Food attractants of Nephrops norvegicus -search for artificial and alternative creel baits

Erika Andersson

Degree project for Master of Science (Two Years) in Marine ecology

Degree course in (BIO760) 30 hec Spring 2014

Department of Biological and Environmental Sciences University of Gothenburg

Examiner: Susanne Baden Department of Biological and Environmental Sciences University of Gothenburg

Supervisor: Anna Sara Krång Department of Biological and Environmental Sciences University of Gothenburg

Assistant Supervisor: Anette Ungfors Department of Biological and Environmental Sciences University of Gothenburg

Sammanfattning Det kommersiella fisket av havskräfta (Nephrops norvegicus) är ett av de mest ekonomiskt viktiga fiskena i Europa och Sverige. Trålning som står för 74% av landningarna i Sverige är emellertid mycket energikrävande och orsakar stora skador på havsbottnen. Burfisket står för resterande 26% av landningar. Samtidigt som det har mindre negativ inverkan på den marina miljön, måste burfisket effektiviseras ekonomiskt för att kunna konkurrera med trålning. Fiske av betesfisk står för omkring 10% av kostnaderna inom burfisket och över 1 kg saltad sill (Clupea harengus) används som bete per kg landad kräfta. Syftet med denna studie var att utveckla ett artificiellt bete som ett alternativ till sill och därmed minska behovet av att fiska efter bete och göra burfisket mer miljövänligt och ekonomiskt effektivt. En bioassay med en tvåvals-flum användes för att testa attraktionen av olika fraktioner av frystorkad sill för N. norvegicus. Fraktionering gjordes först med Kupchan separering. De mest attraktiva fraktionerna: extraherade med butanol (p=0.035) och hexan (p=0.007) separerades sedan ytterligare med SPE- kolumner. Därefter analyserades de mest attraktiva fraktionerna därifrån: extraherade med methanol 100% och hexan 100% (p=0.035 vardera) med GC-MS för att identifiera sammansättningen av ämnen i fraktionerna. Fyra ämnen som lyckades identifieras testades sedan i flumen, dock utan att vara signifikant attraktiva för havskräfta. I en separat fältstudie testades simkrabba (Liocarcinus depurator) som alternativt bete till havskräfta. Resultaten visade att simkrabbor stöts bort av döda artfränder, medan N. norvegicus varken stöts bort eller är attraherad av levande eller döda simkrabbbor. Dock kunde en trend ses mot att levande simkrabbor stör N. norvegicus.

Abstract The commercial fishery of the Norway lobster (Nephrops norvegicus) is one of the most economically important fisheries in Europe and Sweden. However, trawling that stands for 74% of the landings in Sweden is very energy demanding and causes vast damage to the seafloor. The creel fishery stands for the last 26% of the landings. While having far less negative impact on the marine environment, the creel fishery needs to get more economically efficient to be able to compete with trawling. About 10% of the cost for creel fishery is due to the fishing for baits, where over 1 kg salted herring (Clupea harengus) is used as bait per kg landed lobster. The aim of this study was to develop an artificial bait as an alternative to herring, to decrease the need to fish for bait and make the N. norvegicus creel fishery more environmentally and economically efficient. A bioassay using a two-choice flow- through flume was used to test the attraction of N. norvegicus to different fractions of freeze-dried herring. Fractionation was first done by Kupchan extraction. The most attractive fractions: butanol (p=0.035) and hexane (p=0.007) were further separated with SPE-columns. The most attractive fractions methanol 100% and hexane 100% (p=0.035 for both) were thereafter analysed with GC-MS to identify compound constitution. Four of these compounds (phosphoric acid, palmitic acid, myristic acid and pristine) were further tested in the flume, however, without being significantly attractive to the lobsters. In a separate field study, the swimming crab (Liocarcinus depurator) was tested as alternative bait as well to N. norvegicus. The results showed that the swimming crab is repelled by dead conspecifics, while N. norvegicus neither is significantly repelled nor attracted to live or dead swimming crabs, but a trend could be seen towards N. norvegicus being disturbed by live swimming crabs.

Keywords Norway lobster, swimming crab, salted herring, creel fishery, artificial bait, bioassay, fractionation

Image on the front page: Sue Scott-MarLIN, http://www.marlin.ac.uk/speciesfullreview.php?speciesID=38

Contents Sammanfattning ...... 2 Abstract ...... 2 Keywords ...... 2 Introduction ...... 1 The Nephrops norvegicus fishery today ...... 1 Foraging in Nephrops norvegicus ...... 2 Artificial and alternative baits ...... 3 The aim of the study and hypotheses ...... 4 Materials and methods ...... 5 Flume experiments ...... 5 Procedure ...... 6 Fractionations and compound identification ...... 8 Liquid-Liquid separation ...... 8 Column separation ...... 9 Gas chromatography ...... 10 Test concentrations of the compounds identified from the GC-MS ...... 10 Preparation of test odours ...... 13 Data analysis ...... 13 Field experiment ...... 13 Experimental set up ...... 13 Data analysis ...... 15 Results ...... 16 Flume experiments ...... 16 Liquid-liquid separation ...... 16 Column separation ...... 17 Gas chromatography ...... 17 Field experiment ...... 20 Landed lobsters ...... 21 Discarded lobsters ...... 22 Total catch of lobsters ...... 23 By-catch ...... 25 Discussion ...... 26 Flume experiments ...... 26 Field experiment ...... 31 Conclusions ...... 35 Acknowledgements ...... 36 References ...... 36

Introduction

The Nephrops norvegicus fishery today The Norway lobster (Nephrops norvegicus) fishery is with a total allowable catch (TAC) of more than 60000 tons 2014 one of the most important fisheries in Europe (Ungfors et al. 2013; EC, 2014). Sweden stands for just over 1300 tonnes (2%) of the TAC. In 2012 the value of landed lobsters in Sweden was € 12.8 million and the N. norvegicus fishery is ranked as the fourth most economically important fishery in Sweden, where herring (Clupea harengus) is the highest ranked with a value of € 17 million. Trawling is the most common method standing for 74% of the landings in Sweden. Two types of trawls are currently being used: species selective grid trawls and conventional trawls (Ziegler and Valentinsson, 2008; Ungfors et al. 2013). In the life cycle assessment of different fishing methods for N. norvegicus Ziegler (2006) found that even though grid trawling is better than conventional trawling in regards of species caught as by-catch, it is still damaging to the seabed (33000 m2 trawled per kg lobster in conventional trawling and 15600 m2 in grid-trawling) and the fuel used per kg landed lobster is still high compared to creel fishing. About 9 litres of diesel per kg lobster is consumed in conventional trawling and 4.3 litres in grid trawling (Ziegler, 2006). In contrast Hornborg et al. 2012 found grid trawling to be equal to conventional trawling in fuel use and seafloor swept per kilo landed lobster and even higher than conventional trawling per kilo landings (including fish).

Creel fishing that stands for the last 26% of the landings of N. norvegicus affects about 1.8 m2 of sea floor and uses 2.2 litre diesel per kg landed lobster. Despite using 1.1 kg salted herring as bait per kg landed lobster, the creel fishery is still considered less environmentally damaging than both types of trawling (Ziegler, 2006; Ziegler and Valentinsson 2008; Hornborg et al. 2012; Ungfors et al. 2013). There is still room for improvement though. The fishing for baits puts extra pressure on the herring stock as well as being a waste of a good food source for humans (Lindqvist et al. 2007). About 10% of the fuel in creel fishery comes from the herring fishery (Ziegler, 2006). The baiting of creels can also be seen as a semi aquaculture where undersized N. norvegicus able to move in and out of the creels are fed with an external food source which also increase the nutrient load into the ocean (Ziegler and Valentinsson, 2008). An artificial bait or an alternative bait that need not actively be fished for

1

and will decrease the nutrient load into the ocean could both have less negative impact on the environment and be more economic.

Foraging in Nephrops norvegicus N. norvegicus is a benthic, burrow digging decapod crustacean which can grow up to 25 cm in total length (rostrum-uropod). It is distributed from Iceland to Morocco in the northeast Atlantic Ocean and in the Mediterranean Sea to the Adriatic Sea and lives at depths from about 20-800 m on muddy substrates (Sabatini and Hill, 2008). It is an indiscriminate scavenger and predator, foraging on what is available (Cristo, 1998).

Foraging crustaceans sense chemical signals from the surroundings with the help of two different types of organs: the aesthetascs and the non-aesthetascs. The aesthetascs are unimodal chemosensory organs most similar to the vertebrate olfactory receptors. They are located on the antennules (1st antennae) of the animals (figure 1). The non-aesthetascs are bimodal sensilla consisting of both mechano- and chemosensory cells. They are located on the body, antennae, walking legs and mouthparts and are more similar to the gustatory receptors of vertebrates (Hallberg and Skog, 2011; Schmidt and Mellon, 2011). The aesthetascs and non-aesthetascs are both connected to the central nervous system via different chemosensory pathways. The aesthetascs connect to the olfactory lobe in the midbrain through the aesthetasc chemosensory pathway. The non-aesthetascs are in turn connected to various areas of the brain and ventral nerve cord via the non-aesthetasc chemosensory pathway (review by Derby, 2000; Schmidt and Mellon, 2011). In a study on the Caribbean spiny lobster (Panulirus argus) Steullet et al. (2001) found that both aesthetascs and non-aesthetascs had to be removed for the lobsters food searching ability to decrease, indicating that they together are involved in the food search behaviour.

The most attractive compounds to crustaceans are usually small water soluble molecules such as amino acids (McClain et al. 2012), but also amines and organic acids, but some species are also attracted to larger compounds such as proteins. Combinations of substances are often more attractive than single substances (Carr, 1978; Carr et al. 1984; Carr and Derby, 1986a; Carr and Derby, 1986b; Carr, 1988). Also the concentration of the substances relative to each other is important since synergetic effects can occur at certain concentrations while suppressive effects can occur at others. Suppressive substances can even make a solution less attractive even if the attractants are there (Zimmer-Faust et al. 1984; Carr and Derby, 1986b).

2

For example, in a study on the common gulf-weed shrimp (Leander tenuicornis) Johnson and Atema (1986) tested various different compounds and found that the organic acid taurine was the most attractive while the amino acid proline was inhibitory. Since there are high concentrations of taurine in crustacean prey and at the same time low concentrations in the surrounding seawater taurine might be a good food indicator for the predators (Johnson and Atema, 1986). However, Zimmer-Faust et al. (1984) found taurine to be a poor food attractant to the California spiny lobster (Panulirus interruptus). A mixture of L-amino acids and betaine has been suggested to be attractive to the Daggerblade grass shrimp Palaemonetes pugio (Carr, 1978).

