= —————vournal of Cephalopod Biology Volune 2, nunber) —L2Z/— SQUID AND CUTTLEFISH MARICULTURE: AN UPDATED PERSPECTIVE 31 nape in Py Tew iy Ln 2 an eee ; le Copy os a ABSTRACT Etptenber 1992 er ees pee 2 a aa ey ae ee ee ret ce STi panos an Seas coma tern eee a ae eas cae se sa a eee eee Seles ee ere Se ee eet ee ee seine tbe aettes tapes evces rroarome epee Secee n oeshaayaipase ates Pea pone ges ee KEYWORDS: Mariculture - Squid - Cuttlefish - Nutrition - Water Quality INTRODUCTION (ur laboratory has pursued the mariculture of cephalopods for 15 years (¢., Hanlon et al, 1979; Yang et al, 1983; Hanlon and Forsythe, 1985; Turk ef al, 1986; Yang et al, 1986; Hanlon etal, 1987; Hanloa etal, 1989; Yang et al, 1989) and a directed effort has been aimed ‘at culturing foliginid squids for biomedical research. Variable success with Loligo syp. bas resulted but recently there has been coasistent success culturing the loliginid squid Sepioruhis {essoniana through is lifecycle with lite morality and superb growth and reproductifn. This paper is not a review because detailed ones have appeared with regularity (Shevtéors, 1977; Boletzky and Hanlon, 1983; Hanlon, 1987; Hanlon, 1990). This paper presents (1) a synopsis of ‘our most recent culture achievements, (2) continuing and future research priortes that may speed progress in cephalopod mariculture and (3) suggestions for the culture of oceanic squid. [RECENT CULTURE PROGRESS Previous success can be summarized by pointing out that perticula attention was pid to water quality and system design, to feeding and behavior of squids and finally to finding species that are adaptable to the laboratory. Farthermore, the subtropical environment of south Texas ‘enabled collection and evalnation ofa variety of food organisms from estuaries, bays, nearshore and oceanic waters throughout the yea. Cult of the Inde i lesson liginida) This loliginid has proved to be a reliable culture candidate and its neuroanatomical features are equivalent to Loligo spp. which makes the species weful for biomedical research. ‘Furthermore it san important human food species, especially in Japan where it commands «high market price ($40/kg).TE 32. ———— Journal of Cephalopod biciesy 10000 Sepioteutis lessoniana ‘ Se ote WEIGHT(g) ° 100 200 300 400 S00 AGE(days) Figwe 1. Laboary gow of sds Sepithisetonina and Lg fore! sod PH ‘cuttlefish, Sepia officinalis. Note that the Y-axis is logarithmic (from Forsythe ca 191) Seven cent cule e=iments ave ben compe: del are big plied scpantey. As indeed a Figure 1 ts species ows to al si (2-2 iat 6 mono, rik t man insuaaneom gow rie of 728 an msi lraaoeost pow leo at Inpesiv 13% ices in body weigh per day ow). Maly hs ee very tow (ely ‘out 5 ater theft week portathing sn the slew assistant compared 0 Lalo ‘pp. Socal mated sus tave ee Sled wo maid in cach experten ta ,pogey tive bon race fea pencrnton wat acter one, atongh te mamber of cuing ‘tcp wer low. Sou lemted crowding wel wae gully ws god especialy eepiog Bia blow 80mg mucnal ws oats of 25 Egat end 2 age were ono the raceway dese tlow. Thee rel fr rua eying complsbed with Llp 7 inion best growth (2 ia 18 oad) ws demons by Hanlon eal 9B) nue ofthe Eur th Sepia officinalis (Seploidea: Sepidac) Denis of cutefish culture are described by Forsyhe etal (1991). This cuttlefish is also ‘8 superb tlomedica research animal and is marketed for human food. The large hatchlings are easy to rear and morality to adulthood was usually negligible. Cutlefish ea variety of ive and ‘dead food: (DeRusha et al, 1989; Lee etal, 1991) and grow at rates of 3-4% bw/d to 500-1400 ‘gin about 10 mouths at temperatures of 20-24°C (Figure 1). The sedentary cutefish tolerate ‘crowding well and successive generation up to F, have been cultured. Overlapping generations of cutlefuh have been produced