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Applied Animal Behaviour Science 168 (2015) 77–83

Contents lists available at ScienceDirect

Applied Animal Behaviour Science

journa l homepage: www.e lsev ier .com/ locate /applan im

uttlefish in captivity: An investigation into housing and husbandryor improving welfare

elinda M. Tonkinsb, Alexandra M. Tyersa, Gavan M. Cookea,∗

School of Biological Sciences, Bangor University, Deiniol Road, Bangor, Gwynedd LL57 2UW, Wales, UKSchool of Ocean Sciences, Bangor University, Askew St, Menai Bridge, Isle of Anglesey LL59 5AB, Wales, UK

r t i c l e i n f o

rticle history:ccepted 19 April 2015vailable online 28 April 2015

eywords:uttlefishelfare

ehaviourusbandry

a b s t r a c t

The European cuttlefish (Sepia officinalis) is often kept in public aquaria, is becoming more common inaquaculture, and is also the most frequently used cephalopod in European research. Since 1st January2013, all cephalopods (Mollusca) have been protected under UK/EU law (A(SP)A 1986, European Direc-tive 2010/63/EU), following Australia, Canada and New Zealand. Presently, unlike other organisms usedin research, there is no detailed specific guidance available from UK/EU legislators on best practicesfor keeping cuttlefish. In captivity, juveniles can easily become damaged by impacting with tank wallswhen startled. These injuries rarely heal and can have a major impact on growth and survival. Six experi-ments were performed, using juvenile and adult cuttlefish, in which exhibition of thigmotaxis in differentenvironments, responses to simulated husbandry in different scenarios, and responses to typical andnovel forms of enrichment (e.g. photographs of substrates) and refuges was investigated. Refuge use wasalso investigated, including response to husbandry when different refuges were provided. In addition tothigmotaxis, the frequency of negative behaviours (such as those likely indicating stress or preceding

damaging behaviours) was recorded. The results suggest that certain environments, clothing/equipmentand refuges/enrichment can significantly reduce the frequency of negative behaviours. It was also foundfake seaweed and photographs of substrates placed in tanks may be used by cuttlefish with the bene-fit of preventing localised pollution. We conclude by providing an evidence-based guide to improvinghusbandry practices, which could improve the lives of captive cuttlefish.

. Introduction

With the exception of public aquaria, the standard conditionsn which aquatic organisms are kept in most mediums to largeacilities are impoverished. Bare laboratory conditions may aid inhe maintenance of good environmental conditions and thereforeood animal health (Olsson and Dahlborn, 2002), but impoverishednvironments can alter behaviour and reduce the ability of captivenimals to perform natural behaviours for which they are highlyotivated (Dawkins, 1988). This has the potential to reduce health

nd well-being (Dawkins, 1988, 1998), and also limit the reliabil-ty of conclusions drawn from research (Würbel, 2001). There is

substantial body of research which suggests that environmen-al enrichment is beneficial for captive animals (e.g. Brydges and

raithwaite, 2009), and studies have also shown that increasinghe complexity of rearing environment can be beneficial for aquaticpecies kept in commercial aquaculture (Salmo salar Brown et al.,

∗ Corresponding author. Tel.: +44 01248 38 3806.E-mail address: g.cooke@bangor.ac.uk (G.M. Cooke).

ttp://dx.doi.org/10.1016/j.applanim.2015.04.004168-1591/© 2015 Elsevier B.V. All rights reserved.

© 2015 Elsevier B.V. All rights reserved.

2003; Gadus morhua; Salvanes and Braithwaite, 2006) and scientificresearch (Williams et al., 2009; Danio rerio Schroeder et al., 2014)systems. However, many other benefits of enriched environments,such as reducing fear and aggression (Reinhardt, 2004; Kadry andBarreto, 2010); removing stress and negative response to stress-ors (Fox et al., 2006); and reducing negative stereotypic behaviour(Mason et al., 2007) have only been investigated for terrestrial lab-oratory animals.

Cephalopods are increasingly reared in commercial aquaculturesystems and frequently used in research. Due to growing evidencesuggesting that the cognitive abilities of these animals may becomparable to some vertebrates (e.g. Mather and Anderson, 1999;Dickel et al., 2000) these complex invertebrates have the abilityto experience physiological (Broom, 2007; Andrews et al., 2013)and psychological suffering (Andrews et al., 2013), considerationsof welfare are increasing and legislation is being put in place toprovide these animals with the same protection as vertebrates.