Figure 1. The Norway lobster (Nephrops norvegicus). The arrow indicates the antennules of the animal. Drawing by Anna Sara Krång

Artificial and alternative baits A challenge when creating artificial baits is to make them both economically and environmentally profitable at the same time as being attractive to the target species. Both extracts of whole prey (Mackie et al. 1980), pellets (Ungfors A., Krång A-S. and Eriksson S. P. unpublished) or single and mixtures of synthetic substances (Mackie et al. 1980; Harpaz and Steiner, 1990; Mendoza et al. 1997) have been tested as attractants for several different decapod crustaceans, including some commercially fished species such as the Daggerblade grass shrimp (Palaemonetes pugio) (Carr and Derby, 1986a) and the European lobster (Homarus gammarus) (Mackie et al. 1980). Mackie et al. (1980) tested different kinds of artificial baits containing a mixture of compounds (L-amino acids, taurine, glycine and adenosine 5’-monophosphate, (AMP) amongst others) compared to a natural bait both in the field and in the lab. They found that it is not only the compound composition that decides the success of an artificial bait, but also the diffusion time from the bait. The artificial baits made

3

of gypsum in this experiment had a longer diffusion time and could therefore fish for a longer time than another artificial bait made of agar.

Instead of a truly artificial bait, also alternative baits may be of use. Investigations have started to see whether the swimming crab (Liocarcinus depurator) can be used as alternative bait to salted herring or as a complement to it, which would decrease the need to fish for baits separately (Ungfors A., Krång A-S. and Eriksson S. P. unpublished). Like N. norvegicus, the swimming crab is an opportunistic species feeding on dead and live feed (Hill, 2008). It is distributed from the Mediterranean Sea to Norway and lives on slightly harder substrates than N. norvegicus (sand and gravel bottoms) down to below 300 m (Hill 2008; Sabatini and Hill, 2008).

The swimming crab is caught as by-catch while fishing for N. norvegicus (Bergmann and Moore, 2001). In previous trials, fresh dead swimming crab has shown potential as bait for N. norvegicus (Ungfors A., Krång A-S. and Eriksson S. P. unpublished). Baiting with dead swimming crab was shown to give good captures (similar as traditional bait) on harder bottoms and less on softer bottoms. A combination of dead swimming crab and the traditional salted herring was shown to be a promising concept for high capture efficiency of N. norvegicus.

The aim of the study and hypotheses This study was done in collaboration with the EU FP7 SME project “NEPHROPS” (www.NEPHROPS.eu) with the aim to make creel fishery more competitive as compared to trawling and thus decrease the negative impact on the marine environment. The first aim of this study was i) to identify what type of compounds in the Atlantic herring are the most attractive to N. norvegicus and ii) to test these compounds in the field as artificial baits to compare their effectiveness to herring. The hypothesis was that this artificial bait would be comparable to herring in attractiveness. The second aim of this study was to test swimming crab (Liocarcinus depurator) as an alternative bait to salted herring in the field, with the hypotheses that live swimming crab are deterred by dead conspecifics and will avoid entering creels baited with dead swimming crab and also that N. norvegicus avoids live swimming crabs and thus the catch would be higher in creels with less by-catch of swimming crab. The effect would be more visible on harder substrates where there are more swimming crabs.

4

Materials and methods

Flume experiments All experiments were conducted at Sven Lovén centre for marine sciences, Kristineberg, Fiskebäckskil, Sweden.

The animals were caught by a local fisherman at depths around 50-60 m at various locations inside and in the area of the Gullmar fjord entrance in February and April 2014. They were kept in large tanks (≥400 l) free to walk around with cut pipes as shelters. The water temperature was kept constant at 12°C and the room had a light:dark cycle of approximately 12:12 hours.

Derby and Atema, (1981) observed that the American lobster (Homarus americanus) can get used to a certain food species if fed with the same feed for a month and will react to that at lower concentrations than another unfamiliar feed. Therefore the animals in this study were kept on a diet of alternating approximately two blue mussels (Mytilus edulis) and two shrimps (Pandalus borealis) per animal and feeding occasion so as not to get used to one particular feed. Only males were used with a mean carapace length (CL) of 44 ± 3.4 mm.

A two choice flow-through flume (260x46 cm) was used for the bioassay (figure 2). To get a laminar flow several plastic honeycomb bricks (of different length and diameter) were placed at the inflow as well as the outflow part of the chamber. The laminar flow was tested with fluorescein on both sides of the flume. The water level was kept at approximately 21 cm with a water flow of about 0.6 cm s-1. A peristaltic pump delivered the different test solutions from treatment bottles into the compartments of the flume (2.2 cm/s) through a 20 µl pipette stuck into the last brick of honeycomb before the test arena and attached to a tube (inner diameter 3mm) with an approximate length of 1 m connected to the outflow of a smaller tube (inner diameter 1.85mm, length 41 cm) going through the pump. On the other side the pump tube was connected to an 80 cm long tube of the same inner diameter as the tube connected to the pipette (figure 3). A net cage was placed in the middle of the outflow part of the test arena with a closable opening towards the inflow. The water temperature was set to 12°C throughout the experiment, although, some variation in water flow, water level and water temperature occurred at some occasions due to sedimentation in the temperature regulator.

5

Procedure A lobster was collected from its holding tank and put into the cage (with gate opened) in the flume, free to explore the whole test arena for 30 minutes. After this acclimatisation the cage gate was closed, the lobster returned to the cage and the pump started, pumping only filtered seawater into both sides of the flume for 15 minutes. Then the tubes were put into the different treatments. The cage was opened again when the odour would just have reached the lobster about 4 minutes later. The lobster had a maximum of 30 minutes from the opening of the cage to make a choice between the two treatments. Between each lobster the pump tubes were changed back to filtered seawater. The sides and bottom of the flume was also brushed in between lobsters where after the flume was flushed through with seawater for 20 minutes before a new lobster was tested.

The time parameters above were used for the tests: herring vs seawater and herring vs swimming crab. The time parameters for all the following tests were adjusted due to time limitations of the project. The new time parameters were decided based on the results of the first tests and were: 15 minutes acclimatisation (free to explore the flume) followed by 5 minutes inside the cage before onset of treatment odour and finally 15 minutes to make a choice. If the lobsters were on their way towards the chambers when the time was out, they were still allowed to continue as long as they were heading for a chamber and were accounted for if they made a choice without hesitation. The flume was flushed with seawater 10 minutes in between lobsters.

Each treatment was pumped into the same compartment twice before changing sides of the tubes so that each treatment was pumped from both sides equally many times, while at the same time being more time efficient than if the sides had been changed after each run. The start order was determined by randomization. At least ten lobsters were tested per treatment. Treatments where several lobsters responded to the treatment were further tested until at least 15 lobsters had made a choice. Each lobster was only tested once for the same compound. However, each lobster was used approximately three times in the flume experiments in total, but with at least one week’s rest in between each trial. For the test of preference between swimming crab and herring the lobsters had starved for over two weeks while in the other experiments the lobsters were starved at least six days before the start of the experiment, due to time limitations.

6

Figure 2. The flume (viewed from above) used for bioassays, testing food attractants of N. norvegicus.

Figure 3. Simplified drawing of the flume and the pump. Blue arrows indicate the tubes connected to the pump, delivering the test solutions from the bottles with odour and seawater (SW) to the flume. Black arrows indicate the honeycombs connected to the tubes. Pump tubes are marked with red.

7

Fractionations and compound identification To identify what compounds within the herring are attractive to N. norvegicus a series of fractionations were performed in two steps. 1) Liquid-liquid separation creating five fractions with a broad spectrum of polarities and 2) Column separation fractioning the most attractive fractions from the liquid-liquid separation into a narrower polar spectrum.

The most attractive fractions from the column separation were then tested with gas chromatography mass spectrometry (GC-MS) wherefrom four possibly attractive substances were identified.

Liquid-Liquid separation In order to be sure that enough material was extracted, an excess of unsalted freeze-dried herring (176.23 g) was mixed to small fragments and put into a beaker with dichloromethane on a shaking-table for one hour and sieved through a Munktell filter paper 3. The procedure was repeated once. Thereafter the herring mixture was mixed with equal parts dichloromethane and methanol twice and finally with methanol twice following the same procedure as with the dichloromethane extraction. The extracts were combined and the solvents were evaporated using a BÜCHI Rotavapor R-200 at 36°C, until only little remained creating a crude extract which was stored in a fridge overnight.

The following day the crude extract was mixed with different solvents according to the Kupchan separation method (Kupchan, 1970), but with some modification in solvents used. Five fractions (table 1) of different polarities were extracted with six solvents. From nonpolar to polar the solvents used were hexane, chloroform, ethyl acetate, butanol, methanol and MQ water. The MQ fraction will be referred to as the water fraction.

In the beginning the crude extract was poured into a separation funnel together with the polar solvents methanol and MQ in the proportions 9:1 to a total of 367 ml combined with 450 ml of the nonpolar hexane. The mixture was shaken after which it was left to separate for at least one hour or until separation had occurred. The different fractions were poured into separate round bottles. The hexane fraction was subsequently evaporated while the water:methanol fraction ratio was altered to 3:2 to a total of 550 ml and mixed in the separation funnel with 450 ml chloroform. After separation the chloroform was treated in the same way as the hexane fraction, while the water:methanol fraction was evaporated in the rotavapor until only water remained and thereafter refilled with more MQ to a total volume of 450 ml and mixed

8

with 450 ml ethyl acetate for separation. After separation the ethyl acetate fraction was evaporated (but some of the odour still remained as with the chloroform and hexane fractions). The water fraction was put into the rotavapor until almost no smell of ethyl acetate remained, refilled to 450 ml and mixed with butanol in the separation funnel. The two fractions were then evaporated as much as possible. In addition to the rotavapor, all fractions were also evaporated with the help of heating blocks (40°C) with nitrogen gas and/or a Heto vacuum centrifuge.

Since the wet weight (569.8 g) of the freeze dried herring was unknown when the concentrations for each test-fraction was calculated, the dry weight of the freeze-dried herring was considered to be approximately 33% of the wet weight. The proportion of each fraction was calculated to match 10 g wet weight herring l-1 water, (table 1).

