Effects of Demersal Otter Trawls on the Re-suspension of Copepod Resting Eggs and its Potential Effects on Recruitment

Resting eggs are important phases in the life strategy of many coastal and estuarine copepods. The egg mortality in the sediment layers where they are buried, as well as re-suspension from the sediment into the water column where eggs may hatch are factors that affect the success of this life strategy. Considering that fishing effort in some coastal areas is high and trawling leads to re-suspension of the bottom sediments, it is important to understand these effects on the biology of organisms that utilize sediment habitats in part of their life cycle. This study examined the re-suspension and the hatching success of copepod resting eggs in the wake of two different demersal fishing gear components (doors and discs) that are in contact with the seabed, in two areas off the coast of Scotland that are rarely worked by fishermen. Sediment cores were taken and analysed for resting eggs quantity and hatching performance and compared with samples taken in the water column right after re-suspension of sediment by the gear components. This study demonstrated for the first time that although eggs are re-suspended in the water column together with the sediment, providing them with the opportunity to hatch and recruit nauplii to the pelagic, egg viability was reduced by the passage of the gear components. This study also suggests that the viability is dependent on the gear component and accordingly potential effects must be considered for at this level.


Introduction
Resting eggs are produced by around 50 species of calanoid copepods [1,2] and are known to be produced to survive harsh environmental conditions [3]. The roles of these eggs in the life cycle of many ecologically important species are often underestimated despite a seasonal occurrence of species disappearance from the water column [4,5]. Different types of resting eggs have been described in the literature and represent different types of survival strategies. There are: immediate survival strategies (quiescent eggs), short-term resting strategies (e.g. delayed hatching eggs), and long-term survival strategies (diapause) where eggs accumulate in the sediment as an egg bank, saving these genes for the future [6][7][8][9][10]. It is, however, difficult to separate eggs according to these categories when sampled from the sea bed because diapause and delayed hatching eggs for which hatching is maternally controlled may lie in the sediment under quiescence once their refractory period is over. Also, morphological approaches for the identification of eggs often produce contradictory results and therefore must be used cautiously [11][12][13].
Eggs in the sediment may be subject to predation, infection and senescence, [14]. However, these eggs may also be released from their resting stage after re-suspension in the water column following the mobilization of sediment as a result of anthropogenic, physical and biological disturbances [15].
Demersal trawl fishing is one of the most widespread anthropogenic activities that affect the seabed. Towed demersal fishing gears can significantly impact the benthic ecosystem and many previous studies have shown that such gears can damage benthic organisms, alter the benthic habitat and sediment structure [16,17]. The physical interaction of towed demersal gears and the sea bed can be broadly classified as being either geotechnical or hydrodynamic in nature [18]. The penetration, piercing and lateral displacement of the sediment can be considered to be geotechnical [19] and the associated pressure and shearing forces may damage benthic in fauna and habitat. The turbulent shearing in the wake of the gear components are hydrodynamic and these give rise to the mobilization of sediment into the water column (e.g. [20,21]). The mobilization of sediment has been related to the re-suspension of phytoplankton cysts and zooplankton resting eggs [22][23][24], however, the extent to which the passage of a fishing gear affects the viability of these stages has never been assessed. The present study is aimed to obtain an initial understanding of these processes, and to explore how demersal trawl fisheries may affect the re-suspension and viability of copepod resting eggs.

Sample collection
Experimental trials were carried out during November 2007 by the RV Clupea close to Nairn (57°37'N 3°54'W at depths ~ 22 m) and close to Burghead (57°41'N 3°45'W at depths ~ 54 m), two areas within the Moray Firth on the northeast coast of Scotland. To quantify the sediment type and the depth distribution of eggs, 6 core samples (Ø 10 cm) were taken at each site using a Sholkovitz corer and sectioned in three different depth intervals: 0 -2 cm, 2 -6 cm and 6 -14 cm on board the ship.