Octopus vulgaris has been protected under the UK Animals Sci-entific Procedures Act 1986 since 1993, and since the 1st January2013 all other cephalopods have also been given protected statusunder UK & EU law – A(SP)A 1986 & European Directive 2010/63/EU

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8 B.M. Tonkins et al. / Applied Anim

following Canada in 1991, New Zealand in 1999 and Australia in004. In addition to the paucity of studies on the effects of captiv-

ty on cephalopod behaviour and physiology (but see Malham et al.,002), in recent years (2005–2011), a substantial body of researchas been published which if carried out now would require a proce-ural licence because it would likely cause pain or lasting stress (seemith et al. (2013) for a review on previous research). This indicatespressing need for further investigations into the welfare of captiveephalopods (e.g. Smith et al., 2013). Although some studies havenvestigated improving growth conditions in cephalopod aquacul-ure systems (Sykes et al., 2011; Forsythe et al., 1994; Correia et al.,005; Boal et al., 1999), and also for captive breeding (Boal et al.,999; Adamo et al., 2000), the few studies aimed at improvinghe overall welfare of captive cephalopods have been conductedn zoos and are therefore limited to observations on a very smallumber of individuals (e.g. Anderson and Wood, 2001). Addition-lly, suggestions for enrichment for zoo animals may frequentlyot be suitable for other situations in which cephalopods are com-only kept. Therefore, despite environmental enrichment being a

equirement for all protected animals (under annex III of Directive010/63/EU), and the “freedom to express normal behaviour” andfreedom from fear and distress” being incorporated into UK/globalnimal welfare laws (see The Brambell Report, 1965), there are, aset, no guidelines for best husbandry practices for cephalopods keptor scientific research.

Behaviour can be a useful non-invasive indicator of welfareDawkins, 2003) and the ability of cuttlefish to produce complexignal and camouflage to their environment through the use ofapid skin colour/pattern changes and posturing provides greatotential for improving welfare. In cuttlefish, anti-predatory sig-als are well documented (Hanlon and Messenger, 1996) andhese displays, and other behaviours such as thigmotaxis (indica-ive of stress, Schnörr et al., 2012) and jetting and inking (flightesponses, Gilly and Lucero, 1992; Lucero et al., 1994; Hanlonnd Messenger, 1996; Oestmann et al., 1997), may allow us toecipher how a situation or stimulus is interpreted and aid inur ability to reduce stress and reduce the likelihood of lastingnjury (which can occur after impact with tank walls when flee-ng perceived threats, Boletzky and Overath, 1991; Oestmann et al.,997).

Having been used fairly widely in research in Europe (Smitht al., 2013), Sepia officinalis (Linnaeus 1758), the common Euro-ean cuttlefish, is a relatively well-understood cephalopod. Like allephalopods they are thought to be semelparous with two repro-uctive phenotypes: depending on foraging success during theirrst year, individuals may breed either in their first or second yearf life (Le Goff and Daguzan, 1991), although, if prevented fromreeding, adults may live up to 4 years in captivity (Cooke, per-onal observation). Juveniles are epibenthic, burying in, or sittingn, the substrate and will only move when threatened or to pur-ue prey (Hanlon and Messenger, 1988). It has been suggestedhat cuttlefish strongly prefer a substrate (Boal, 2011; Forsythet al., 1994) but due to the potential for increased cleaning timer localised environmental pollution, evidence as to the benefitsf certain types of enrichment may be necessary before institu-ions change husbandry practices. Interestingly, it has been shownecently (Schroeder et al., 2014) that the zebra fish (Danio rerio)ill accept images of a substrate, which removes some costs associ-

ted with enrichment of environments for aquatic organisms. It haseen proposed (John Rundle, personal communication, MBA, Plym-uth) that dark figures leaning over tanks may be interpreted asredators by captive cuttlefish, and that the use of lighter clothing

uring husbandry has the potential to reduce frequency of negativeehaviours.

To our knowledge this is the first study to investigate how differ-nt environments (i.e. impoverished, enriched) of captive cuttlefish

aviour Science 168 (2015) 77–83

affect responses to typical stimuli encountered in captivity, such ashousing environment, care staff and husbandry activities.