The concentration of the water fraction represents slightly less than 10 g herring l-1 due to a calculation error. However, due to uncertainties of the precision of the scale used and the inevitable fact that some material disappeared in each step of handling despite efforts to prevent this, all concentrations used are approximations.

Column separation The butanol fraction extracted with liquid-liquid separation was further separated into four new fractions (table 1) through a reversed phase SPE SUPELCO ENVITM – 18 packing column, 60 ml, 10 g. The solvents used to obtain the new extracts were methanol:MQ (1:19), methanol:MQ (1:4), methanol:MQ (1:1) and 100% methanol. Before adding the samples, the carbon-chains in the column were activated by pouring one column length of 100% methanol through the column, after which one column volume of the first solvent (5% methanol) was run through once before adding the sample. Approximately 60 ml solution went through the column for each fraction. Since not the entire butanol fraction dissolved or even entered the column even in 100% methanol, a try was made to dissolve the remains with MQ instead (without success). In addition to the four planned fractions, this “MQ fraction” was also tested in the flume.

The hexane fraction was separated into five new fractions (table 1) to be tested in the flume using a normal phase SPE TELOSTM Flash silica column, 70 ml, 20 g. The solvents used for

9

the separation were 100% hexane, hexane:ethyl acetate (49:1), hexane:ethyl acetate (19:1), hexane:ethyl acetate (4:1) and 100% ethyl acetate. Since the extract volume was too large to put all at once in the column, the separation was done in several rounds. The column was rinsed with approximately 210ml ethyl acetate and 70 ml hexane after each round. Between 70-140 ml solution went through the column for each fraction per round.

All fractions were dried using a BÜCHI Rotvapor R-200 and a Heto vacuum centrifuge.

Gas chromatography The two fractions from the column separations that were found to be most attractive to N. norvegicus (methanol 100 % and hexane 100 %), were prepared for gas chromatography mass spectrometry (GC-MS) by adding a small portion of each fraction into 1 ml of methanol or hexane respectively. These samples were then diluted to concentrations of 1/100 and 1/10. 200 µl of each concentration was transferred into small vials and were evaporated in 31°C by nitrogen gas. Derivatization of the molecules in the fractions was done to make the polar compounds in the samples more volatile and thermally stable so as to ease their transport through the GC-MS system (Fluka Chemica, 1995). This was done in two steps: First by methoxymation: 60 µl of O-methoxylhydroxylamine hydrochloride in pyridine (20 mg methoxyamine HCl per ml pyridine) was added to each sample and incubated in 31°C for 30 minutes. Thereafter by silylation: 60 µl of N-Methyl-N-(trimethlsilyl)trifluoroacetamide (MSTFA) was added as well and incubated in 37°C for 1.5 hours. An additional sample containing 50 µl alkane solution C8-C20 and 50 µl alkane solution C21-C40 was prepared as a retention time control. Also a hexane blank as a negative control was prepared. All samples and controls were transferred into GC vials with inlets and run through a Mid-polarity DB200 column in an Agilient Technologies 6890N Network GC System and Agilient Technologies 5973 inert Mass Selective Detector. The initial temperature was set to 60°C and the maximum temperature to 300°C. The total length of each measurement was 27 minutes. The outcome of the data was analysed using NIST MS Search 2.0 software.

Test concentrations of the compounds identified from the GC-MS Out of all peaks found in the chromatograms of the methanol 100% and the hexane 100% fractions (figures A1 and A2 respectively, appendix), four compounds were identified. These were phosphoric acid, myristic acid, palmitic acid that were present in both fractions and also pristane, which was only present in the hexane 100% fraction. They were ordered from Sigma

10

Aldrich (phosphoric acid (85%), myristic acid (≥98%), palmitic acid, (free acid) and synthetic pristane) to test their attractiveness to N. norvegicus.

Phosphoric acid was the first compound to be tested for attractiveness, since it is the cheapest of the four substances ordered. It was not possible to see an exact concentration of each substance in the chromatograms and synergetic effects can also make combined substances more attractive at lower concentrations than one of the substances tested on its own (Zimmer- Faust et al. 1984; Carr & Derby, 1986b) making it harder to decide which concentration to use. In the end the concentration used was based on the total hexane 100% concentration. However, the final concentration (2E-3 M) used was slightly lower due to a calculation error.

Thereafter three mixtures containing all four compounds in different concentrations (table 1) were prepared and tested in the flume. For Mix 1 all substances had the same concentration as the phosphoric acid when tested on its own (2E-3M). When Mix 1 did not turn out to be attractive to N. norvegicus, the concentration in Mix 2 was set to correspond to the relative abundance between the substances seen in the hexane 100% chromatogram. The hexane 100% chromatogram was chosen over the methanol 100% chromatogram since all substances were represented in the former. The same concentration as before was used for the phosphoric acid. Even though the relative abundance of the substances differed between the methanol 100% and the hexane 100% fraction and it is impossible to see an exact abundance of the substances in the chromatograms, phosphoric acid had the highest concentration in both, followed by palmitic acid and myristic acid in the methanol 100% fraction. Pristane had a higher abundance than palmitic acid in the hexane 100% fraction though. In the third mix (Mix 3) the concentration of all four compounds was set to 10-2 M since previous studies have found organic and amino acids attractive to decapods at this concentration to crustaceans (Shelton and Mackie, 1971; Coman et al. 1996; Krång A-S. personal communication).

The calculation of the phosphoric acid concentrations alone and within the mixtures was based on a 100% solution while the solution used only contained 85% phosphoric acid in water. Therefore the actual concentrations of phosphoric acid that were used were slightly lower than written in the text (table 1).

11

Table 1. Concentrations used for the different fractions. The concentrations were calculated to be proportional to 10 grams (wet weight) of herring per litre water. Actual concentrations of phosphoric acid used in the experiment are written within brackets.

12

Preparation of test odours The swimming crab was killed and crushed and the salted herring was cut into pieces the day before the experiment and diluted in filtered (0.5 µm) seawater over night with the concentration 10 g swimming crab or herring per litre filtered seawater. The odour water was filtered the next morning through a 200 µm filter and put on ice throughout the experiment.

The fractions and compounds tested were prepared by adding the right concentration into filtered (0.5 µm) seawater the same day as the experiment. Those not directly soluble in seawater were mixed by a magnetic stirrer between the tests. Fractions and compounds still not dissolving were put in an ultrasonic water bath (30°C) until dissolved or for maximum one hour.

Data analysis All data from the flume experiments were tested with exact One-Sample Binomial Tests in IBM SPSS Statistics 22, which does not take into account the number of lobsters not making a choice. This test was chosen over more common tests like Chi-square and G-test due to small sample sizes (McDonald, 2014) Alpha was set to 0.05 and the confidence interval was 95%.

Field experiment The swimming crab (Liocarcinus depurator) was tested in the field, both fresh dead crabs as alternative bait to salted herring and deterrent to L. depurator, as well as live crabs to test if they are deterrent to N. norvegicus.

Experimental set up Seven links of creel cages were put out at seven locations inside the Gullmar fjord on two occasions (first occasion 3rd to 6th of February and second occasion 6th to 10th of February i.e. three and four soak-days respectively). Three out of seven links were put on sandy clay bottom (“hard bottom”) and four on clay bottom (“soft bottom”), (figure 4: table 2). Four different baits were used: 1. salted herring combined with three live swimming crabs individually marked with cable ties around chela, 2. salted herring, 3. Salted herring combined with fresh dead crushed swimming crab (approximately 3 individuals) within a bait box and 4. fresh dead swimming crab (approximately 3 individuals) within a bait box. The baits were put into the creels in a predestined, randomized order. Two of the hard bottom links contained 40 creels while one contained 26 creels. The soft bottom links contained 26, 27, 25 and 28 creels

13

respectively. D-shaped single cages (58Lx42Wx32H cm) were used for all links except the two longest links, where rectangular parlour chamber creels (70Lx40Wx28H cm) were used.

Since there was not an equal amount of creels for each bait type on every link, excess creels were randomly excluded from the analysis. The link on the first location (Kolvik) on the first occasion was excluded from the analysis due to a mistake with the notation of the bait orders. After random exclusion to balance the data 43 creels per bait type on the first occasion and 51 creels per bait type on the second occasion were left for analysis.

2

Figure 4. Map over the locations for creel fishery field tests in the Gullmar fjord, Sweden (Google Maps). Locations used on both occasions are marked with red, locations only visited the first occasion are marked with blue and the ones used only on the second occasion are marked with yellow. The marine research station Kristineberg is marked with green.

14

Table 2. Detailed information of the locations used for the field test on the two occasions February 3rd -6th and February 6th -10th.

Occasion 1 Location Latitude Longitude Depth (m) Substrate Cage type No. of cages No. of cages (corrected) Kolvik 58°20.352N 011°34.749E 40-60 Soft bottom Single cage 26 0 Jordfall 58°19.834N 011°34.215E 39-48 Soft bottom Single cage 27 24 Fossen 58°18.954N 011°32.773E 49-65 Soft bottom Single cage 25 20 Döns bukt 58°18.738N 011°32.565E 36-65 Soft bottom Single cage 28 28 Gåseklåvan 58°18.328N 011°32.122E 44-60 Hard bottom Single cage 26 24 Essvik 58°17.452N 011°31.321E 34-60 Hard bottom Chamber cage 40 36 Yttre Essvik 58°16.867N 011°30.610E 36-58 Hard bottom Chamber cage 40 40 Occasion 2 Location Latitude Longitude Depth (m) Substrate Cage type No. of cages No. of cages (corrected) Kolvik 58°20.359N 011°34.745E 31-65 Soft bottom Single cage 26 24 Jordfall 58°19.814N 011°34.123E 43-60 Soft bottom Single cage 27 24 Fossen 58°18.934N 011°32.731E 40-65 Soft bottom Single cage 25 24 Döns bukt 58°18.739N 011°32.499E 40-60 Soft bottom Single cage 28 28 Gåseklåvan 58°18.300N 011°32.069E 40-65 Hard bottom Single cage 26 24 Essvik 2 58°16.723N 011°30.120E 44-54 Hard bottom Chamber cage 40 40 Stadsportsdammen 58°15.869N 011°27.143E 31-41 Hard bottom Chamber cage 40 40

Data analysis All statistical tests were conducted in IBM SPSS Statistics 22.