year-round by controlling temperatures berween 13 and 25°C (Figure 2). These cutlefish are also extremely tolerant of handling and they have been shipped ‘vceessfuly throughout North America. aac Head Ha = e+es Journal of Cephalopod Biology ———————-—33 Grom ED spomning YY Tonp. 2528 © gs ow Temp. 1618.6 2m Tomp. 2:28 6 é g om Tomp. 2126 6 1 Temp. 176 Gear ea seer tet eee aore tive teiag Months Figure 2, Duration of hatching, grow-out and spawning for Sepia officinalis in relation to rearing temperature. ‘The hatching phase represents the period fom fist hatching to last hatching for a generation while the spawning period was measured from first egg-laying until deat ofthe last cuefish in that generation. RESEARCH PRIORITIES Development of Pelleed Diets ‘A major limitation in culturing larger numbers of cephalopods bas been providing the ‘proper nutron. Live foods are expensive because of the time and equipment required for Personne! to collect and maintain them. The ultimate aim isto develop pelleted diets that ae ‘utritous for squids during all phases of the life cycle. The approach has been (1) 10 tse behavioral conditioning to enhance the inal reactions to new diets, (2) 10 identify chemical fauractants that enhance caprure and palatability of pelleted diet and (3) 10 determine the Aigestbilty of diets as well asthe dietary requirements of te target cephalopod species. Inial ‘cost analyses indicate thatthe use of pelleted diets has lowered food costs by 40% iaially and eventually could lower the costs by as much as 80%, ‘Thus far we have conducted tials in which various dead but natural foods have been resented to octopuses, eutlefishes and squids (DeRusha et al, 1989; DiMarco etal, pers. comm late juvenile cuttefsh and squid grew well on frozen shrimp and marine ish, respectively, Behavioral contitioning enhanced the response to new diets in octopuses; however twas foun thatthe immediate feeding history of the octopuses influenced the degre to which they responded to conditioning techniques and new diets (Hanlon et al, pers. comm). Nevertheless, oclopset could be induced to eat pellets and chicken meat within a8 lite as 2 days of raining. Recealy, ‘squid were conditioned to take food off rods held by the experimenter, andthe food was changed from live shrimp, 10 dead shrimp, to pellets over a 18-day period. Further experiments proved that squid fed freshly dead or frozen fish will grow atthe same rate as squid fed live fish aad Unt their digestive gland histology fs similar (DiMarco et al, pers. comm) — Hanlon et ai. Squid ang Cutttefieh culture—Holyrood Syspostun —- TT 34 Journal of Cephstopod Biolozy ‘Chemoatraction in cephalopods bas been demoastated experimentally with octopuses using a Y-maze chamber and monitoring the locomotion of the octopuses (Lee, in press). The ‘octopuses responded positively to minute quantities of amino aids (10* M) entering ane ofthe ‘Y-maze arms. This method can be wed to identify chemical that might act as feeding stractants Gilly and Lucero (in press) have verified that the presumed “olfactory pit" behind the eye of Ioliginid squids actually does function in detecting “odors,” and thelr experimental design (based ‘upon neurobiological responses that fire the giant 2x00 for jet propulsion) wil allow ancter assay to test reactions to chemical. In growth wials, tre pelleted det formulations were fed to octopuses and cutefish (Lee tal, 1991). Adult cutefish survived and grew on penaeidshrimp-bated pellets for 60 days, although some cannibalism occured and growth rate was les than 50% of normal. Juvenile ‘cutlefish (13 g) were conditioned to penaeidshrimp-based pellets within 4 days and aftr 57 days ‘they had doubled their weight (instantaneous growth rate of 1.5% Bw/A), although abou oe third had been cannibalized. Thus artificial