2. Materials and methods

2.1. Experimental animals, rearing and housing

Eggs were obtained from The Marine Biological Association(Plymouth, UK) and hatched in Bangor University School of Biolog-ical Sciences marine aquaria facilities, where all experiments werecarried out. From hatching they were fed Mysid spp., then main-tained on a mixed diet of common shore crabs (Carcinus maenas)and frozen sand eels (Gymnammodytes spp. or Ammodytes spp.).Small prey items (Mysid spp.) were emptied into tanks using smallcontainers and larger items (C. maenas, Gymnammodytes spp. orAmmodytes spp.) were presented individually by hand. After feed-ing, remains of crabs were removed from tanks by hand. Juvenileswere between 6 and 10 months old during the experimental period(February–May 2014), and had yet to show full adult behavioursor patterns (i.e. courtship behaviours or Intense Zebra Patterning),it was therefore not possible to sex them at this stage. Duringeach experiment, individuals were housed in individual grey plas-tic tanks (with a mixed substrate of sand and gravel) between trials.Adults were between 20 and 24 months old but had not mated priorto being used in these experiments.

2.1.1. Experimental tanks and facilitiesWith the exception of the glass tank (L 0.7 m × W 0.4 m × 0.76 h,

volume = 21.56 l) used in experiment 1, all experimental tanks werepart of a 75k l recirculating system with an average water tem-perature of 13.0 ◦C. All of the smaller grey tanks used for juvenileexperiments measured L 0.57 m × W 0.37 m × H 0.11 m (giving awater volume 21.65 l) and were positioned on benches above waistheight. Where glass and grey plastic tanks were used in the sameset of experiments, glass tanks were filled to a level to keep totalwater volume the same as the grey tanks. In-flow to tanks wasalways turned off for 5–10 mins before each experiment. Negativebehaviours were recorded following Hanlon and Messenger (1988,1996), with additional observations defined by us (Table 1). Asresponse to husbandry (and being kept in captivity generally) wasthe focus of this study, no attempt was made to hide the observer.

2.2. Experiment 1: substrate preference

In this experiment, 12 juvenile cuttlefish were used to com-pare time spent displaying thigmotaxis (defined as touching theside of the tank with any part of the mantle fin, but facing inwardstowards the centre of the tank) in different tank environments. Intotal, six tank types were set up: two with no substrate (bare glasstank with a white polystyrene base, and a bare grey plastic tank);and four enriched grey plastic tanks with four alternative types ofsubstrate/enrichment (sand, gravel, synthetic seaweed, laminatedphotograph of gravel). Gravel was chosen as the artificial substratebecause, in contrast to sand in which they bury, a photograph ofgravel still provides a background against which camouflage maybe achieved by alteration of appearance rather than physical inter-action. After release into the centre of an experimental tank, a 1 min

acclimatisation period was allowed before behaviour observationsof the cuttlefish began. Observations lasted 5 min. Each of the 12cuttlefish experienced each of the six tank types (bare glass, bareplastic, sand, gravel, photograph of gravel, synthetic seaweed).

B.M. Tonkins et al. / Applied Animal Beh

Table 1Behaviours recorded during the study (* denotes descriptions taken from Hanlonand Messenger, 1988, 1996).

Behaviour Definition/description

Locomotor Thigmotaxis Touching side of tank with mantel fin,but facing away from tank wall.

Moving From stationary to stationary or onsetof another recorded behaviour.Included reorientation and attempts tobury. Did not include movement froman area occupied during simulatedhusbandry.

Jetting Sudden rapid movement away fromstimulus.*

Pushing Pushing or pressing against side of tankwith body or folded arms/tentacles.

Postural Flattened body Flattening of body* on thedorsal/ventral plane (i.e. pressingdown into tank floor).

Raised arms Raising of middle pair of arms.*Buried Burying in substrate (motion

attempted even in tanks with nosubstrate).

Defence Ink pseudomorph Release of small amount of ink.*Ink fully Release of large amount of ink (tank

blacked out).*

Texture Papillated skin Rapid changed to rough textured skin*(used out of context of camouflage).

Chromatic Blanche Change to all white colouration.*Colour oscillation Rapid or moving colour/pattern

changes (but not the same as “passingcloud”* display observed in the contextof predation).

Dark eyes Intense dark bands around the eyes.*Dark spots Dark striping on sides of the body.*Deimatic spots One to four dark spots on the back.*Disruptive pattern Common camouflage pattern* used out

of camouflage context.

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Mottle Common camouflage pattern* used outof camouflage context.