The relative effects of the factors bait, substrate and location (table 3), as well as the interaction between bait and substrate and between bait and location on the dependent variables were tested with three factor nested ANOVAs. Dependent (test) variables were: 1) number of landed lobsters (carapace length CL ≥ 40 mm), 2) number of discarded lobsters (undersized, CL < 40mm), 3) total numbers of lobsters and 4) by-catch of swimming crabs (numbers). The two occasions were tested separately. Location was nested under substrate since the same locations were not present on both hard and soft bottom and was set as random in the analysis.

The post-hoc test Tukey HSD was used to see which levels differed within the factor bait, when it was significant (no post-hoc test was done for location (since it was a nested factor) or for substrate since it only had two levels). The homogeneity of variances was tested with Levene’s test of equality of error variances while the normal distribution of all levels within each factor was tested with Shapiro Wilk’s test of normality.

Since none of the test variables were normally distributed except landed lobsters (p=0.141) and the total number of lobsters caught (p=0.090, with log transformation) on the first occasion at the location Fossen, and because only landed (p=0.068 first occasion and p=0.053 second occasion) and the total catch of lobsters (p=0.220 first occasion and p=0.064 second

15

occasion, with log transformation) had homogenous variance, the conditions for parametric testing were not fully met and nonparametric tests were conducted as comparison. Also the effect of cage type (single cages or chamber cages) was tested as a factor here, although both cage types did not exist on both substrates or on every location.

The two occasions (February 3rd-6th and February 6th-10th) were tested separately also with the nonparametric tests, with the same dependent variables as in the parametric tests. The relative effects of bait and location on catch of lobsters and by-catch were tested with Independent- Samples Kruskal-Wallis (KW) tests and the effects of substrate and cage type on catch of lobsters and by-catch were tested with Independent-Samples Mann-Whitney U (MW) tests.

Table 3. The factors tested in the three-factor nested ANOVA and their levels. Factor Level Bait (four levels) Salted herring and live swimming crab Salted herring Salted herring and dead swimming crab Dead swimming crab Substrate (two levels) Hard bottom (Sandy clay) Soft bottom (Clay) Location (seven levels) Kolvik Jordfall Döns bukt Gåseklåvan Essvik Yttre Essvik Essvik 2 Stadsportsdammen

Results

Flume experiments The results from the flume experiment tests show that N. norvegicus significantly preferred herring over seawater (p=0.017; figure 5A). However, it had no preference for either herring or swimming crab compared to each other (p=0.815; figure 5B).

Liquid-liquid separation Of the fractions separated by Kupchan separation, the butanol and hexane fractions were both significantly more attractive compared to seawater: 12 out of 15 lobsters chose butanol over seawater (p=0.035) and 13 out of 15 preferred hexane (p=0.007; table 4; figure 6). However, only one lobster did not respond at all in the butanol test, while 14 did not respond in the 16

hexane test, indicating that the butanol fraction was more attractive than the hexane fraction despite the higher significance in the latter.

The rest of the fractions were not significantly attractive to the lobsters (table 4; figure 6). However, the lobsters tended to react to the water fraction since most lobsters chose this fraction over seawater (11 vs. 4; p=0.118), but only 15 out of 27 lobsters responded to the stimuli. Also the chloroform fraction showed some potential as attractant to N. norvegicus, but did not differ significantly from seawater (p=0.302).

Column separation Of the fractions deriving from the original butanol fraction separated in the SPE columns, the methanol 100% fraction was undoubtedly the most attractive (p=0.035; table 4; figure 7A). 12 out of 15 lobsters chose this fraction over seawater, while 8 lobsters did not respond. None of the other fractions deriving from butanol were significantly attractive. No lobsters chose seawater in the Methanol 50% test and therefore no statistical test could be made for this fraction. However, only three chose the fraction and 12 did not respond.

Of the fractions deriving from the original hexane fraction, hexane 100% was the most attractive one (p=0.035; table 4; figure 7B). The same number of lobsters responded to the hexane 100% fraction as to the methanol 100% fraction (12 out of 15 choosing the fraction over seawater), but in contrast 14 lobsters did not make a choice. The rest of the fractions deriving from the hexane fraction were not significantly attractive.

Gas chromatography Out of the three different mixtures (Mix 1, 2 and 3) made out of the compounds identified with GC-MS, Mix 3 (10-2 M) showed most potential as attractant compared to seawater, although not significantly so (p=0.388). Mix 1 and 2 were not attractive compared to seawater (p=1.000 for both). Phosphoric acid seemed promising at first, but as more lobsters were tested the fraction was not very attractive compared to seawater after all (p=0.727; table 4; figure 8).

17

Table 4. Summary of all results from the flume experiments, showing the number of N. norvegicus choosing the odour, the seawater control and not making a choice and the total number of lobsters tested for each test solution as well as the p-values of the Binomial-tests. Choice Name Odour Seawater No choice Total p-value Herring 17 5 3 26 0.017 Liquid-liquid separation Water 11 4 12 27 0.118 Butanol 12 3 1 16 0.035 Ethyl acetate 3 1 11 15 0.625 Chloroform 10 5 13 28 0.302 Hexane 13 2 14 29 0.007 Column separation (butanol fraction) MQ 3 2 5 10 1.000 Methanol 5 % 9 6 17 32 0.607 Methanol 20 % 2 1 7 10 1.000 Methanol 50 % 3 0 12 15 - Methanol 100 % 12 3 8 23 0.035 Column separation (butanol fraction) Ethyl acetate 100 % 1 3 6 10 0.625 Ethyl acetate 20 % 2 5 3 10 0.453 Ethyl acetate 5 % 3 1 6 10 0.625 Ethyl acetate 2 % 1 3 6 10 0.625 Hexane 100 % 12 3 14 29 0.035 Compounds identified with GC-MS Phosphoric acid 5 3 12 20 0.727 Mix 1 2 1 7 10 1.000 Mix 2 2 2 6 10 1.000 Mix 3 8 4 11 23 0.388 Swimming Herring vs Swimming crab Herring crab No choice Total p-value Herring vs Swimming crab 8 10 6 24 0.815

20 12

10 15 8 10 6 4

5 2 Numberoflobsters Numberoflobsters 0 0 Herring Swimming No choice Herring Seawater No choice crab Choice A Choice B

Figure 5. The number of Norway lobsters (Nephrops norvegicus) choosing herring compared to seawater (A) and the number of lobsters choosing herring compared to swimming crab (B).

18

16

14

12

10

8

6

Numberoflobsters 4

2

0 Water Butanol Ethyl acetate Chloroform Hexane Treatment

Figure 6. The attractiveness of the Norway lobster (Nephrops norvegicus) to the different fractions from the liquid-liquid separation compared to seawater control. The bars show the number of lobsters choosing the odours (blue), seawater (red) and the number of lobsters that did not make a choice (purple) for each of the fractions.

18 16

16 14 14 12 12 10 10 8 8 6 6 4

4 Numberoflobsters Numberoflobsters 2 2 0 0 MQ MethanolMethanolMethanolMethanol Ethyl Ethyl Ethyl Ethyl Hexane 5 % 20 % 50 % 100 % acetate acetate acetate acetate 100 % 100 % 20 % 5 % 2 % Treatment A Treatment B

Figure 7. The attractiveness of the Norway lobster (Nephrops norvegicus) to the fractions deriving from the butanol fraction (A) and the ones deriving from the hexane fraction (B) compared to seawater control. The bars show the number of lobsters choosing the odours (blue), seawater (red) and the number of lobsters that did not make a choice (purple) for each of the fractions.

19

A

14

12

10

8

6

4 Numberoflobsters 2

0 Phosphoric acid Mix 1 Mix 2 Mix 3 Treatment

Figure 8. The attractiveness of the compounds identified with GC-MS: phosphoric acid and the different mixtures of phosphoric acid, myristic acid, palmitic acid and pristane compared to seawater control to the Norway lobster (Nephrops norvegicus). The bars show the number of lobsters choosing the odours (blue), seawater (red) and the number of lobsters that did not make a choice (purple) for each of the fractions.

Field experiment The results from the field study testing swimming crab as alternative bait is presented separately for each catch variable (landed lobsters, discarded lobsters, total catch of lobsters and by-catch). Both parametric and nonparametric statistical results are summarised in table 5. None significant interactions are not mentioned in the text. Additional statistical results are presented in the appendix.

20

Table 5. The main results of the field study for both occasions. The results of the parametric three-factor nested

ANOVA to the left and the nonparametric results from the Kruskal Wallis tests (Bait and Location) and the

Mann Whitney U tests (Substrate and Cage type) to the right. df = Degrees of freedom, Standard error (SE) shown within brackets. Significant p-values are shaded grey. Parametric (three-factor nested ANOVA) Nonparametric tests Occasion 1 Occasion 2 Occasion 1 Occasion 2 Landed lobsters p-value F-value df p-value F-value df p-value Test statistic df (SE) p-value Test statistic df (SE) Bait 0.052 3.356 3 0.070 2.888 3 0.189 4.775 3 0.070 7.063 3 Substrate 0.180 2.627 1 0.054 6.314 1 0.000 2235.5 (306.820) 0.001 3917.0 (387.128) Location 0.000 7.790 4 0.155 1.628 5 0.000 41.077 5 0.004 18.876 6 Cage type ------0.000 1790.0 (308.859) 0.046 4205.0 (378.089) Bait*Substrate 0.287 1.401 3 0.085 2.681 3 ------Bait*Location 0.37 1.093 12 0.657 0.818 15 ------Discarded lobsters Bait 0.101 2.559 3 0.226 1.623 3 0.257 4.045 3 0.077 6.832 3 Substrate 0.190 2.481 1 0.038 7.825 1 0.000 2529.0 (218.040) 0.000 3677.0 (269.784) Location 0.000 7.993 4 0.001 4.384 5 0.000 47.481 5 0.000 48.974 6 Cage type ------0.000 2760.0 (219.488) 0.000 3736.5 (263.484) Bait*Substrate 0.853 0.26 3 0.38 1.101 3 ------Bait*Location 0.216 1.315 12 0.071 1.628 15 ------Total catch of lobsters Bait 0.030 4.047 3 0.025 4.167 3 0.234 4.265 3 0.021 9.778 3 Substrate 0.102 4.459 1 0.017 12.473 1 0.000 3958.5 (254.148) 0.000 3099.0 (396.604) Location 0.000 9.359 4 0.011 3.053 5 0.000 60.293 5 0.000 39.298 6 Cage type ------0.000 1382.0 (312.605) 0.000 3504.5 (387.344) Bait*Substrate 0.244 1.560 3 0.050 3.316 3 ------Bait*Location 0.808 0.636 12 0.386 1.072 15 ------By-catch Bait 0.050 3.45 3 0.005 6.351 3 0.003 13.841 3 0.000 23.114 3 Substrate 0.803 0.071 1 0.203 2.152 1 0.158 1804.5 (310.542) 0.009 5988.0 (299.742) Location 0.000 7.353 4 0.005 3.463 5 0.000 32.127 5 0.001 23.217 6 Cage type ------0.000 4582.5 (255.837) 0.000 6169.5 (292.743) Bait*Substrate 0.839 0.28 3 0.078 2.775 3 ------Bait*Location 0.072 1.699 12 0.54 0.923 15 ------Landed lobsters Bait had no significant effect on the number of landed lobsters on any occasion (p=0.052 and 0.070 respectively, three-factor ANOVA), although on both occasions a trend could be seen with slightly more lobsters being caught in average with salted herring in combination with dead swimming crab, as well as slightly fewer being caught with only dead swimming crab (figure 9A). Substrate had no significant effect on the number of landed lobsters on any occasion (p=0.180 and p=0.054 respectively), while the number of landed lobsters differed between locations on the first occasion (p=0.000) but not on the second (p=0.155; table 5; figure 9B).