diets such as pellets can be wed 19 maintain growing cephalopods. Seawar ‘Squid grow-out systems. Systems for culturing four species of squids and two species of cutlefishes have been published (Yang et al, 1989; Forsythe et al, 1991). While many cogincering and biological issues remain tobe resolved, the methodology is suiciently developed to warrant aggressive pursuit of production cost reduction. The costs identified as mest realy ‘reducible through improved system design fall nto the categories of labor and ilies (Turk and Lee, 1991). Our recent atention has focused on maintaining the required culture enviroameat ‘sing passive filtration driven by airlift pumps. Airlift pumps have been used for aeation and circulation within ponds and tanks (Parker and Sule, 1987) but their poteaial for pumping the large volumes associated with recirculating systems has not been fully appreciate. A side-by-side comparison ofthe mechanical pump-drven raceway system versus sift. eiven raceway system was reported recently (Turk and Lee, 1991; Figure 3). Water weatmest ‘was similar forthe asif-driven Figure 3A) and pump-driven raceways (Figure 3B; described ‘by Yang etal, 1989) except that all but one circulation loop was removed for the iit driven riceway. For the arif-driven raceway, water from the raceway (Figure 3A) was aiifed (B) to a reservoir (C) then lifted again by 4 arts (D) into a particle fitereader tank (E). Gravity forced the water through 3 protein skimmers (F, venturi style) and 3 upwelling activated carbon ‘chambers (G) into the filter tank where it was consiauously recirculated by aii, Tested water ‘vas then drawn from beneath the filter bed (H; crushed oyster shel) by three ailits () into 2 Ieader tank (J) and flowed by gravity through an ultraviolet strlizer(K) back into the raceway ‘opposite the end where water was removed The pump-driven protein skimmer loop (M) Wat ‘elained for increased foam stipping capability during periods whea the squids ink. Waer quality ata for the side-by-side comparison is displayed in Figure 4. The airift-riven raceway ‘maintained water quality conditions (Figures 4B, 4D and 4F) equivalent to the punp-riven raceway (Figures 4A, 4C and 4B) while supporting similar biomass through 200 days of culture. ‘Thus airif-driven recirculating systems have the potential 10 reduce high labor costs associated with pump-diven systems with a concomitant reduction in energy usage. The airlift system equaled or outperformed the pump-driven systems in terms of; (1) water tunover, (2) ‘water quality, (3) rearing squid (4) labor for maintenance (5) equipmeat and material relecemeat and (6) energy usage. Based on our costs for labor and electricity, the savings per system forthe Ibatchery phase (fist 2 months) was $180/yrhank and for the grow-out phase (4-6 ments) was, ‘2023/yrraceway. While the actual savings are modest when expressed in terms ofa side-by-side system comparison, these savings would become significant fora larger production operation (e8., 20.50 systems). — anton at al. Sautd and Cuttlefish culture—Holvracd Syaposta a Journst of Cephalopod Biology Volune 2, nusber 1————— 35. “tas A iol oot B t Feel fe hes ie Foe 3 ae Lea oa Ee, ee, de c ] eine! lees i 7 of og Po Fz ag ilutah + aaftoeny or E wafer oor F bas Figwe 4. Water quality dat comparing pump driven (A, C,B) and ait-eiven (B,D, F) recirculating raceways containing similar numbers and biomass (kg) ofthe squid Seploteuthis leczoniana. The broken lies represent the point at which squids were ‘wansferred from Hatchery (raceway systems. Computer automation of recirculating seawater systems. Aquaculture industies ‘worldwide have been tlow to uilize automation and process contol technology, whereas many Industries (eg. food processing, chemical xod petrochemical plats) use process contol