.3. Experiment 2: negative behaviours in response to husbandryn different tank environments and with differentusbandry-related stimuli

The following trials were carried out using the same 12 juvenileuttlefish that were used in experiment 1 (Section 2.2). In each sub-xperiment (a–c) the same “standardised simulated tank cleaningusbandry” procedure was used to test reaction to husbandry

n different tank environments or with different coloured cloth-ng/equipment. Simulated husbandry consisted of the removal ofve glass marbles (standardised objects rather than, for example,rab carapaces), from the corners and centre of the tanks usingn aquarium net. The standardised procedure was as follows: (1)pproach tank, (2) stand facing tank for 10 s, (3) retrieve the fivebjects (taking 20 s to collect each object), (4) stand facing tankor 10 s, (5) move away from tank. Individuals were tested onet a time and allowed to acclimatise for 10 min after release intohe centre of the experimental tank. During the following 2-minbservation period, one person simulated cleaning husbandry andelayed observations to a scribe. For all experiments, individual cut-lefish trial order was randomised. Negative behaviours are listedn Table 1 and cover the behaviours which may be signs of stress orrecede damaging behaviours.

.3.1. Experiment 2a – negative behaviours and reactions tousbandry in different tank environments

For this initial experiment to test for differences in behavioururing husbandry in different tank environments, the same six

aviour Science 168 (2015) 77–83 79

experimental tank environments used in experiment 1 (Section 2.2)were used, e.g. a bare glass tank; a bare plastic tank; a plastic tankwith sand in; a tank with gravel and tanks with a photograph ofgravel and synthetic seaweed. All cuttlefish underwent one trialand the order that each cuttlefish was tested in each tank environ-ment was randomised. A small green aquaria net and a white labcoat were used throughout.

2.3.2. Experiment 2b – reactions to different coloured clothingworn during husbandry

In this experiment clothing colour of staff was investigated tosee if it influenced the frequency of negative behaviours duringhusbandry. Each cuttlefish underwent three trials within one day(spaced 2–3 h apart), different colour clothing consisting of: light(white lab coat); intermediate (grey clothing); dark (black cloth-ing). All trials were carried out in a randomised order for eachcuttlefish. A single environment type was used in this experiment,a grey bare plastic tank.

2.3.3. Experiment 2c – reactions to different coloured aquariahusbandry equipment

For this experiment, tank environment and clothing colour werekept the same throughout (bare tank and white lab coat), only netcolour was changed. Standard blue, white and red fine mesh aquarianets measuring 127 × 152 mm were used. Cuttlefish are thoughtto be colour-blind (e.g. Marshall and Messenger, 1996) and so thewhite (light), blue (intermediate) and red (dark) nets were cho-sen to provide a similar colour/shade comparison to that used inthe white/grey/black clothing colour experiment (exp 2b, Section2.3.2). The only difference in standardised simulated husbandrywas that the net was presented at an approximate 30◦ angle overthe tank during the “stand in front of tank phase.” Each cuttlefishunderwent three trials within one day (spaced 2–3 h apart), dif-ferent colour net (white, blue, red in a randomised order for eachcuttlefish) was used during each trial. A single environment typewas used in this experiment, a grey bare plastic tank.

2.4. Experiment 3: refuge use and response to husbandry bysolitary cuttlefish

2.4.1. Experiment 3a – juvenile and adult refuge usePlastic pipes are commonly provided as refuges for captive

cephalopods. However as cuttlefish generally bury into substrateor hide by camouflage, and are not known to make use of con-fined spaces for hiding in their natural environment, pipes maynot provide the same environmental enrichment for cuttlefishas they do for octopuses. In this experiment we compare use ofpipes with use of an alternative “non-substrate” environmentalenrichment against which it is possible for cuttlefish to camouflage(seaweed). A scan sampling approach was used; after an accli-matisation period of 24 h, 10 observations were made over thenext 5 h (once every 30 min). For each observation three differentbehaviours were recorded; whether the cuttlefish was using therefuge or not, whether it was attempting to camouflage against itor not and whether it was displaying thigmotaxis or not. After thefirst 10 observations of thigmotaxis for each cuttlefish, refuge typewas switched and a further 10 observations of thigmotaxis weremade the following day.

