THE VELIGER @ CMS, Inc., 1996 The Veliger 39(4):342-347(October I, 1996)

Ventilation Rate of , officinalis, ~ in Response to Visual Stimuli

I' f by

J. G. BOAL AND J. N. NI

Marine Biomedical Institute, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-1163, USA

Abstract. Ventilation rate can provide information about the detectability and salience of stimuli. Octopus ventilation rate responds to chemical stimuli; here we tested whether cuttlefish ventilation rate is sensitive to visual stimuli. We measured the changes in ventilation rate of juvenile Sepia oificinalis in response to a general disturbance (being moved to a new tank), to the sight of prey items (live fish and live ), and to the sight of conspecifics (familiar and unfamiliar). Ventilation rate increased relative to controls in all cases, but most to general disturbance. Responses to prey items were stronger than reponses to conspecifics, a finding consistent with the semi-solitary lives of free-living juvenile cuttlefish. Although cuttlefish prefer crabs to fish and hunt them differently, no differences in responses were found between types of prey. Responses to familiar and unfamiliar conspecifics also did not differ. We conclude that analysis of ventilation rates is a good method for measuring the perception and relative significance of broad classes of visual stimuli in cuttlefish. INTRODUCTION alanine, lactic acid, and serine) (Boyle, 1986). Chase & Wells (1986) observed respiratory changes in O. vulgaris It is not always easy to discern what perceive. in response to weak solutions of glutamic acid, glycine, The appearance of stimuli important to an can and adenosine 5'-monophosphate. cause increased alertness, with a possible concomitant in- The feasibility of using ventilation rate to measure re- crease in ventilation rate (hereafter, referred to as "arous- sponses to chemical stimuli was demonstrated by Boyle aI"). A directly observable behavior, ventilation rate has (1983), using two different methods: (1) direct observation the potential for providing a convenient measure of the by an experimenter, and (2) automated measurement of relative salience of different stimuli. To investigate this the electrical impedance of the water surface, which fluc- possibility, we measured changes in ventilation rates of tuates with the disturbances caused by ventilation move-

cuttlefish in response to visual stimuli. ments. Automated measurements were sometimes dis- change the rate or volume of water respired rupted by movements of the subject within the enclosed in response to changes in activity level. The common oc- space or by spontaneous bursts of hyperventilation. In the topus, Octopus vulgaris Cuvier, 1797, increases its venti- experiments reported here, we measured ventilation rates " lation rate with even small increases in activity level (Boyle, by the simpler method of direct observation.

\ 1983]..The common cuttlefish, SePia oificinalis Linnaeus, We measured the ventilation rates of Sepia oificinalis in ~ 1758, Increases ventilation volume, with a reduction in response to visual stimuli: live fish, crabs, shrimp, and l; ventilation rate, when it jets (Trueman & Packard, 1968). familiar and strange conspecifics. We addressed four ques- Cephalopods also change the rate or volume of water tions: is ventilation rate affected by (1) general disturbance respired in response to chemical stimuli. In Octopus vul- (moving the animal into the test tank); (2) the presence of garis, ventilation rate increased in response to extract a food item (live shrimp); (3) the sight of a conspecific and to water from a crab holding tank (Boyle, 1983). (familiar or unfamiliar); and (4) the type of live stimuli

Ventilation rate in the octopus Eledone cirrhosa Lamarck, (fish, crab and other cuttlefish)? We discuss the possible 1798, changed in response to several common chemical relationship between relative magnitudes of ventilation constituents of arthropod flesh (betaine, glycine, proline, changes and significance of stimuli to the cuttlefish. I J. G. Boal & J. N. Ni, 1996 Page 343

i ; MATERIALS AND METHODS

B I B Subjectscohort of juvenile (7.5-10.5 laboratory-cultured cmmantle length) SePia were officinalis taken from(For- a

Tank 3 Tank 4 sythe et al., 1991). This cohort of approximately 120 an- I imals was maintained in groups of about 10 cuttlefish per ~ I 1.52 m diameter round tank. All 12 holding tanks, as well as the test tank, were interconnectedto the samerecir- I culating water system (see Forsythe et al., 1991). Water

B I B was obtainedfrom the Gulf of Mexico, and rangedfrom ., - 19-21°C with a salinity of 35 ppt. Light was provided Tank 2 I Tank 1 with a combination of natural light and artificial light on