routinely to reduce cost, eliminate failures and maximize production (Lee, 1991). Cephalopods require high stndards of water quality while feeding at high rates 20-30% bw/d) and producing copious (quantities of ammonia and sometimes ink. The management and operation of the cephalopod Culture systems is labor-intensive and requires well-ained persoanel, resulting in high production costs. These costs can be reduced significantly through the pragmatic design of an automated ‘contol system. A functional automated culture system should be capable of coatolling Aemperatue, water movement, water quality, photoperiod and feeding: it should be economical, — Wanton et al. Squid and Cuttlefish culture—Holyrood Symposium —-_ee- essay 36————— Journal of Cephalopod Biology Volune 2, nusber 1 userviendly, redundant, flexible, maiatenance-fee, and compatible with a wide range of instrumentatica, The use of aificial intelligence (1. expert computer progam) 1 integrate the fanetion of the aquaculture tanks ac well se predict perhations and coneet fr them fle possible. The advantages of ineligen aviomaed systems fall into four extegories: (1) online ‘monitoring of system funetion so tat a manager has instantaneous satus of system operation; (2) historical data and trending of system functions for use in predictive management, (3) 24-hour ‘monitoring and alarm capabilities 1 reduce the labor required to manage the systems; and (4) ‘rapid development and duplication of contol models improve the uniform implementation of proven management schemes and asit in the rapid transferral of these methods to oer potential ‘A micocomputer-based automated system has been designed and the intial performance indicates that tis a logical platform for accomplishing the objectives above (Lee, 1991). A data acquisition anf control system was installed on a closed recirculating seawater culture system (Turk and Lee, 1991). “Sensors monitor dissolved oxygen, pH, conductvity/alinity, ‘oxidaton/reducton potential exmperature, water level, low rte, and prfiter head presse, The final system controls ertical components, wigges alarms and performs the laoriou tasks of data collection, computation and summarization, The microcomputer is connected to the MBI's ‘microcompote local area network (ethernet LAN) that is composed of 19 peronal computers (BM-compatbles, Maclnoshes and Digital Equipment MicroVax), Thus, data on aquaculre system function and alarm conditions can be accessed readily from the LAN tnd many culture routines are controlled remotely from the manager's desk or automated entirely. The development of costeffectve, automated culture systems for cephalopods holds the poteaial for expansion of, ‘cephalopod cutie to laboratories wit less experence in culture than the MBI Reduction of nitrate in recirculating seawater systems. Curent tess ae being conducted (on an automated dentrifying bioreactor (filter) that was designed a8 a direct rel of advances in contol technology (Lee, 1991). Aquarium/aquaculture sysiems have econoaical snd ‘manageable mechanisms (ie. aerobic biological filters) fornication (reduction of ammonia) and mineralisation of metabolic wastes. NH, —> NO, —> NO, ammonia nite mirate However, an economical means for limiting the accumulation of nitrate (NO,), the product of sitrifcation, has not been available, Nitrate (80 mg L) isnot as toxic as either ammonia (0.1 amg L') or niite (<0.1 mg L') but it causes morality thigh levels and sub-ethal levels cause aberant behavior, depression of growth and decreased fecundity. As a resilt one it forced to replace some or all of the system water to dilute the nitrate: these water exchanges can be expensive andor cause unwanted perturbation. The alterative is to operate a denitifying bioreactor that convers nitrate to nitrogen fas. which thea escapes