Neither in the nonparametric test was there any significant effect of bait on any occasion (p=0.189 and p=0.070 respectively, KW; figure 9A; table 5). In contrast to the parametric results substrate had an effect on the catch of landed lobsters though on both occasions. More landed lobsters were caught on soft (clay) bottom than on hard (sandy clay) bottom (p=0.000 and p=0.001 respectively, MW). Also cage type had an effect, with more lobsters being

21

caught with single cages compared to chamber cages on both occasions (p=0.000 and p=0.046 respectively, MW). The location affected the number of landed lobsters on both occasions (p=0.000 and p=0.004 respectively, KW). On the first occasion, fewer landed lobsters were caught in Yttre Essvik than in Gåseklåvan, Jordfall (p=0.001 for each) and Fossen (p=0.000). There was also fewer landed lobsters caught in Essvik than in Gåsklåvan (p=0.017), Jordfall (p=0.011) and Fossen (p=0.001; figure A3, appendix). On the second occasion there was a higher catch of landed lobsters in Jordfall than in Gåseklåvan (p=0.014) and Essvik 2 (p=0.006; figure A4, appendix).

A B

Figure 9. Mean number of landed Norway lobsters (Nephrops norvegicus)caught (A) with different baits and (B) at different locations. Blue bars show the mean number of landed lobsters on the first occasion and green bars show the second occasion. Error bars: ± SE.

Discarded lobsters Although bait had no significant effect on the number of discarded lobsters caught on any occasion (p=0.101and p=0.226 respectively, tree-factor ANOVA; table 5) slightly more discarded lobsters were caught with salted herring in average than with any of the other baits (figure 10A). The substrate had no effect on the first occasion (p=0.190), but on the second occasion (p=0.038), with more lobsters being caught on soft bottom than on hard bottom (figure 10B). The locations differed significantly in number of discarded lobsters caught on both occasions (p=0.000 and p=0.001 respectively).

As in the parametric test bait did not significantly affect the number of discarded lobsters caught on any occasion (p=0.257 and p=0.077, respectively, KW; figure 10A). However the catch of discarded lobsters differed between substrates on both occasions (p=0.000 for both, MW; figure 10B). More lobsters were caught on soft bottom than on hard bottom. On both

22

occasions also cage type, with more lobsters being caught in single cages than chamber cages (p=0.000 for both, MW) and location had a significant effect on the catch of discarded lobsters (p=0.000 for both, KW). On the first occasion significantly more discarded lobsters were caught in Jordfall than at the other locations (p=0.000 for each; figure A5, appendix), while on the second occasion more lobsters were caught in Döns bukt (p=0.008), Fossen (p=0.001) and Jordfall (p=0.000) than in Stadsportsdammen. Also in Fossen and Jordfall significantly more lobsters were caught than in Gåseklåvan (p=0.012 and p=0.004 respectively) and Kolvik (p=0.015 and p=0.006 respectively; figure A6, appendix; table 5).

A B

Figure 10. Mean number of discarded Norway lobsters (Nephrops norvegicus) caught (A) with different baits and (B) on different substrates. Blue bars show the mean number of discarded lobsters on the first occasion and green bars show the second occasion. Error bars: ± SE.

Total catch of lobsters Bait had a significant effect on the total number of lobsters caught on both occasions (p=0.030 and p=0.025 respectively; three-factor ANOVA; table 5; figure 11A). However the bait types did not differ significantly in the post-hoc test on the first occasion. On the second occasion creels baited with dead swimming crab caught a smaller number of lobsters than both salted herring (p=0.014) and salted herring combined with dead swimming crab (p=0.010; table A1, appendix). The trend was similar on the first occasion. More lobsters were caught on soft bottom (p=0.017) than on hard bottom on the second occasion, while no significant effect was observed on the first occasion. Also the interaction between substrate and bait, had a significant effect on the catch of lobsters on the second occasion (p=0.050) and a trend could be seen where salted herring combined with dead swimming crab fished relatively better than the other baits on hard bottom compared to soft bottom where all baits containing herring

23

fished better than dead swimming crab (figure 11B). Location had a significant effect on the total catch of lobsters on both occasions (p=0.000 and p=0.011, respectively).

In the nonparametric test the total number of lobsters did not differ between the baits on the first occasion (p=0.234, KW), but on the second occasion (p=0.021). The relative effect of the baits was the same as in the parametric test. Creels baited with salted herring combined with dead swimming crab and salted herring alone both caught a higher total number of lobsters than dead swimming crab (p=0.046 for both; figure A7, appendix). More lobsters were caught on soft bottom than on hard bottom on both occasions (p=0.000 for both, MW; table 5). Also the effect of location on the total catch of lobsters was significant on both occasions (p=0.000 for both, KW). On the first occasion fewer lobsters were caught in Yttre Essvik than in Döns bukt (p=0.007), Gåseklåvan (p=0.000), Fossen (p=0.000) and Jordfall (p=0.000). Also in Essvik fewer lobsters were caught than in Gåseklåvan (p=0.007), Fossen (0.001) and Jordfall (p =0.000), (figure A8, appendix). On the second occasion the total number of lobsters caught in Essvik 2 was lower than in Döns bukt (p=0.025), Fossen (p=0.012) and Jordfall (p=0.000). In Jordfall more lobsters were caught than in Gåseklåvan (p=0.000) and Stadsportsdammen (p=0.003) as well (figure A9, appendix).There was also more lobsters caught with single cages compared to chamber cages (p=0.000 for both, MW) on both occasions (table 5).

A B

Figure 11. (A) The mean total number of Norway lobsters (Nephrops norvegicus) caught with different baits.

Blue bars show the mean total number of lobsters on the first occasion (February 3rd-6th) and green bars show the second occasion (February 6th-10th). (B) The mean total number of N. norvegicus caught on different substrates (soft bottoms shown with blue bars and hard bottoms shown with green bars) and with different baits. Only data from the second occasion are presented here. Error bars: ± SE.

24

By-catch Bait had a significant effect on by-catch of swimming crabs on both occasions (p=0.050 and p=0.005 respectively, three-factor ANOVA), (figure 12A, table 5). Creels baited with salted herring caught a higher by-catch than those baited with salted herring combined with dead swimming crab (p=0.036) on the second occasion while the same was only a trend on the first occasion (p=0.052). Creels baited with salted herring caught a higher by-catch than those baited with dead swimming crab on both occasions (p=0.000 on both occasions). Also creels baited with salted herring combined with live swimming crabs caught a higher by-catch than dead swimming crab alone on both occasions (p=0.029 and p=0.009 respectively; tables A2 and A3, appendix). There was no significant difference in by-catch between creels baited with salted herring and salted herring combined with live swimming crab (p=0.576), dead swimming crab and salted herring combined with dead swimming crab (p=0.295) nor between salted herring combined with dead swimming crab and salted herring combined with live swimming crab (p=0.475). Location had a significant effect on by-catch on both occasions (p=0.000 and p=0.005, respectively), (figure 12B), while substrate had no effect on any occasion (p=0.803 and p=0.203 respectively; table 5).

Bait had a significant effect on by-catch on both occasions (p=0.003 and p=0.000 respectively, KW). The relative effect of bait on by-catch was the same in the nonparametric test as in the parametric test. On the first occasion creels baited with dead swimming crab had a significantly lower by-catch than those baited with salted herring combined with live swimming crab (p=0.019). Dead swimming crab also caught a lower by-catch than salted herring (p=0.004; figure A10, appendix). On the second occasion creels baited with salted herring caught a higher by-catch than those baited with dead swimming crab (p=0.000) as well as salted herring combined with dead swimming crab (0.030). Salted herring combined with live swimming crab also caught a higher by-catch than creels baited with only dead swimming crab (p=0.030; figure A11, appendix; table 5). By-catch did not differ between the substrates on the first occasion (p=0.158, MW), but on the second occasion a higher by-catch was caught on hard bottom than on soft bottom (p=0.009). The by-catch was higher in chamber cages than in single cages on both occasions (p=0.000 for both, MW). Also location had a significant effect on by-catch on both occasions (p=0.000 and p=0.001 respectively, KW). On the first occasion there was a higher by-catch of swimming crabs in Essvik than in Gåseklåvan (p=0.000), Döns bukt (p=0.001), Fossen (p=0.015) and Yttre Essvik (p=0.003), (figure A12, appendix). On the second occasion there was a higher by-catch in

25

Stadsportsdammen than in Kolvik (p=0.030), Jordfall (p=0.030) and Gåseklåvan (p=0.030), (figure A13, appendix; table 5).

A B

Figure 12. The mean number of by-catch of swimming crab (Liocarcinus depurator) caught (A) with different baits and (B) in different locations. Blue bars show the mean number of by-catch on the first occasion and green bars show the second occasion. Error bars: ± 1 SE.

Discussion

Flume experiments The main aim of this study was to find an alternative bait to salted herring in creel fishery of N. norvegicus. In addition to an increased or a similar capture efficiency of N. norvegicus of the novel bait, the new bait also has to be cheaper (more cost-effective) compared to salted herring, but especially the bait needs to have less environmental impact than the herring fishery has at present.