I a 12 hr light/12 hr dark cycle. The animals were fed frozen shrimp twice a day ad libitum. I The test tank was a glass-bottomed aquarium (109 x 90 x 90 cm). Stout timbers set on building blocks sup- Permanent Divider ported the tank along the outside perimeter and allowed . . an experimenter to view the tanks from beneath. The outer - - Movable PartitIons side walls of the tank were covered with dark green cam- F. 1 ouflage cloth. Four identical smaller aquariums (51 x 25 19ure x 40 cm) were placed within the test tank in a 2 x 2 Schem~tic of test tanks. Tanks had glass bottoms, ~nd.cuttlefish array (Figure 1). A lid made from plastic mesh fit over were vlewed!rom below In order to observe the ventilation move- the four smaller tanks. A permanent opaque divider sep- ments of their funnels. arated the tanks into two pairs; movable opaque partitions could be placed betweenthe two tanks of each pair. A 0) 100 ~ = = .- ~ . ~ 80 \ ~ 1\ G 1~ § 60 1 1"-!-1'--l -..c: 1~ oS 1"-. -..c: "'.

. >= 40 "' e ,., L 0 20 40 60 80 Minute Figure 2 Experiment 1: Ventilation rate of cuttlefish (n = 20) after being transferred from holding tanks to test tanks (mean :f: SE). Page 344 The Veliger, Vol. 39, No.4 -+ - Shrimp e Control Shrimp in Jar

..-~ = 85 = .- T '" T T...... i: ~ - 75 " ' 1 ~ f :.1 1 J .

rIl 6 1 ' .:: ~ 5 / ~ . . ~. ~ '(:' . , x' '-i: ~ 55 /: oJ- - ~ .- = --= 45 ~ .- ..- = > 35 -10 0 10 20 30 40 Minutes

Figure 3 Experiment2: Ventilation rate in responseto the appearanceof a prey item at minute 0 (mean:t SE), small desk lamp (60 watts) was placed below the test tank, were placed into the test tanks, Sixteen subjects were ob- shining upward, The experimenter lay quietly beneath the served for 1 hour; an additional four subjects were observed tank, observing the ventral sides of the subjects, for 1 hour and 20 minutes, Subjects were selected at random from the 12 holding tanks and placed in the test tank 1 hour before trials, When 2. Presence of Food trials began, the experimenter recorded at 5 minute inter- W e recor d e d th e response 0 f cu ttl e fi s h t 0 th e presence

f f ' I ' .. d ' A ' vals the total time for 30 inhale/exhale cycles for each t

. 0 ami lar 100 I ems, n mve rt e d PVC cap, a b ou t 5 cm subject, Cycles could be observed, directly from the ventl- in diameter, was placed over a live shrimp in two of the lati~n mo~ement of the fu~nel with the pressure c~an~es small aquariums; in the other two aquariums the cap was of InhalatIon and exhalation, We compared v~ntllatlon empty (control), Subjects were again placed into each of th t k ' , I ' I t ' rates before and after each event for each subject, Pre-

e an s, m vlsua ISO a Ion, Aft er 1 h our h a d e I apse, d event rates were determined , by averaging the three mea- caps were mve , rt e d b y means 0 f an a tt ac h e d s t ring, ' V en- t ' l t ' t d t 5 ' t ' t I t rt ' 4 surements immediately prior to the event, Post-event rates I a Ion ra e was measure a mmu e m erva s s a mg were determined by averaging highest recorded" rate after 15 mmu ' t es b e f ore th e cap was mver ' t e d , ..lor a t 0 t a I 0 f 1 the event with the two recorded rates that Immediately h d 15 ' t T I b ' ct d ' '

.. II d h k our an mmu es, we ve su ~e s were use, SIXWI th

10 owe t e pea rate, h h ' d " h h I d ' , t e s nmp an SIX Wit t e contro con Itlon, F or an ' additional two subjects,the cap and shrimp were confined I, General Disturbance within a glass jar in order to determine the effect of solely W d d h f I fi h h I visual stimulation, e recor e t e responses 0 cutt e s to t e genera disturbance of being moved tb the test tank, One cuttlefish 3 S' ht f C 'fi was placed into each of the four small tanks, Subjects were ,lg 0 onspecl cs in visual isolation with all barriers in place, Ventilation In this experiment, we recordedthe responseof cuttlefish rate was measured starting immediately after the subjects to the sight of familiar and unfamiliar conspecifics, One n J. - G. Boal & -J. N. Ni, 1996 Page 345

-+ - Unfamiliar 0 Control Familiar ~

I ] 55 ':- ~

\ '- ~ 53 j-l ~ .1- . - .! ..:r .' ~ 51 .1- - 1\

U 1 1 --T Ii = ... '.