to the atmosphere, NO, —> NO, —-> NO —>N,0 —> N, irate nite nic nitrous nitrogen oxide ‘oxide "gar ‘The major problems associated with the bioreactors operation are the nee forthe bioreactor to bbe anaerobic tc accommodate the deniifying bacteria and the type and level ofthe carbon souree ede to feed the bacteria (usually methanol in seawater systems). Aquariste bave built and maintained such filters but they are labor-intensive to monitor andthe potential production of toxic nitrite and hydrogen sulfide exits if the iter isnot operated optimally. — Hanior et a1. Squid and Cuttlefish culture—Holyrood Sympoaiue —ase eR Se er RA Journal of Cephaloped Biology Volume 2, nuaber 1————37 ‘The denitiying bioreactor has been completely automated using sensors and outpy: devices a described above forthe automated culture system (Lee, 1991). A compartment mode! was developed based upon our insights into the physiological and chemical processes involved “The amount of nitrate produced by a standard biomass of squids (with 2 knowa instntaneot _growth ric) has been modeled quantiaively and included inthe model. As a result, the leve ‘of nite inthe tank canbe regulated simply by adjusting the rate of methanol addition andthe {low rate through the binreactor. The automated bioreactor is as an example ofa filration system, ‘that is too complicated to control manually but when automated and refined may become bot ‘cost-effective and dependable for a wide usergroup. IDEAS FOR OCEANIC SQUID CULTURE ‘The central preblems are (1) the tiny size of hatchlings compared to Sepia, Sepioteuthie _and even moet Loligo spp, (2) unknown mode of feeding with fused tentacles and no observation: ‘on feeding bebaviot an! (3) highly variable egg availablity. Considering that all oceanic squid feem to bave tiny hatchlings (eg, <1.5 mm), there must be some adaptive reson that fuse tentacles have evolved. Understanding this mechanism of feeding and how it affects erly survival ‘would have far-reaching ramifications for squid biology and fisheries. Fused tentacles are the primary feature of the “Yhynchoteuhion lava" (Roper and Lu, 1978) that in ler is about 1.0 mm manile length (ML) at hatching and about 8 mm ML atthe largest size before the entacles Separate and the squid transforms into a juvenile with all 4 arm pals and the tentacle similar i form to typical adults (O"Dor, 1983). This is obviously atime of specinlized feeding (Balch et al, 1985; O'Dor etal, 1985). ‘Based upon experience with larger equi hatchlings the published repons on Ilex (ibid) and numerous discussinas, the following ideas are presented for future consideration in culture fatempts, ‘Temperate of eggs should be Kept close t that found in nature; fluctuating {emmperatures appeat to cause many problems during organogenesis in early stages, or inthe late sages these flactmations induce premature batching, which has disastrous consequences. Most fmportanly, Inter stage eggs and hatchlings cannot be moved or bandied in any way because physical disruption of the delicate skin and its mucus covering lead to bleeding, secondary {nection and morality (Hanloo, 1990). For example, Balch et al (1985; see their Fig. 1) described an incubatioe tank in which hatched squids were drawn though a tube into a collecting cone: the turbulence and contact with robe sides would be sufficient to damage hatchlings ‘eriously. I is advisatle to suspend or float the eggs in the same tank in which rearing will be tempted (see Arpold, 1990 for other techniques), and most importantly, the rearing tank should be large in dimension snd volume so that excellent water quality (NH-N'< 0.1 mg/L, NON < (0.05 mg/L, NO,-N < 50 mg/L.) can be maintained end hatchlings have litle opportunity to contact, wall Balch eal (1985) wsed 4 L containers. Increasing this to 400 L