Eleven juvenile cuttlefish (from the same batch of eggs used inthe first experiments) were tested in individual grey tanks withno substrate and were provided either a plastic pipe measuring

70 × 100 mm or plastic seaweed replicate measuring 160 × 80 mmin a randomised order. Ten adult cuttlefish (again, reared fromeggs provided by the MBA, Plymouth) were also tested, these wereisolated in bare tanks measuring L 1.2 m × H 0.5 m D × 0.3 m and

80 B.M. Tonkins et al. / Applied Animal Behaviour Science 168 (2015) 77–83

Table 2Substrate comparison, Z and p-values for post hoc Wilcoxon matched pairs tests for time spent exhibiting thigmotaxis (experiment 2a, Sections 2.3.1 and 3.2.1), and Z andp-values for Wilcoxon matched pairs tests for negative behaviours (i.e. behaviours that may be related to stress or precede damaging behaviours) displayed during simulatedhusbandry, in tanks containing different types of substrate (experiment 1, Sections 2.2 and 3.1). N = 12 cuttlefish.

Substrate comparison Thigmotaxis (time in s) Negative behaviours (n)

Means Z p Means Z p

Bare glass vs bare plastic 279 vs 249 −2.65 0.008 13 vs 12 −0.757 0.449Bare glass vs sand 279 vs 109 −2.63 0.009 13 vs 02 −2.950 0.003Bare glass vs fake seaweed 279 vs 066 −3.03 0.002 13 vs 04 −2.666 0.008Bare glass vs gravel 279 vs 279 −0.2 0.443* 13 vs 4 −2.581 0.01Bare glass vs photo of gravel 279 vs 184 −2.53 0.011* 13 vs 4 −2.708 0.007Bare plastic vs sand 249 vs 109 −2.55 0.011* 012 vs 02 −2.943 0.007Bare plastic vs fake seaweed 249 vs 0066 −2.85 0.004 012 vs 4 −2.995 0.003Bare plastic vs gravel 249 vs 279 −0.30 0.706 ** 012 vs 4 −3.061 0.002Bare plastic vs photo of gravel 249 vs 184 −0.17 0.091 ** 0112 vs 4 −2.703 0.007Gravel vs sand 279 vs 109 −2.16 0.031* 4 vs 02 −1.068 0.286Gravel vs fake seaweed 279 vs 066 −2.87 0.004 4 vs 04 −0.286 0.789Gravel vs photo of gravel 279 vs 184 −1.60 0.109 ** 4 vs 04 −0.307 0.759

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Sand vs fake seaweed 109 vs 066 −0.95Sand vs photo of gravel 109 vs 184 −1.37Fake seaweed vs photo of gravel 066 vs 184 −2.10

rovided with either a plastic pipe measuring (0.25 m diameter,.45 m long) or seaweed anchored to a rock of a similar volume.

.4.2. Experiment 3b – negative behaviours during husbandry inanks with different refuge types

To test whether refuge type affected the frequency of negativeehaviour during husbandry, each juvenile underwent a standard-

sed simulated husbandry trial (outlined in experiment 2, Section.3) with each of the two different refuge types. A white net and ahite lab coat were used throughout.

.5. Statistical analysis

Data from all experiments were tested for homoscedasticity andormality before parametric statistics were used for data analysis.

f assumptions for parametric testing were not met, attempts toectify the problem were made by transformation. If data still vio-ated the assumptions of parametric tests, either non-parametriclternatives, or generalised linear models were used. Although mul-iple post hoc testing was used during analysis of experiments 1 and, due to the relatively small sample size, Bonferroni correctionas not applied (see Perneger, 1998; Nakagawa, 2004). All statis-

ics were carried out in SPSS v.20 or R v.3.0.2 (R Development Coreeam, 2011).

.5.1. Experiment 1: substrate preferenceA Friedman test was used to test for overall differences in the

ime spent displaying thigmotaxis in tanks with different sub-trates. Post hoc Wilcoxon Matched Pairs tests were used to identifyetween which substrates the significant differences were found.

.5.2. Experiment 2: negative behaviours in responses tousbandry in different tank environments and with differentusbandry-related stimuli

Friedman tests were used to test for differences in the number ofegative behaviours displayed in response to husbandry (in tanksontaining different types of substrate and during husbandry usingifferent colour clothing and equipment). Wilcoxon Matched Pairsests were used to identify between which substrates or colours theignificant differences were found.