My results from the flume experiments show that N. norvegicus is attracted to both polar and nonpolar fractions of the Atlantic herring (Clupea harengus), since the butanol- and the hexane fraction were the most attractive fractions. After the column separations of the most attractive fractions from the Kupchan separation (the butanol- and hexane fraction) the least polar fractions from both separations were the most attractive ones (methanol 100% and hexane 100% respectively), although the methanol 100% fraction was still very polar, while the hexane 100% fraction was the absolute most nonpolar fraction. Since so many fractions were tested in this study, the replicates in the lab had to be kept at a minimum. Consequently,

26

only promising fractions were tested beyond ten animals. Since the binomial test used to analyse the choices of the lobsters does not take the ones not making a choice into consideration, the butanol- and the hexane 100% fraction had the same significance level although they had very different amounts of responsive lobsters. This could seem misleading, but it was preferable to have the same amount of lobsters responding for the comparison of the results. It is likely though that the significance level of the butanol-fraction would have been higher than that of the hexane fraction had the same number of lobsters been tested.

It is interesting that both polar and nonpolar substances seem to attract N. norvegicus. In previous studies especially small molecules, like water soluble amino acids (i.g. glycine), organic acids (i.g. taurine) and nucleotides (i.g. adenosine 5’-monophosphate, AMP) have elicited attractive responses in other crustaceans. Mixtures of substances seem to be more attractive than single compounds (Carr, 1978; Carr et al. 1984; Zimmer-Faust et al. 1984; Carr and Derby 1986a; Carr, 1988; McClain et al 2012). Krång et al. (2012) saw that the terrestrial hermit crab (Coenobita clypeatus) is only attracted to water soluble compounds and Zimmer- Faust et al. (1984) found that both molecule size and charge affect the attraction of compounds to the California spiny lobster (Panulirus interruptus). Amino acids with less charged R-groups and with smaller molecular weight were more attractive. The amino acids glycine, alanine and serine as well as the organic acid (succinic acid) and betaine were the most attractive compounds. Only the latter two elicited a locomotion response in P. interruptus, while the others elicited antennule flicking and wiping as well as leg probing, but no locomotion. The organic acid taurine did elicit an increase of antennule flicking and leg probing, but no antennule wiping and was thus not a strong enough attractant to elicit feeding behaviour. In contrast McClain et al. (2012) found that taurine was present in high concentration in waters containing fresh pogy, an attractive bait to crawfish and could thus be an important indicator of food to crawfish. Also Johnson and Atema (1986) found taurine to be attractive to the common gulf weed shrimp (Leander tenuicornis). Carr et al. (1984) found that amino acids (i.e. glycine) in combination with other substances such as trimethylamine oxide, AMP, betaine, homarine, hypoxantine, inosine and lactic acid were attractive to the daggerblade grass shrimp (Palaemonetes pugio). AMP contributed with most of the attraction of the mixture and a synergetic effect could be seen, since AMP within the mixture was more attractive than expected based on its attraction ability alone. A similar synthetic mixture of a squid extract containing glycine and taurine as well as L-amino acids, AMP and other substances such as inosine and homarine, was also found attractive to the European lobster

27

(Homarus gammarus), (Mackie, 1973). Amino acids alone were not so attractive as the full mixture of compounds. Mixtures containing L-amino acids and betaine also elicited a feeding response in P. pugio, although betaine on its own was not as attractive (Carr, 1978).

The compounds found in this study, however, were three fatty acids (palmitic acid, myristic acid and pritane) and one organic acid (phosphoric acid). None of these compounds have, to my knowledge, been found as attractants in previous studies on crustaceans. However myristic acid was found to attract adult Hide beetles (Dermestes maculatus), (Cohen et al. 1974). Phosphoric acid was found in the squid extract in the study on attractants to H. gammarus (Mackie, 1973), but due to the low concentration it was not included in the synthetic mix created based on the squid extract. Molecules containing phosphoric-, palmitic- and myristic acid have also been tested as insect attractants, but with none to moderate attraction ability (Beroza and Green, 1963). Palmitic acid is, however, attractive to the German cockroach (Blattella germanica) according to a study by Wileyto and Boush (1983). The reason for choosing these four substances was first and foremost because they were the only ones that could be clearly identified in the chromatograms. Since all but pristane were surprisingly (due to their very different polarities) found in both fractions it was also possible that they would be the attractants causing the attractiveness of both fractions. Pristane was also chosen since it would be interesting to see whether a really nonpolar substance not tested previously would be attractive. The fact that they were not significantly attractive at the concentrations tested may be that they are the wrong compounds, which would agree with earlier studies where mostly small water soluble compounds seemed to be attractive (Carr, 1978; Carr et al. 1984; Zimmer-Faust et al. 1984; Carr and Derby 1986b; Carr, 1988; Krång et al. 2012). Since water soluble molecules disperse easier in water it is more likely that polar substances will be attractive to water living animals from a longer distance, however, maybe nonpolar substances will remain longer at the source and can work as an additional attractant at a shorter range. Therefore it is possible that a bait containing both polar and nonpolar substances will work better, than one containing only water soluble compounds. The lack of significant attraction of the compound mixtures tested could also be due to the possibility that the wrong concentrations were used. As was seen in the study by Carr and Derby (1986b) on P. pugio, both synergetic and suppressive effects can occur in the same mixture of compounds at different concentrations. Therefore it is possible that the compounds suppressed the attractiveness of each other at the concentrations used. Another possible explanation is, however, that the attractive compounds could not be identified in the chromatograms. Even if

28

some of the nonpolar compounds could be found in the methanol 100% fraction, there were most likely more polar substances as well that could not be detected. Although the derivatization of the fractions before injecting them into the GC-MS was done to make the substances more volatile (Fluka Chemica, 1995), one explanation for the lack of polar compounds found could be that the polar substances were larger and thus less volatile than the nonpolar ones and therefore may not have been be gaseous at the temperatures (in this case 60-300°C) used in the GC-MS (Hašlová and Čajka, 2007). In such a case they would not have travelled through the GC-column, where the moving phase is a gas.

That the same substances were found in both the hexane 100% and the methanol 100% fraction indicates that the separation of the fractions were incomplete. Otherwise phosphoric acid, which is polar and water soluble, would not have been found in the very nonpolar hexane fraction. It is hard not to get some overlap of the fractions though since the division line between two fractions are judged by vision while separating. It is possible that substances in the butanol fraction that should have been in a less polar fraction originally gathered in the methanol 100% fraction since it was the least polar of the fractions deriving from the butanol fraction. Why phosphoric acid was found in the hexane 100% fraction is harder to explain. Possibly due to contamination.

As the concentration used in grams per litre for each fraction is relative (table 1) and represent ten grams wet weight herring per litre water (as described in materials and methods) the absolute concentrations (g l-1) differed between the fractions which may have affected the results. For instance the water fraction (0.1 g l-1) might have had a higher attractiveness had the concentration been as high as for the hexane fraction (0.8 g l-1). Ethyl acetate had the lowest absolute concentration of all fractions (0.01 g l-1). However, ethyl acetate by itself has shown attractiveness to insects in other studies (Smilanick et al. 1978; Jaffé et al. 1993), so extra care was taken to evaporate all solvent from the ethyl acetate fraction so as not to get a false positive response to this fraction due to the solvent. However, that was obviously not a problem here since very few lobsters responded at all to this fraction. A possible reason for the low response could be that the active compounds might have degraded in the extra process of evaporating the solvent or due to that the absolute concentration of the ethyl acetate fraction was too low to attract the lobsters. However, the butanol fraction with the second lowest concentration (0.03 g l-1) was still the one fraction which seemed most attractive to N. norvegicus considering that almost all tested lobsters reacted to this fraction (only one did not

29

respond; tables 1 and 4). What also needs to be considered is that some substances are only attractive within a certain range of concentrations and can lose their effect both in too low and too high concentrations. Harpaz et al. (1987) found that Adenosine 5'-monophosphate (AMP) decreased in attractiveness to the fresh water prawn (Macrobrachium rosenbergii) at concentrations higher than 10-2 M. Carr and Derby, (1986b) found that synergetic effects occurred at low concentrations (<800µM) of a crab extract and suppressive effects at higher concentrations of the same extract tested on P. pugio. Therefore I wanted the concentrations of each substance to be as similar as possible to what they are naturally occurring in the herring to make sure that the attractive substances within the herring would be so even after fractionation. Thereby the relative concentrations used for each fraction in this study.

Since the relative concentrations of the four substances found with GC-MS were unknown, the concentration used for them was based on the concentration of the hexane 100 % fraction and on concentrations used in other studies as described in the materials and methods. The concentration of phosphoric acid and the first mix of the four compounds was within the range (10-3-10-2 M) where some compounds (a mixture of glycine, lactic acid, betaine and AMP was significantly attractive at 10-2 M) have been found to be attractive to N. norvegicus in the same experimental set up as used here (Krång A-S., Ungfors A. and Eriksson S. P. unpublished) and also above the concentration Harpaz et al. (1987) found the amino acid glycine and the organic acid taurine amongst others (attraction occurred at concentrations 10-7 -10-8 M) to be attractive to M. rosenbergii.

A difference in response was noted between lobsters from different holding tanks. Many of the lobsters tested had lost one of both of their 2nd antennae. However, many lobsters without 2nd antennae found their way when tested previously in this study and since they have chemosensory sensilla all over their body, while the olfactory sensilla are mostly situated on the antennules and not the 2nd antennae, they should be able to detect water bound chemicals even without 2nd antennae. Though, maybe not as accurate (Steullet et al. 2001; Hallberg and Skog, 2011).

Future research should include further investigations of the substances found in this study using more replicates to find out if they are truly attractive or not. Phosphoric acid could also be tested alone but at a concentration of 10-2 M, since it seemed to activate some response at the lower concentration. It is also the cheapest of the four substances and thus more likely to

30

be a relevant constituent in an artificial bait. Considering the high cost of pristane (357 SEK ml-1) this compound is not relevant to test further. It would, however, be interesting to test one or some of the other substances by themselves and in combination with other compounds such as taurine, AMP, glycine or betaine which have shown potential before as attractants to other crustaceans (Mackie, 1973; Carr, 1978; Carr et al. 1984; Johnson and Atema, 1986) to see whether the combination will be more attractive to the lobsters. To test the polar methanol 100% fraction using another method than GC-MS or possibly another derivation method (to get more of the polar substances to go through the GC-column) would also be interesting or preferably fractionate both of the most attractive fractions until the effect is lost to truly find the active compounds and then test them in the field. Due to time limitations of this project, the planned field experiment testing an artificial bait made of compounds found in the bioassay had to be excluded.