as a starting point would be highly recommends, Success with rearing the small 1.6 mm ML hatchlings of Loligo pealei ‘was liited (Yang ets, 1980) until we enlarged tank size manyfold (Hanlon etal, 1987). [No unoquivoes! observations of natural feeding have been made on any oceanic squt hatching o our knowledge. The suggestion of O'Dor etal (1985) about the possibility of mucus feeding in lexis provocative and worthy of consideration. These paralarvae inhabit seas that are seasonally rich with plankton; thus morphological studies of their fused tentacles, arms and the digestive tract may discover analogs wilh other moluscan filter feeders. An addtional suggestion fs tothe use dissolve organics in sea water as « potential source of nutrition for bathlins. —— Wanton et ai. Squid and Cuttlefish culture—Holyreod Symposiumay 38 “— Journal of Cephaleped Biology Volume 2, nusber 1————— Other motiusan larvae utilize this source (Manahan, 1989) and the microvllar epidermis of cephalopod hatchlings might be capable of thie function. Experiments with dissolved organics met with variable sults. Survival of unfed hatchlings under coatolled enviroamenal conditions was extended (.e.,5 days without dissolved organics versus 10 days with them) wih solved orga levels of 10.0 mg Carboa(L, but similar levels did not improve survival of hatchlings in mass culture experinents. Digestive gland histology of te hatchlings given dissolved organics wae ifferent from that of fed and ufed laboratory cultured hatchlings but similar to that of fild- collected hatchlings and adults (Vecchione and Hand, 1990). Thus, increased survival and more Iealiny appearance ofthe digestive gland issue warrants further experimentation with diesolved ‘organics i squid culture systems. To understand fully the benefits of dissolved organics, an ‘axenic culture would be required to prove that dissolved organics are taken up by the hatchlings irety, since bacteria and other microorganisms ean fist uilize the organics and secondarily be consumed by the hatchlings (Manahan, 1989). “The recent techniques developed for atic insemination of oceanic squid eggs (Armold sand O'Dor, 1990) may enable researchers to produce hatchlings for prelimioary rearing wials Fiaally, it may be worthwhile to coasider tht these very small ad specialized atchlings may have some microscopic organ feature (comparable othe lateral ine analogue recently described {in squids and culefisbes; Budelmann and Bleckman, 1990) that complements feeding withthe fused tentacles and thes peculiar arrangement of suckers. Careful morphological analytes ofthe tentacles, arms and head region (by LM, TEM and SEM) may give important clues to their fonction (op. cit; Kier, 1985) and together with deuiled behavioral observations of living Iatchlings some progress may ensue CONCLUSIONS ‘Significant progress has been made toward culturing cephalopods of particular importance to zoological and biomedical research and much of this information is relevant to fur large- sale culture for human food. Progress in developing less expensive storable diets for these active camivores is 4 mandatory step that would have far-reaching effects in the expanded use of cephalopods for research, and recent experiments indicate that tis goal is attainable. System aviomation and improved re-use of seavater trough development of an anaerobic bioreactor ‘would improve the reliability and cost-efTecivenes of cephalopod cultre for biomedicine, and ‘would have broad applications throughout the aquaculture induty. ‘There is an imporant need to acquire basic life history information for the many ‘commercially important oceanic squid species worldwide, and its essential to lear how these specialized hachlings feed and grow daring the critical first few weeks of life. Our work with loliginid squids