.5.3. Experiment 3: refuge use and response to husbandry by

olitary cuttlefish

To investigate which factor (refuge type/age category) has thereatest influence on refuge use, we conducted a Multivariate Bino-ial Generalised Linear Mixed Effect Model. We used two response

0.340 ** 02 vs 04 −1.662 0.0960.172 ** 02 vs 04 −1.430 0.1530.035 * 04 vs 04 −0.51 0.959

variables (number of observations where refuge was in use and notin use) and included individual as a random factor to account forthe repeated use of the same cuttlefish in the two experimentalconditions (see Bolker et al., 2008). Analysis was carried out usingthe statistical software package “lme4” in R v.3.0.2 (R DevelopmentCore Team, 2011) and the model based on a maximum likelihood(“glmer”) test with a binomial distribution and a logit link.

Wilcoxon Matched Pairs test was used to test for differencesin the number of negative behaviours during husbandry in tankscontaining different types of refuge.

2.6. Ethical considerations

The experiments were subjected to ethical review by BangorUniversity’s Named Animal Welfare and Care Officer and ethicalreview process. Before the experiments began, it was decided thata trial would end if jetting into the side of the tank occurred, if“non-impact” jetting occurred three times, or if one full ink or threepseudomorph inking events occurred. This allowed the cuttlefishto in some way determine the end of the experiments.

3. Results

3.1. Experiment 1: substrate preference

There was a significant difference in the amount of time that the12 juvenile cuttlefish spent exhibiting thigmotaxis in different tankenvironments (Friedman test �2 = 27.448, d.f. = 5, n = 12, p < 0.001).Post hoc (Wilcoxon Matched Pairs) testing (Table 2, Fig. 1) foundsignificant differences between (by substrate, ranked in order ofdecreasing duration of stress display (thigmotaxis)): bare glass vsmost other substrate types (bare plastic p = 0.008, sand p = 0.008,fake seaweed p = 0.002, photograph of gravel p = 0.011); gravel vssand (p = 0.031) and fake seaweed (p = 0.004); bare plastic vs sand(p = 0.011) and fake seaweed (p = 0.004); fake seaweed vs photo-graph of gravel (p = 0.035).

3.2. Experiment 2: negative behaviours and responses tohusbandry in different tank environments and with differenthusbandry-related stimuli

3.2.1. Experiment 2a – negative behaviours and reactions to

husbandry in different tank environments

Significant differences in the number of negative behavioursbetween some substrates was found (Friedman �2 = 36.425, d.f. = 5,p < 0.001). Post hoc (Wilcoxon Matched Pairs) tests (Table 2,

B.M. Tonkins et al. / Applied Animal Beh

Fig. 1. In juvenile cuttlefish, there was (A) a significant difference in the amountof time spent displaying thigmotaxis (Friedman test �2 = 27.448, d.f. = 5, p < 0.001:Table 2) and (B) the number of negative behaviours displayed during husbandry(Friedman �2 = 36.425, d.f. = 5, p < 0.001: Table 2), in tanks with different substrates.The number of negative behaviours displayed during husbandry was reduced with(C) the use of light vs dark clothing by staff and (D) the use of light vs dark colouredaquaria nets. (E) Significantly less negative behaviours were observed during hus-bandry in tanks containing seaweed, rather than pipes, as a refuge (WilcoxonMatched Pairs Z = −2.586, p = 0.01). (F) Both refuge type and age had a significant

aviour Science 168 (2015) 77–83 81

Fig. 1) revealed significant differences between (ranked in orderof decreasing number of negative displays/behaviours); bareglass vs gravel (p < 0.01), fake seaweed (p = 0.008), photograph ofgravel (p = 0.007); gravel vs bare plastic (p = 0.002); bare plasticvs sand (p = 0.007), fake seaweed (p = 0.003), photograph of gravel(p = 0.007); bare glass vs sand (p = 0.003).

3.2.2. Experiments 2b and c – reactions to different colouredclothing and aquaria equipment used during husbandry

Significant difference in total number of negative behavioursduring trials in which different colour clothing was worn dur-ing simulated husbandry was found ((Friedman �2 = 25.48 d.f. = 1,p < 0.001), Fig. 1). There was a strong trend but non-significant dif-ference between light (white) and dark (black) clothing (WilcoxonMatched Pairs Z = −1.915, p = 0.055) and no significant differencebetween light and intermediate (grey) clothing (Wilcoxon MatchedPairs Z = −1.026, p = 0.305) nor between dark and intermediateclothing (Wilcoxon Matched Pairs Z = −1.022, p = 0.307).