Fisheries & Livestock Production
For the assessment of the fishing gear effects on the sea bed and the resting eggs buried into it, two different components of a demersal trawl were used: a trawl door, which is used to spread the mouth of the trawl net and ensure it maintains contact with the seabed (rectangular 1.37 m/0.88 m, 101 kg) ; and a rubber disc section of the ground gear, which is used to protect the fishing net and maintain contact with the seabed (Ø 15 cm /3.5 cm thick, 1.36 m long, 20.6 kg) [24]. The gear components were towed individually from the vessel at a speed of ~ 1 ms -1 (2 knots) during 15 min tows and a benthic sledge was attached behind the components to sample the sediment plume at 35, 55 and 80 cm above sea bed (for the trawl doors) or 35 cm above seabed (for the rubber discs) by using a 20 µm mesh size plankton net with a Ø 6.0 cm opening [24]. Two trawls (3 mesh samples for each) were carried out at each station to evaluate the effects of doors and six trawls (1 mesh sample for each) were carried out at each station after the rubber discs.
All samples (plankton net and core) were kept at 5°C on the research vessel and transferred to the laboratory after the cruise.

Analysis of cores and net samples
Sediment samples from the cores and the plankton net were processed in the laboratory the week following the cruise. Cores from each sampling sites were processed as following: two samples were fixed in buffered formalin 4% final concentration for counting, three were used for hatching experiments and the last sediment sample was analysed using a Malvern Instruments Mastersizer E Particle Size Analyser.
The first set of mesh samples (3 samples) harvested after the doors and three sets of mesh samples harvested after the rubber discs were used for counting the number of eggs re-suspended in the water column (formalin 4%). The second set of mesh samples (3 samples) harvested after the doors and three other sets of mesh samples harvested after the rubber discs were used for the hatching experiment. There was no particle size analysis done on the sediment harvested in the mesh after re-suspension.
Particles less than 20 µm are partly lost through the meshes during trawling and therefore, comparing the egg density in the cores and the mesh samples was done by pondering the counted number of eggs in all samples with the weight of the 50-200 µm sediment fraction (successive filtrations of a known sample volume). For fixed samples, the 50 -200 µm fraction containing resting eggs was transferred into LUDOX solution 40%, mixed and centrifuged at 900 g [25]. The upper fraction containing the eggs was rinsed on a 50 µm mesh and eggs were enumerated under a dissection microscope at 40X magnitude.
The live samples (cores and plankton net) were rinsed through 200 and 50 µm filters with 10 µm filtered sea water (FSW) and the 50-200 µm fraction containing eggs was incubated in the dark at 12°C in 100 ml cell culture flasks in FSW on a rocking table to ensure gentle movement of water and sediment and thereby oxygen to the resting eggs in the flasks. Every three days, the water was exchanged; nauplii were harvested and fixed in formalin for later identification. The hatching experiment lasted for 35 days and at the end of the incubation, the remaining eggs were counted and the weight of the sediment fraction was obtained.

Statistical analysis
For statistical analysis, ANOVAs were carried out and followed with pairwise comparison tests (Holm-Sidak method with p=0.05) using Sigma plot ( ® V.12.3).

Sediment structure and egg distribution
The sediment was classified as a mixture of poorly sorted 'muddy sand' and 'sandy mud' at Nairn and poorly sorted 'sandy mud' at Burghead [26]. The percentage silt (<63 µm) in the sediments was 54 and 66% by volume, respectively (   The eggs density across the three layers of sediment varied from 15.1 to 30.8 eggs cm -3 in the sediment of Burghead and from 6.5 to 13.9 eggs cm -3 at Nairn (mean of two cores). The density of eggs in the surface and bottom layers of Burghead was lower than in the middle  (Figure 1). The density of eggs m -2 of surface area can be estimated by integrating the egg density in all three layers of sediment and gives 3.08 x 10 6 eggs m -2 and 1.71 x 10 6 eggs m -2 in the Burghead and Nairn sediments, respectively.