Field experiment In the field study testing swimming crab as alternative bait to salted herring I found that the total catch of N. norvegicus was lower in creels baited with dead swimming crab compared to creels baited with salted herring or with salted herring in combination with dead swimming crab (on the second occasion). Also on the first occasion the N. norvegicus captures using different baits was similar to that on the second occasion and bait had a significant effect in the ANOVA, although the baits did not differ significantly in the post-hoc test. No significant difference in catch could be seen between creels baited with salted herring in combination with live swimming crab and the other bait types. However, on both occasions a trend could be seen with a lower catch of lobsters in creels baited with salted herring combined with live swimming crab compared to creels baited with salted herring combined with dead swimming crab as well as creels baited with only salted herring (figure 11A).

I also found that there was a significant lower by-catch of swimming crabs in creels baited with dead swimming crab than both creels baited with salted herring and creels baited with salted herring combined with live swimming crab. One reason for the lower by-catch found in creels baited with only dead swimming crab could be the absence of salted herring, the presence of dead conspecifics or more likely a combination of both. On the second occasion also creels baited with salted herring combined with dead swimming crab had a significantly lower by-catch than those baited with only salted herring, while the same was nearly

31

significant on the first occasion. This makes it probable that dead swimming crab works as a deterrent to live swimming crabs, since the by-catch was higher in creels not containing dead conspecifics. Several other studies have found similar results for other crab species (Hancock, 1974; Chapman and Smith, 1978; Richards and Cobb, 1987; Moore and Howarth, 1996). In a field study done by Moore and Howarth (1996) creels baited with fish and dead conspecifics of the shore crab (Carcinus maenas) decreased the catch of this crab species compared to creels baited only with fish. The catch of other decapod species tested remained unaffected by the dead C. maenas. Also Chapman and Smith (1978) saw a reduction in catch of the edible crab (Cancer pagarus) when creels were baited with dead conspecifics and fish compared to only fish as did Richards and Cobb (1987) for spider crabs (Libinia spp.) Similar behaviour has also been observed for e.g. Carcinus maenas, Cancer pagarus and the spider crab Maia squinada (summarized by Hancock, 1974). These studies highly support the results of my study where the by-catch of L. depurator was significantly decreased by the presence of dead conspecifics, while the number of N. norvegicus caught did not differ between creels baited with salted herring compared to dead swimming crab and salted herring. In contrast Hall et al. (1990) found L. depurator in the gut contents of the same species and although most individuals found in the stomachs were juveniles they can obviously be cannibalistic as well., However, it seems like the odour of dead species of the same species might trigger a defence mechanism rather than a feeding response.

Despite that baiting with dead swimming crab should not have an effect on the N. norvegicus capture directly I hypothesised that it might have a positive effect indirectly via biological interaction, due to a release of competition of bait between the target and by-capture species. However, since there was no significant difference in catch of lobsters between creels baited with salted herring and those baited with salted herring in combination with dead swimming crab, while there was a significant higher by-catch of swimming crabs in creels baited with salted herring compared to those baited with salted herring in combination with dead swimming crab on the second occasion, it is possible that N. norvegicus is more attracted to herring than swimming crab and also prefers creels containing herring no matter the presence of live swimming crab i.e. baiting with swimming crab has no effect on the catch of lobsters directly or indirectly. It should be noted, however, that the live swimming crabs used as creel baits were not counted as by-catch, so the total actual number of live swimming crabs within those creels could in fact be higher than in the ones baited with salted herring alone or in combination with dead swimming crab despite the fact that none of them differed

32

significantly in by-catch in the tests. And although there was no significant difference between creels baited with herring and live swimming crab from any of the other treatments in total lobster catch, the catch was lower in average in creels baited with herring and live swimming crabs than in creels baited with salted herring combined with dead swimming crab and salted herring alone on both occasions, (figure 11A). Therefore it is still possible that N. norvegicus does get affected by the presence of live swimming crabs within and around creels.

The significant interaction between bait and substrate for the total catch of lobsters on the second occasion showed an interesting trend where salted herring combined with dead swimming crab seemed to gain a higher catch of lobsters relative to the other baits on hard bottom compared to soft bottom (figure 11B). A likely reason would be the significantly (in the MW test, but not in the ANOVA) higher by-catch of swimming crabs caught on hard substrate on that occasion. Therefore it is possible that dead swimming crab as bait does affect the catch of N. norvegicus more on harder substrate where there are more live swimming crabs than on softer substrates (Hill, 2008; Sabatini and Hill, 2008; Ungfors A. personal communication). However, since this interaction was not significant for any other variable or for the total catch of lobsters on the first occasion, further investigations need to be conducted before any conclusions on the matter can be made.

The parametric and nonparametric tests deviated in some aspects. Substrate was more often significant in the nonparametric tests, as was location, while the opposite was true for bait. A possible reason could be that in the nonparametric tests each factor was tested separately without regards for the other factors. Thus location was treated as a factor of its own and not nested under substrate as in the parametric test. An overestimation of the significance of the effect of location, cage type and substrate could therefore be possible in the nonparametric test, although an overall trend towards a higher catch of lobsters (all categories) could be seen on soft substrate as well as a trend towards a higher by-catch of swimming crabs on harder substrate on both occasions. This was expected due to the different substrate preferences of the two species (Hill, 2008; Sabatini and Hill, 2008). However, location seems to have a higher impact than substrate on the catch, since it had a significant effect on all different catch variables except landed lobsters on the second occasion in the three-factor ANOVA and on all categories on both occasions in the Kruskal Wallis test. In the nonparametric test it could be seen that locations differed greatly in catch of both N. norvegicus and swimming crab. Fossen

33

and Jordfall (soft bottom) seem to yield a high catch of N. norvegicus and a low catch of swimming crabs (at least on the first occasion), while e.g. Essvik (hard bottom) had a generally low catch of lobsters and a high catch of swimming crabs, but the variance was also high within many of the locations (e.g. Gåseklåvan) between the occasions. The cage type was significantly affecting all catch variables. A higher by-catch was found in two-chamber cages while a higher catch of lobsters was caught with single cages. However, since two chamber cages were only present on hard bottom and all but one link of single cages were laid on soft bottom, the difference is more likely to be caused by the bottom substrate than the cage types. Especially since chamber cages have proven to yield a higher catch of N. norvegicus (Ungfors A., Krång A-S. and Eriksson S. P. unpublished), as well as of European lobster (Homarus gammarus) and edible crab (Cancer pagarus), (Lovewell et al. 1988) as compared to single cages in previous studies. However, it is not possible to exclude the effect of cage type with these data.

There were also some dissimilarities in the effect of the different factors on the catch of landed, discarded and total number of lobsters (table 5). However the total catch of discarded lobsters was much lower than that of landed lobsters and the heterogeneous variances of the discarded lobsters making those results more uncertain than those of the landed and the total catch of lobsters. Also, the interaction bait and substrate would have gone amiss had not total catch of lobster been regarded. A larger sample size of undersized lobsters would be preferable when comparing them to larger specimens. Although bait had no significant effect on neither landed nor discarded lobsters in this study, the average of landed lobsters was slightly higher in creels baited with salted herring in combination with dead swimming crab on both occasions (figure 9A), while slightly more discarded lobsters were caught in creels baited with only salted herring (figure 10A). One possibility for this could be that small and larger specimens of N. norvegicus have different feed preference, or possibly larger specimens scare of smaller ones as they are known to be a territorial species (Katoh et al. 2008). However, more research is needed.

In the future, further investigations should eliminate unnecessary factors of variance (such as different cage types and locations) for a more powerful analysis. Locations should be totally random or the same locations should be used on all occasions. Here the same locations were not used on both occasions since the fisherman wanted to change places of two locations not fishing very well. Also the same type of cages should be used, as we the plan but due to

34

practical circumstances this could not be met in the field. More replicates would also be preferable to be able to see any differences between the bait types more clearly.

Conclusions This study found that N. norvegicus is attracted to both polar and nonpolar substances since both a highly polar and the most nonpolar fraction of freeze-dried herring initiated food search and were significantly attractive to lobsters. This is interesting since previous studies indicate that small polar molecules are among the most attractive ones. None of the mixtures of substances identified in the GC-MS chromatograms (phosphoric acid, palmitic acid, myristic acid and pristane) were significantly attractive to the lobsters at the concentration range tested, i.e. 10-5 to 10-2 M. Nor was phosphoric acid alone attractive at 10-3 M. However, at the highest concentration tested most lobsters chose the mixture over the seawater control, even though many did not choose at all. Thus, more replicates are needed before any definite conclusions can be made of these substances’ potential as inclusion in a future artificial bait. A combination of polar and nonpolar substances will possibly become the most effective bait. However, the cost and environmental impact of the future bait compared to that of the herring fishery will be the main factors deciding the contents of the bait.

The field experiment showed that dead swimming crabs work as a deterrent to live swimming crabs, but my results show that N. norvegicus is not significantly affected by the presence of dead or live swimming crabs in creels, although a trend towards N. norvegicus being affected could be seen, but further investigations are needed to confirm the results. At present there seems to be no point in using swimming crab as alternative bait. Nevertheless, the local fishermen fishing for N. Norvegicus in the Gullmar fjord has started using a combination of herring and swimming crab as bait especially on harder fishing grounds (Roysson T. personal communication) so maybe it is effective after all, although this could not be seen in this study. If so baiting with swimming crab could be a release on the herring stock as well as lower the costs for the fishermen as the swimming crab is captured as by-catch. However, simultaneous investigations must be made on the swimming crab population so as not to overexploit this species.

35

Acknowledgements To Anna Sara Krång and Anette Ungfors for helpful comments and for the opportunity to do this time consuming but very interesting project, Gunnar Cervin and Stina Jakobsson for their help with the fractionations, Göran Nylund for explaining the GC-MS, Tony Roysson and Jannicke for pleasant company and for providing me with lobsters, Tove Andersson for helpful comments and for telling me what I did not want to hear but needed to hear and to Amandine Vuylsteke for good ideas and pleasant company during long hours in the lab.