Similarly, there was a significant difference in the numberof negative behaviours during trials in which different colouredaquaria nets were used during simulated husbandry (Friedman�2 = 23.51, d.f. = 1, p < 0.001) Fig. 1). Post hoc (Wilcoxon MatchedPairs) tests revealed a significant difference between light (whitenets) vs dark (red nets) (p = 0.02), a strong but not significant trendbetween light vs intermediate (blue nets) (p = 0.054), and no differ-ence between intermediate vs dark nets (p = 0.422).

3.3. Experiment 3: refuge use and response to husbandry bysolitary cuttlefish

3.3.1. Experiment 3a – refuge use by juvenile and adult cuttlefishBoth refuge type (seaweed vs pipe) and age (adult/juvenile) had

a significant effect on refuge use (Table 3). Cuttlefish were signifi-cantly more likely to be observed using seaweed than pipe refuges(GLMM: n = 21, z = 9.99, p < 0.001), and that juveniles were signif-icantly more likely to be observed using refuges than adults are(GLMM: n = 21, z = 3.23, p = 0.001).

3.3.2. Experiment 3b – negative behaviours during husbandry intanks with different refuge types

Significantly less negative behaviours were displayed duringhusbandry in tanks where seaweed was provided as a refuge com-pared to tanks where pipes were provided (Wilcoxon Matched PairsZ = −2.586, p = 0.01, Fig. 1).

4. Discussion

It is perhaps unsurprising that cuttlefish prefer some substratesand tank environments over others (experiments 1 and 2). Herewe found that the amount of time spent displaying thigmotaxisand the number of negative behaviours displayed during simulatedhusbandry were reduced when cuttlefish were housed in tankscontaining a substrate/enrichment (sand or seaweed) compared tobare tanks. It is of little surprise that bare tanks (both glass andopaque plastic) resulted in far more negative responses; cuttlefishare highly adapted for camouflage and are unlikely to be at ease

in an environment which limits the expression of this protectivebehaviour. It is interesting that gravel ranked lower than the pho-tograph of gravel for both thigmotaxis and frequency of negativebehaviours. The cuttlefish were adept at pattern matching to the

effect on refuge use: seaweed was significantly more likely to be used that pipes(GLMM: n = 21, z = 9.99, p < 0.001), and juveniles were significantly more likely tobe observed using refuges than adults (n = 21, z = 3.23, p = 0.001). Box plots showmedian and interquartile range, error bars display minimum/maximum variation,asterisk (*) denotes outliers.

82 B.M. Tonkins et al. / Applied Animal Behaviour Science 168 (2015) 77–83

Table 3Cuttlefish refuge use study: use of different refuge types by cuttlefish in a sequential choice test. Mean use (total use count for each individual/number of individuals), GLMMlogistic parameter estimates (Est.), standard errors (SE), z and p-values from Chi squared test, and significance levels of main variables (**p < 0.005, ***p < 0.001).

Parameter Mean refuge use Est. SE z p

Intercept −3.16 0.67 −4.69 2.68e−06***Refuge type (seaweed vs pipe) 17 vs 5 4.18 0.47 9.99 <2.00e−16***Age group (juvenile vs adult) 14 vs 9 2.76 0.86 3.23 0.00122**

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ravel but displayed thigmotaxis repeatedly. It may be that theravel was insufficiently fine for the small cuttlefish to bury, ashey would in sand, and felt uncomfortable on this substrate. But,s they could at least pattern match, this type of substrate rankedlightly higher (compared to glass) when negative behaviours dur-ng husbandry were investigated (Fig. 1a and b). These results alsohow that cuttlefish will attempt to camouflage against 2D hetero-eneous backgrounds (e.g. a photograph of gravel), which suggestshat this sort of substitute may prove to be a suitable enrichmentlternative in systems where the use of substrate may not be prac-ical. Although all of the heterogenous tank environments reducedhe time spent in thigmotaxis (compared to bare glass), only sandnd seaweed (not gravel or photo of gravel) resulted in a significanteduction compared to bare plastic tanks. However, compared tooth of the bare tanks, all heterogeneous environments did result

n less negative behaviours during simulated husbandry.Factors other than immediate tank environment can have an