.,g 49 1 1 " = ~ .- ... ~ 47 > -10 0 10 20 30 Minute Figure 4 Experiment 3: Ventilation rate in response to the appearance of a conspecific (familiar or unfamiliar) or an empty tank (control) at minute 0 (mean :t SE). "- cuttlefish was placed into each of the four small tanks. 1896) or a gulf killifish (Fundulus grandis Baird & Girard, Subjects were in visual isolation. After 1 hour, the movable 1853). The movable partition separated the tanks. After partitions were raised by means of an attached string. Each 1 hour, the movable partition was raised. Ventilation rate animal could thus see one other subject. For five pairs of was measured statrting 15 minutes before the barriers were subjects, both animals came from the same holding tank raised, for a total of 1 hour and 15 minutes. The fish was (familiar conspecific); for five other pairs, each individual used for four subjects; the crab was used for four other " came from a different holding tank (unfamiliar conspe- subjects. Results were compared with those of Experiment cific). For a further eight subjects, the adjacent tank was 3 with familiar and unfamiliar conspecifics. empty (control). Ventilation rate was measuredstarting 15 minutes before the barriers were raised, for a total of 1 hour and 15 minutes. RESULTS 1. General Disturbance - 4. Differences between Stimuli Immediately after the subject was moved to the test tank We tested whether cuttlefish arousal, as measured by (the disturbance, time 0), ventilation rates averaged 83 ventilation rate, differed between similar-sized stimuli. We ventilation cycles per minute. After 50 minutes, average ~ compared responses to a crab and a fish, each roughly the rates had slowed to 45 cycles per minute, a decrease of samesize as the subjectwhen viewedfrom the cuttlefish's about 45%. Ratesthen stabilized(Figure 2). perspective (fish total length, crab carapace width, and cuttlefishmantle length), with the responseswe found in 2 Pre enceof Food Experiment 3 to familiar and unfamiliar conspecifics. . s For each pair of tanks, one cuttlefish was placed in one Treatment (control, live shrimp, live shrimp in glass jar) of the small tanks; in the other, a large glass jar was placed had a significant effect (F = 157.51; df = 2,13; P < 0.001) containing either a blue crab (Callinectes sapidus Rathbun, (Figure 3); ventilation rates increased about 40% in re- Page 346 The Veliger, Vol, 39, No, 4 ~ 20 = .-= ~ ~ 15 0.1 ~ Q. rIl ~ 10 ~ U = Q ~ 5

.--- = ~ >- 0 Control Conspecifics Prey Items Unfamiliar Familiar Crab Fish Figure 5 Experiment 4: Ventilation rate increase in response to stimuli, each of the same overall size as the subjects (mean :t SE). sponse to the presence of the shrimp, as compared to con- DISCUSSION trois. A priori tests between means showed that responses with the glass jar were significantly different from those Disturbance had an effect on ventilation rates of cuttlefish, without the glass jar (F = 21.36; df = 1,13; P < 0.001). as did the sight of conspecifics and other animals. These results are consistent with and extend those of Boyle (1983) 3, Sight of Conspecifics and Chase & Wells (1986) on the effect of chemical stimuli . on octopus ventilation rate. Ventilation rate thus appears In the test for response to conspeclfics, treatment (con- b d 11 . d . , . . , . ., to e a goo overa In Icatlon 0 f cep h a 1 opo d arousa. 1 trol, familiar conspeclfic, unfamiliar conspeclfic) had a very Th 1 . fl .1 . 11 . . 11 . . fi ff (F 3 53 df e argest In uence on ventI atlon rate was t h at 0 f t h e sma, yet statIstIca y slgm cant e ect =.; = . 0 05) (F . 4) A .. b h general disturbance. After the transfer to the test tank 2 25 . P " . Igure. pr!on tests etween t e h < d h h d ' (E . 1) . 1 ' d 11 d d h ff . xperlment , ventI atlon rate gra ua y ecrease to t e

means f . s owe t at t ere was no I erence In response to. 1 ' d f ' I ' . fi (F 0 80 df restIng rate, a pattern consistent with an interpretatIon of a ami lar an an un ami lar conspeci c =. . = ... . 1 25. P - 0 45 ' habituatIng to their new surroundings, .. " .). When presented with prey items (Experiment 2), cut-