Egg re-suspension and incubation
Fewer eggs tend to accumulate per gram of the 50-200 µm sediment fraction in the plankton net samples of Burghead than in the cores at the same location but no trends are observed at Nairn (Figure 1).
Eggs in the incubation experiment start hatching after 3 days and most of the eggs hatched within the first ten days of incubation independently from their origin. Only eggs from Nairn (6-14 cm) and Burghead (after Discs) presented a less clear and more delayed pattern ( Figure 2).
Most eggs hatching were from the Centropages spp. and Temora spp. genera, only a few Acartia spp. nauplii emerged from the 35 days incubation period at Nairn. The occasional occurrence of harpacticoid copepods was ignored from the study (Figure 3). The hatching rates of eggs in the cores ranged from 2.1% to 8.7% of the incubated eggs ( Figure 4). Interestingly, the hatching success of these eggs, which were harvested directly from the sediment, was higher than those sampled from the water column after the passage of the gear components (ANOVA with a Holm-Sidak post hoc test, p<0.05). Furthermore, the hatching success of those eggs taken in the wake of the rubber discs tended to be higher than those taken in the wake of the heavier door.

Discussion
Eggs from calanoid copepods were present at both stations, in the different sediment layers, within the range of egg densities previously reported in other sediments around the world [27,28].
There were more eggs found present in all layers of the finer Burghead sediment than in the coarser Nairn sediment, supporting what has been reported elsewhere [23,24]. These observations are likely to reflect differences of the local hydrodynamic processes whereby both the finer sediments and the comparatively lighter eggs (with a relative density of about 1.05 -1.2 as opposed to approximately 2.65 for quartz based sediments) will have a greater tendency to settle and remain in less dynamic areas. Furthermore, the upper sediment layers (0 -2 cm) at both Burghead and Nairn contained the fewest number of eggs, while the most were found in the 2 -6 cm layer which is possibly due to the eggs in the top layers being more available for resuspension and subsequent hatching [29].
Interestingly, there were fewer eggs in the suspended sediments of Burghead than in the cores at the same site but this trend was not obvious at Nairn. This probably reflects that it is only the top layer of sediment (where there are fewer eggs) that is re-mobilized and that it is the finer fraction of this layer that is more likely to be re-suspended and sampled in the plankton nets.
The hatching rates values found in the present study are slightly lower than those described by Marcus [30] where up to 21% of the eggs hatched and far from what is reported in other sediments from the Baltic Sea (~ 80% [31]). There were close to no hatching from Acartia spp. in the present experiment. This could be due to that eggs may have not received the cues for hatching in the present experiment (such as light; [32] but which could also be dependent on temperature, salinity etc.). Considering that Acartia spp. are found in the pelagic in the area [33] during the summer and spring and its disappearance from the water column is likely to be linked with the production of resting eggs [34], it is very likely that eggs are present in the sediment but have not been source of hatching in the present study. Hypothetically, Centropages spp. and Temrora spp were not affected by the lack of light during the present incubations.
Nevertheless, even if all cues that may have allowed a higher hatching success were not tested during the present study, the hatching success was higher for eggs harvested directly from the sediment than those sampled from the sediment plume in the wake of the gear components, demonstrating the potential immediate lethal effect caused by trawling of heavy fishing gears. Although, the passage of the fishing gear re-suspends resting eggs making them more available for hatching, it may also damage the eggs and reduces their hatching success. Additionally, the hatching success of those eggs taken in the wake of the rubber discs was higher than those eggs taken in the wake of the door suggesting that the forces in the sediment associated with the heavier door damages the eggs more than those from the lighter discs. Consequently, when assessing the effect of trawling on copepod resting eggs, it is important to consider it at the level of the individual gear components that comes in contact with the seabed. It is also important to take into account the intensity of the fishing effort, as in certain areas where the effort is high, the seabed may be passed by a trawl many times a year [35] and the cumulative effect on the copepod eggs will need to be considered in recruitment perspectives.

Conclusions
This study has for the first time demonstrated that fishing with demersal otter trawls is both a source of mortality of the copepod eggs but also a source of re-suspension, which has the potential to increase recruitment to the water column. To fully assess the trade-off between mortality and recruitment on copepod ecology it is necessary to assess the extent to which these eggs would otherwise be disturbed, re-enter the water column and hatch. Therefore, it is also necessary to estimate the re-suspension due to bioturbation and storm events under different environmental conditions. We propose that more studies looking at the effect of trawling on the survival, re-suspension and overall recruitment from resting stages in marine sediments should be carried out.