and cutlefishes has highlighted many basic requirements of very small equide (several mmm ML) and this information is applicable to oceanic squid culture ooce the squids grow ‘beyond from the rhynchoteuthion sage. Nevertheless, a novel approach is required to make this, . which would have far-reaching ramifications forall squid biology and ACKNOWLEDGMENTS ‘Our stccess in culturing cephalopods is a result of the labor of many present and past, ‘members ofthe MBI's technical staff. Research Associates John Forsythe, Paul DiMarco and Kay Cooper; Research Assistants Bill Browning, Randy DeRusha, John Dobias and Bobby Dayi Research Tectnician, Vivian Kabla; and Latoratory Technical Assistants Rich Toth and Mike Hiarrelson have all played essential roles in ou receat research programs. Our colleague and —— Manton et a1. Squid and Cuttlefish culture—Holyrood Syeposiue —Journal of Cephsleped Biology Volune 2; nusber 1— Professor Emeritus at UTMB, Dr. Won Tack Yang pioneered research in squit culture. Financial support has been provided by the National Iastiutes of Health, Division of Research Resources (Grants RR 01024 and RR 04226), Texas A&M Sea Grant College Program and the MBI's Marine Medicine account. RTH thanks the late Professor Frederick A. Alsi and hs staff for ‘memorable Newfoundland hospitality and a fine symposium, LITERATURE CITED ‘Arnold,3.M. 1990. Embryonic development ofthe squid. In: Gilben, DL, Acelman, J, W.. and "Arnold, 1M. (eds), Squid as Experimental Animals, Plenum Publishing Corp. New Yor: 7150. ‘Arvold,1.M. and O'Dor, RK. 1990. vitro ferilizaion and embryonic development of oceanic ‘squid. Journal of Cephalopod Biology 1(2): 21-36, Balch, N., O'Dor, RK. and Helm, P. 1985. Laboratory ressing of thynshoteuthions of the aamastrephid squid Mex illecebrosus (Mollusca: Cephalopoda). Ve Miliew 35(3/):243- 246. oletzy, Sv. and Hanlon, R.T. 1983. A review of the laboratory maintenance, rating and ‘culture of cephalopod molluscs. Memoirs ofthe National Museum, Victoria 44: 147- 187, ‘Budelmano, B.U. and Bleckmana, H. 1988. A lateral lie analogue in cephalopods: water waves ‘generate mcrophonic potentials in the epidermal hea lines of Sepia and Lolliguncule, ‘ournal of Comparative Physiology, A 164: 1-5. Dekusta, RL, Forsythe, LW, DiMarco, PF. and Hanlon, R-T, 1989. Alerative diets for ‘maintaining and rearing cephalopods in captivity. Laboratory Animal Science 39(4): 306-312. DiMarco, FP, Turk, PE., Browning, WJ, Scimeca, IM. and Lee, P.G.(pers.comm) The effects ‘of a non-living det on the laboratory survival, growth and digestive tact histology ofthe squid, Seploteuthis lessoniana. Journal of Zoology. Forsythe, LW. Hanloa, FLT. and DeRusha, R. 1991. Pilot large-scale culture of Sepia in biomedical research. In: E. Boucaud (ed), The Cutlefish: Acta I. ht. Symp. Cutefish Sepia. Center Publ. Uni, Caen, France: 313-323. Gilly, WM. and Lucero, MP. (ia press) Behavioral responses to chemical stimulation of the olfactory organ inthe squid, Lollge opalescens. Journal of Experimental Biology. Hanlon, RT. 1987. Mariculure. in: PR. Boyle (et), Cephalopod Life Cycles Vol. Tl ~ ‘Comparative Reviews. Academie Press, London: 291-305. Hanlon, -T, 1990, Maintenance, rearing, and culture of teuthoid aad sepiid squids. ln: Gilber, DL, Adelman, J, W.J. and AmoldJ.M. (ds), Squid as Experimenal Animals. Plenum Publishing Corp. New York: 35-62. Hanlon, RT. and Forsythe, JW. 1985. Advances in the laboratory cultue of octopuses for biomedical research: Laboratory Animal Science 35: 33-40. Hanlon, RT., Hixon, RF, Hulet, WH. and Yang, W-T. 1979. Rearing experiments on the (California market squid Loligo opalescens Berry, 1911. Veliger 21(: 428-431. Hanlon, R.T. Turk, PE., Lee, .G. and Yang, WT. 1987. Laboratory eating of te squid Lotigo peale to the juvenile stage: growth