ffect on reaction to regular husbandry activities (experiment) and the results of these experiments are in agreement withuggestions (Rundle, personal communication) that the use ofighter coloured clothing during husbandry may reduce the fre-uency of negative behaviours in captive cuttlefish: strong buton-significant trends in negative behaviours were observed in tri-ls using a lighter colour compared to darker colours. Although,rom the present study, it is not possible to elucidate the cause ofhis difference in reaction, we speculate that predators of cuttlefishsuch as large fish or marine mammals) may appear as partial sil-ouettes, and thus, leaning over a tank in dark clothing may provokesimilar escape/fear response as to a natural predator (e.g. sea lion,ee Hanlon and Messenger, 1996). Caution should be taken whenransferring the results of this study from well-lit research labora-ory systems to commercial aquaculture systems, which may usearker tanks to increase contrast of background with light colouredrey (e.g. Mysid spp.) to increase prey capture rates and reduce

uvenile mortality (Sykes et al., 2011). In such systems there is theotential that the contrast of a light-coloured figure standing overtank could elicit the same response as a dark coloured figure overell-lit tanks.

Unlike octopuses, cuttlefish are not known to use solid overheadover as a form of refuge in the wild, preferring to hide by bur-ing in the substrate or camouflaging against their environmentHanlon and Messenger, 1996). It is therefore not surprising thatipes, which are commonly provided as a refuge for captive cuttle-sh, were rarely used. Providing seaweed as a form of enrichment

n otherwise bare tanks, on the other hand, gives a backgroundgainst which camouflage can be achieved and has great potentialor reducing negative behaviour. It is also possibly not surprisinghat juveniles used refuges significantly more than adults. Juvenilesre epibenthic in nature in the wild, compared to adults who pos-ibly much more active during their migration to breeding watersnd during breeding courtship (Hanlon and Messenger, 1996). Our

xperiences in this study suggest that as long as the cuttlefish thinkhey are camouflaged, or are attempting to camouflage, then theyenerally exhibit less negative behaviours during husbandry, feed-ng, etc. As cuttlefish are colour blind (e.g. Marshall and Messenger,

1996), it is worth bearing in mind that complexity of contrasts andtextures is likely to be more important that colour.

Although juvenile cuttlefish are solitary in the wild (Hanlonand Messenger, 1988) and are frequently found alone in publicaquaria (Cooke personal observation), they are often stocked inhigh densities in aquaculture and research facilities (Sykes et al.,2011, 2012, 2014). Therefore, as this study focused mainly on thereaction of individually housed cuttlefish to different environmen-tal conditions and stimuli, we suggest that further work is neededto test how our suggestions for environmental enrichment and hus-bandry practices influence the frequency of negative behaviour anddamaging behaviour in animals living in groups in larger systems.Additionally, as behaviour is only one component of measuringwelfare, it would be beneficial if future studies could also includephysiological or immunological measures of stress (e.g. fish, Scottand Ellis, 2007; octopus, Larson and Anderson, 2010).

From this study, and in our experience of keeping large numbersof cuttlefish, we strongly advise against the use of bare glass tanks,and recommend that bare plastic tanks be avoided wherever possi-ble. If glass or bare tanks must be used then they should only be usedtemporarily. Ideally substrates for cuttlefish are those in which theycan bury or camouflage against. However, as substrates and realseaweed have been suggested to pollute the local environmentthrough rotting of food waste and the negative effects of anoxiczones, we recommend providing natural looking 2D images or arti-ficial seaweed which may be a suitable alternative. Our currentunderstanding of the effects of different environmental conditionsand stimuli that captive cuttlefish are commonly exposed to leadsus to suggest that an optimum system for keeping cuttlefish incaptivity would consist of dark tanks (Nabhitabhata and Nilaphat,2000) with rounded sides (Hanley et al., 1999) and a texturallycomplex baffle to disrupt visual contact and provide environmen-tal heterogeneity against which camouflage can be achieved (thisstudy). Additionally, using lighter coloured clothing and husbandryequipment would appear to be a simple way of reducing the fre-quency of occurrence of negative behaviours.

Finally, in our experience we observe a very different reper-toire of (particularly predation) behaviours when providing livefood compared to frozen and it is therefore possible that food typewhich promotes more natural “positive behaviours” could be incor-porated into considerations of enrichment for cephalopods. Weconclude that different environments that captive cuttlefish areheld in may influence the frequency of thigmotaxis and negativebehaviours. By considering providing environmental richness wemay be able to improve the lives of these complex animals.

Acknowledgements

We are thankful for the funding provided to Belinda M. Tonkins

through the paid internship scheme at Bangor University. We arealso grateful to Jonathan England for providing advice on statisticalanalysis. We thank the two reviewers for helping us improve theoriginal manuscript.

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