b ' . tlefish became quite active and focused intently on the 4 D 'LE t St 1 , Illerences e ween Imu I h . h . h . 1 . . s nmp at t e same tIme t at ventI atlon rates Increase. d In the experiment on the reaction of cuttlefish to other Response to the shrimp in the glass jar differed signifi- J living animals of the same size as themselves, we found a cantly from that without the jar. This difference was prob- small but significant difference in ventilation rate between ably a result of sustained responses by the subjects unable presentation of other cuttlefish (familiar or unfamiliar) to reach the shrimp rather than any effect of the absence and other animals (crab or killifish) (F = 14,39; df = 2.25; of chemosensory information. Initial response to the sight P < 0.001) (Figure 5), There was no difference between of the shrimp did not differ between the two groups (Figure their response to a crab and a killifish (t = 2.10, df = 3, 3). P > 0.05). Response to the sight of another cuttlefish (Experiment 1 II J. G. Boal & J. N. Ni, 1996 Page 347 ~ 3) was quite different from that to prey items. Subjects ACKNOWLEDGMENTS were less active and their activity was less focused in any. . . . particular direction. Ventilation rate rose only slightly more We :would like to th~n.k the staff of the ~arme Biomedical than that of controls. We found no significant difference Institute: D: L. Brlmng, V!. J. ~ardmer, V. E. Kahla, between the response to familiar and to unfamiliar con- and F. P. DIM~rco for their tec~mcal support; P. G. Lee specifics. Although results appear suggestive that a differ- for the use of his wonderful.Sepla; and B. U. Budelman.n " ence might be found had we used a larger sample size or and R. !. Hanlon for. their helpful comments on this more sensitive method (Figure 4), Boal (in press) has found m~nuscrlpt. We appreciate the. helpful comments of the similar lack of evidence for social recognition in adults of edl~or and two .anonymous reviewers: We also thank B. ~ the same species. CaIn of Ball High Scho?l f~r her ~ctlve support and en- Response did not differ between the crab and fish pre- couragement of young sclentls~s. This work was supported sentations (Experiment 4). Both are prey items when they by NIH postdoctoral fellowshl~ NRSA# 5F32HD07686- are smaller than the cuttlefish. Cuttlefish will attempt to 02 to J.G.B. and by the National Resource Center .for capture same- or even larger-sized crabs and fish if they Cephalopods (DHHS grant # RR01024) at the Marme are hungry enough. In the laboratory, the cuttlefish appear Biomedical Institute, University of Texas Medical Branch, to prefer crabs over fish. They also approach them differ- Galveston, Texas. ently when preying upon them (Nixon, 1987). It may be LI that our results did not reflect these differences because TERA TURE CITED

our sample size was quite small (n = 8). Another possible BOAL, J. G. In press. Absence of social recognition in labo- explanation is that the two live prey items were equally ratory-reared cuttlefish, Sepia officinalis L. (: Ce- arousing when presented alone to hungry cuttlefish ac- phalopoda). Animal Behavior.

customed to receiving only dead food. Satiated animals BOYLE,P. R. 1983. Ventilation rate and arousal in the octopus. . ht d d . ff tl The Journal of Experimental Marine Biology and Ecology mlg respon I eren y. 69:129-136. The cut:lefish did not resp~nd equally to all live stimuli BOYLE, P. R. 1986. Response to water-borne chemicals by the of approximately the same size; they were clearly more octopus Eledone cirrhosa (Lamarck, 1798). The Journal of interested in food than in each other (Experiment 4). Our Experimental Marine Biology and Ecology 104:23-30. subjectswere juveniles; results with sexually mature adults CHASE,R. & M. J. WELLS. 1986. Chemotacticbehaviour in might be different. Octopus. The Journal of Comparative Physiology A 158: W fi d . h h . h h . . I . 375-381. e n It notewort y t at Wit t IS simp e experl- F ORSYTHE J W R T H & R D R 1991 P .

l d . b . ,..,..ANLON .EUSHA. .lot l m~nta eslgn ,;e. were a Ie. to support results obtaIned large-scale culture of Sepia in biomedical research. Pp. 313- with more sophisticated equipment (Boyle, 1983, 1986). 323 in E. Boucaud-Camou (ed.), The Cuttlefish: First In- ventilation rate is indeed responsive to visual ternational Symposium on the Cuttlefish Sepia. Centre de stimuli, as well as to chemical stimuli. The magnitudes of Publications de l'Universite de Caen: France. changes in ventilation rates were consistent with interpre- NIXON, M. 1987. Cephalo~d diets. Pp. 201-219 in P. .R. tations of differing salience. Ventilation rate provides a Boy~e (ed.), Ceph~lopod Life Cycles, Vol. II. ComparatIve fi bl b h . I b b .. d . RevIews.AcademIc Press: New York. ro ~st, 0 ~ectlve, an ~uantl a. e e. ~vlora measure for TRUEMAN, E. R. & A. PACKARD. 1968. Motor performances testtng anImal perception, and IS sensItive enough to dem- of some Cephalopods. The Journal of Experimental Biology on strate discrimination between classes of stimuli. 49:495-507. , I r