comparisons with fishery dat. Fishery Bulletin B5(I): 163-167. Hanloo, R-T., Yang, W-T., Turk, PE, Lee, PG. and Hixon, RF. 1989. Lavoratory culture and estimated ie span ofthe Eastern Alantic squid Lolig forbes! (Motsca: Cephalopod). ‘Aquaculture and Fisheries Management 20: 15-33. Hanlon et al. Squid and Cuttlefish culture—Holyrood Symposium —40 ———— Journai of Cepratoped Biology Volume 2, number 1 Hanlon, R.T., DiMarco, FP., Lee, P.G, and CR. Gustavson. (pers. comm) Bebavionl ‘conditioning of octopuses as 2 mechanism of introducing novel allemstive det Laboratory Animal Science. Kier, W.M. 1985, The musculature of squid arms and tentacles: ulrastuctural evidence for Tunetional differences. Journal of Morphology 185: 223-239. Lee, PG. 1991. Automation of aquaculture systems: a review and practical guide to ‘implementation. I: Proceedings of the Symposium on Engineering Aspects of Intensive “Aquaculture, Northeast Regional Agricultural Engineering Service, Ihaea, NY: 284-300. Lee, PG. (in press) Chemotaxis by Octopus maya in a Y-maze. Jouraal of Experimental Marine Biology and Ecology. Les, PG., Forsythe, JF, DiMarco, FP., DeRusha, RD. and Hanlon, RT. (1591) Iniial palatability and growih tals on a pelleted diet for cephalopods. Bulletin of Marine Science 49:656-667. ‘Manahaa, D.T. 1989. Amino acid fluxes to and from seawater in axenic veliger larvae of 2 bivalve (Crassosrea gigas). Marine Ecology Progressive Series 53: 247-25. OvDor, RK. 1983. Ilex ilecerosus. In PR. Boyle (e4.) Cephalopod Life Cycles; Volume 1:Species Accounts. Academic Press, Loodoa: 175- 19. O'Dor, RX, Helm, P. and Balch, N. 1985. Can rhynchoteuthions suspension feed? (Mousa: Cephalopoda). Vie Milieu 35(3/4): 267-271. Parker, N.C. and Sutle, M.A. 1987. Design of arift pumps for water recirculation an aeration in aquaculture. Aquacultural Engineering 6: 97-110. Roper, CFE. and Li, CC. 1978. Rhynchoteuthion larvae of ommastephid squids of the Western North Aantic, withthe first description of larvae and juveniles of Mex Mlecebrosus. Fisheries and Marine Service Technical Report No. 833. pp. 1411-1426. ‘Shewtsova, V.D. 1977, Cephalopod ass potential object of rearing. Central Research lnstite, technology, Economics, Research and Information, Fisheries, Moscow. Review NS Information Series Fish Resources World Ocean: 1-46 (i Russian). ‘Turk, PIT. and Lee, P.G. 1991, Design and economic comparsoa of airlift versus electric pump “riven recirculating systems. In: Proceedings of the Symposium on Engineering Aspects of Intensive Aquaculture: Northeast Regional Agricultural Enginering Service, Mhaca, NY: 271.283. ‘Turk, PT, Hanlon, RT, Bradford, LA. and Yang, W-T. 1986. Aspects of feeding, growth and ‘survival ofthe European squid Loligo vulgaris Lamarck, 1799, reared trough the early ‘gromh stages. Vie Milew 36: 9-13. Vecchione, M. and Hand, V. 1990. Digestive-gland histology in paralaval squid (Cephalopods:Loliginiae). Fishery Bulletin 87: 995-1000. ‘Yang, W-T., Hanlon, RT. Hixon, RF. and Hulet, W-H. 1980. Culture of California market squid from hatching - frst rearing of Loligo to subadalt stage. Aquabiology 2:412-418, (in Japanese) ‘Yang, W-T., Hixon, RF, Turk, PE. Krejcl, ME., Hanlon, RT. and Hulet, W.H. 1913. Culture of California market squid from hatching - completion of the rearing cycle to second generation hatchlings. Aquabiology 5(5): 328-339. (in Japanese) Yang, W-T., Hixon, RF., Turk, PE. Krejcl, ME., Hule, WH. and Hanlon, RT. 1996. Growth, ‘eltavior and sexual maturation of the market squid, Loligo opalescens,culteed through the lifecycle. Fishery Bulletin 84: 771-798. ‘Yang, W-T., Hanloo, RT, Lee, P.G and Turk, PE. 1989. Design and function of clossd seawater systems for culturing lolginid squids. Aquacultural Engineering &: 47-65. — Hanion et al. Senta and Cuttierieh evlture—Holyre Srepsciun —