductions in feeding efficiency. Due to their feeding strategy, where Dab feeds more upon crustaceans, mostly appendages, normally out of the substrate - decreases the necessity to spend much energy digging individuals preys. PLAICE DIET AFTER FISHING Braber and De Groot (1973) stated that Polychaetes and mollusc are the most important phyla in Plaice s diet. Rijnsdorp et al., (2001) also observed that the stomach contents of both Plaice and Sole were mainly dominated by Polychaetes. Our results are in accordance with these findings, as Annelida and Bilvalvia were the most dominant and important taxon in Plaice s diet at all the time steps. Rijnsdorp et al., (2001) further showed that Plaice s stomach fullness index increased between 2 to 4 times within 12h of trawling, while our results showed a much faster increase of the stomach fullness, happening around four hours after trawling (T1). This faster increase can be due to the differences in the type of gear used. Rabaut el al. (2008) showed that Lanice conchilega reefs are not as severely affected by trawling as the associated species, mainly due to their adaptation to natural disturbances. Suggesting, that although the seabed disturbance is limited, the L. conchilega tubes are not intact. Contrasting the type of gear used in these studies (Rijnsdopr et al., 2001 and Rabaut et al., 2008) we can suggest that Shrimp Beam trawl has a lower impact on the seabed as 35

38 flatfish beam-trawls have a more intense contact with the seabed (Depestele et al., 2016). It was concluded that Plaice mainly fed on Polychaetes, since a notorious increase on the biomass of some species was observed at T1 (after fishing), in particular for Lanice conchilega, Phyllodoce mucosa, Glycera tridactyla, Stenothoe marina and Nepthys sp., having Lanice conchilega shows further a large contribution to the differences between T1 and the other time steps. Nereis sp. was not observed in the stomach contents before fishing, appearing just afterwards; however its biomass was not high. It was observed that Nereis spp. live semi-permanently burrowed in the sediment, extending between 20 to >40 cm depth (Kristensen, 2001). Probably due to this, Nereis spp. are only observed after fishing disturbance. Approximately, 24h after the fishing disturbance (T3), a big abundance and biomass of Abra alba was observed, so that this species was the main contributor (with more than 30%) to the significant differences between T3 and T0 (p=0,0002), T1 (p=0,0009) and T2 (p=0,0001). Vallet (1993), reported that Abra alba can regulate its position in the water column, by opening their valves in different extensions. This behaviour supports the idea that A. alba may escape from the trawl disturbance. Later in time, due to daylight and to the fact that the tubes have been damaged to some extent, A. alba is more susceptible to predation. All things considered, A. alba is therefore not directly damaged by fishing disturbance (Teal et al., 2014). Nevertheless, it is possible indirectly affected through increased predation. COMPARISON BETWEEN DAB AND PLAICE S DIET AFTER FISHING Dab s and Plaice s diet composition revealed significant differences between times, for both abundance and biomass of prey species as observed on the MDS-plots (Fig 20 and 21). Some differences in the fullness of the stomachs were found between the two species, although they were not significant. A highest stomach fullness was observed at T0 and T0 on Dab stomachs, and at T1 and T3 on Plaice. At T1 both fish species showed the highest values. Bels and Davenport (1996) observed that Plaice has a quicker post capture debris ejection comparing with Dab. This coincides with our results, as Plaice 36

39 showed a smaller stomach fullness at T2, even though it was higher than Dab s at T1. Moreover, a higher biomass of digested debris was also observed in Dab s diet at T1 and T2. Dab s and Plaice diet composition was different, even though it seems that shortly after fishing (T1) and in the morning (T2), the diets are more similar. Dab fed more upon more species of annelids after fishing, decreasing the differences between both fish species diets. Dab is classified as a general feeder with a relative wide prey spectrum (Steven, 1930; Jones, 1952; Braber and De Groot, 1973; Wyche and Shackley, 1986; Knust, 1996; Saborowski and Buchholz, 1996; Beare and Moore, 1997). The results by Hinz et al. (2005) support this classification, as they showed that the relative abundance of prey species in the environment is the main factor influencing Dab s prey choice. It is known that Dab feeds mainly upon crustaceans. However, some of these species are more vulnerable to fishing disturbances (e.g Pariambus typicus) and others can be deeply buried or even be fast-moving species, which can influence their abundance in the environment. Therefore, increasing annelids biomass in Dab s stomachs, makes the diet of both fish species more similar. A study made in Carmarthen Bay indicates that while Plaice is limited to feed on commonly occurring species, Dab is able to use any available food source, taking a wider range of organisms (Wyche and Shackley, 1986). Our results agree with this finding, as a higher species diversity was observed in Dab s diet over time. However, a higher number of individuals were found on Plaice s diet. Which could be a result of the fact that Plaice has faster jaw movements, capturing food more quickly than Dab (Bels and Davenport, 1996). Dab and Plaice s diet composition was significant different between all times analysed, before and after fishing. Withal, it was observed that the diets of both species had more similar composition at T1 and T2, after fishing. These similarities were mainly due to the large contribution of the high biomass of Lanice conchilega and digested debris. 37