References Bergmann M. and Moore P. G. (2001). Survival of decapod crustaceans discarded in the Nephrops fishery of the Clyde Sea area, Scotland. ICES J. Mar. Sci. 58:163–171.

Beroza M. and Green N. (1963). Materials tested as insect attractants. Agriculture Handbook No. 239, pp. 1-148, Washington D.C., Agricultural research service, United States department of agriculture

Carr W. E. S. (1978). Chemoreception in the shrimp Palaemonetes pugio: The role of amino acids and betaine in elicitation of a feeding response by extracts. Comp. Biochem. Physiol. 61A:127-131

Carr (1988). The molecular nature of chemical stimuli in the aquatic environment. In J. Atema, R. R. Fay, A. N. Popper and W. N. Tavolga (eds.), Sensory Biology of Aquatic Animals, pp. 3-27. New York, Springer

Carr W. E.S. and Derby C. D. (1986a). Behavioral chemoattractants for the shrimp, Palaemonetes pugio: identification of active components in food extracts and evidence of synergistic mixture interactions. Chem. Senses. 11:49-64

Carr W. E. S. and Derby C. D. (1986b). Chemically stimulated feeding behavior in marine animals. Importance of chemical mixtures and involvement of mixture interactions. J. Chem. Ecol. 12:989-1011

Carr W. E. S., Netherton III J. C. and Milstead M. L. (1984). Chemoattractants of the shrimp, Palaeamonetes pugio: Variability in responsiveness and the stimulatory capacity of mixtures containing amino acids, quaternary ammonium compounds, purines and other substances. Comp. Biochem. Physiol. 77A:469-474

Chapman, C. J, and Smith, G. L. (1978). Creel catches of crab, Cancer pagurus L. using different baits. J. Conseil. 38:226-229.

Cohen, E., Stanić, V. and Shulov, A. (1974), Olfactory and gustatory responses of Trogoderma granarium, Dermestes maculatus and Tribolium castaneum to various straight‐chain fatty acids, Z. Angew. Entomol. 76:303-311.

36

Coman, G. J., Sarac, H. Z., Fielder, D., and Thorne, M. (1996). Evaluation of crystalline amino acids, betaine and AMP as food attractants of the giant tiger prawn (Penaeus monodon). Comp. Biochem. Physiol. 113A:247- 253

Cristo M. (1998). Feeding ecology of Nephrops norvegicus (Decapoda, Nephropidae). J. Nat. Hist. 32:1493- 1498

Derby C. D. (2000). Learning from spiny lobsters about chemosensory coding of mixtures. Physiol. Behav. 69: 203–209

Derby C. D. and Atema J. (1981). Selective improvement in responses to prey odors by the lobster, Homarus Americanus, following feeding experience. J. Chem. Ecol. 7:1073-1080

European commission (EC), (2014). Fishing tacs and quotas 2014, available from: http://ec.europa.eu/fisheries/documentation/publications/poster_tac2014_en.pdf

Fluka Chemika, Silylating agents, (1995). Fluka Chemie AG, Buchs, available from: http://www.sigmaaldrich.com/img/assets/21120/Silylation_overview.pdf

Hall S. J., Raffaelli D., Robertson M. R. and Basford D. J. (1990). The role of the predatory crab, Liocarcinus depurator, in a marine food web. J. Anim. Ecol. 59:421-438

Hallberg E. and Skog. M. (2011). Chemosensory Sensilla in Crustaceans. In T. Breithaupt and M. Thiel, (eds), Chemical communication in crustaceans, pp. 103-121. New York, Springer

Hancock D. A. (1974). Attraction and avoidance in marine invertebrates - their possible role in developing an artificial bait. J. Cons. Int. Explor. Mer. 35:328-331

Harpaz S., Kahan D., Galun R. and Moore I. (1987). Responses of freshwater prawn, Macrobrachium rosenbergii, to chemical attractants. J. Chem. Ecol. 13:1957-1965.

Harpaz S. and Steiner J. E. (1990). Analysis of betaine-induced feeding behavior in the prawn Macrobrachium rosenbergii (DE MAN, 1879) (Decapoda, Caridea). Crustaceana. 58:175-185

Hašlová J.and Čajka T. (2007). Gas chromatography-mass spectrometry (GS-MS). In Y. Picó (ed.) Food Toxicants Analysis, pp. 419-473. Oxford, Elsevier

Hill J. (2008). Liocarcinus depurator. Harbour crab. Marine life information network: Biology and sensitivity key information sub-programme [on-line]. Plymouth: Marine biological association of the United Kingdom.

37

Available from: http://www.marlin.ac.uk/specieshabitats.php?speciesID=3697 and http://www.marlin.ac.uk/generalbiology.php?speciesID=3697

Hornborg S., Nilsson P., Valentinsson D. and Ziegler F. (2012). Integrated environmental assessment of fisheries management: Swedish Nephrops trawl fisheries evaluated using a life cycle approach. Mar. policy. 36:1193- 1201

Johnson B. R. and Atema J., (1986). Chemical stimulants for a component of feeding behaviour in the common gulf-weed shrimp Leander tenuicornis (Say). Biol. Bull. 170:1-10

Jaffé K., Sánchez P., Cerda H., Hernandez J. V., Jaffé R., Urdaneta N., Guerra G., Martínez R. and Miras B. (1993). Chemical ecology of the palm weevil Rhynchophorus palmarum (L.) (Coleoptera: Curculionidae): Attraction to host plants and to a male-produced aggregation pheromone. J. Chem. Ecol. 19: 1703-1720.

Katoh E., Johnson M. and Breithaupt T. (2008). Fighting behaviour and the role of urinary signals in dominance assessment of Norway lobster, Nephrops norvegicus. Behaviour. 145:1447-1464

Krång A-S., Knaden M., Steck K., and Hansson B. S. (2012). Transition from sea to land: olfactory function and constraints in the terrestrial hermit crab Coenobita clypeatus. Proc. R. Soc. B. 279:3510.3519

Kupchan S. M. (1970). Recent advances in the chemistry of terpenoid tumor inhibitors. Pure Appl. Chem. 21:227-246.

Lindqvist H., Langkilde A. M., Undeland I., Rådendal T. and Sandberg A. S. (2007). Herring (Clupea harengus) supplemented diet influences risk factors for CVD in overweight subjects. Eur. J. Clin. Nutr. 61:1106-1113.

Lovewell S. R., Howard A. E., Bennett D. B. (1988). The effectiveness of parlour pots for catching lobsters (Homarus gammarus (L.)) and crabs (Cancer pagurus L.). J. Cons. Int. Explor. Mer. 44:247–252.

Mackie A. M. (1973). The chemical basis of food detection in the lobster Homarus gammarus. Mar. boil. 21:103-108

Mackie A. M., Grant P. T., Shelton R. G. J., Hepper B. T. and Walne P. R. (1980). The relative efficiencies of natural and artificial baits for the lobster, Homarus gammarus: laboratory and field trials. ICES J. Mar. Sci. 39:123-129.

McClain R., Romaire R. P., Gregory L. C., Gatlin III D. M., Davis D. A. (2012). Development and evaluation of cool-water crawfish baits. SRAC Twenty-fifth Annual Progress Report. 73-80

38

McDonald, J. H. (2013). Handbook of Biological Statistics (3rd ed.). pp. 80-83. Baltimore, Sparky House Publishing. Available from: http://www.biostathandbook.com/small.html

Mendoza R., Montemayor J. and Verde J. (1997). Biogenic amines and pheromones as feed attractants for the freshwater prawn Macrobrachium rosenbergii. Aquacult. Nutr. 3: 167–173

Moore P. G., and Howarth J. (1996). Foraging by marine scavengers: effects of relatedness, bait damage and hunger. J. Sea Res. 36:267-273.

Richards R. A. and Cobb J. S. (1987). Use of avoidance responses to keep spider crabs out of traps for American lobsters. Transactions of the American Fisheries Society. 116:282-285.

Sabatini M. and Hill J. (2008). Nephrops norvegicus. Norway lobster. Marine life information network: Biology and sensitivity key information sub-programme [on-line]. Plymouth: Marine biological association of the United Kingdom. Available from: http://www.marlin.ac.uk/specieshabitats.php?speciesID=3892 and http://www.marlin.ac.uk/speciesfullreview.php?speciesID=3892

Shelton R. G. J. and Mackie A. M. (1971). Studies on the chemical preferences of the shore crab, Carcinus maenas (L.). J. Exp. Mar. Biol. Ecol. 7:41-49

Smilanick J. M., Ehler L. E. and Birch M. C. (1978). Attraction of Carpophilus spp. (Coleoptera: Nitidulidae) to volatile compounds present in figs. J. Chem. Ecol. 4:701-707.

Schmidt M. and Mellon Jr. D. (2011). Neuronal processing of chemical information in crustaceans. In T. Breithaupt and M. Thiel, (eds), Chemical communication in crustaceans, pp. 123-147. New York, Springer

Steullet P., Dudar O., Flavus T., Zhou M. and Derby C. D. (2001). Selective ablation of antennular sensilla on the Caribbean spiny lobster Panulirus argus suggests that dual antennular chemosensory pathways mediate ordorant activation of searching and localization of food. J. Exp. Biol. 204:4259-4296

Ungfors A., Bell E., Johnsson M. L., Cowing D., Dobson N. C., Bublitz R. and Sandell J. (2013). Nephrops fisheries in European waters. In M. L. Johnson and M. P. Johnson (eds), M. Lesser (series ed.), Advances in marine biology vol 64: The ecology and biology of Nephrops norvegicus, pp. 247-306. Oxford, Elsevier

Wileyto P. E., and Boush M. G. (1983). Attraction of the German cockroach, Blattella germanica (Orthoptera: Blatellidae), to some volatile food components. J. Econ. Entomol. 76: 752-756.

Ziegler F. (2006). Environmental Life Cycle Assessment of Norway lobster (Nephrops norvegicus) fished by creels, conventional and species-selective trawls along the Swedish west coast. A data report, SIK report 746:1- 19

39

Ziegler F. and Valentinsson D. (2008). Environmental life cycle assessment of Norway lobster (Nephrops norvegicus) caught along the Swedish west coast by creels and conventional trawls-LCA methodology with case study. Int. J. Life Cycle Assess. 13:487-497

Zimmer-Faust R. K., Tyre J. E., Michel W. C. and Case J. F. (1984). Chemical mediation of appetitive feeding in a marine decapod crustacean: the importance of suppression and synergism. Biol. Bull. 167: 339-353

40