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Physiological and behavioral responses of tanner crabs (Chionoecetes bairdi) to handling, emersion and temperature

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(C H I O N O E C E T E S BAIRDI) T O H A N D L I N G , E M E R S IO N A N D T E M P E R A T U R E

By

S onya Y. El M ejjati

H ead, G raduate P rograni in M arine S ciences and L im nology

A P P R O V E D : \ ■ ________

D ean, School o f F isheries and O cean S ciences

D ean o f the G raduate School

J - f 2~c7<9<~>_________ Date

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( CHJONOECETES BAIRDT) TO HANDLING, EM ERSION AND TEM PERATURE

A THESIS

Presented to the Faculty o f the U niversity o f A laska Fairbanks

in Partial Fulfillm ent o f the Requirem ents for the Degree o f

M A ST ER OF SCIENCE

By

Sonya Y. El M ejjati, B.A.

Fairbanks, Alaska D ecem ber 2006

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C om m ercially harvested Tanner crabs (Chionoecetes bairdi) are exposed to physical stressors during capture and sorting including changes in tem perature and oxygen availability. This study characterizes the physiological and behavioral responses o f Tanner crabs exposed to air (em ersion) at 8 and -15°C for various durations.

Concentrations o f glucose and lactate in hem olym ph m easured betw een 30 and 120 min following em ersion for 45 m in differed betw een anim als exposed to 8 or -15°C. Glucose concentrations were higher am ong anim als em ersed at 8°C than those exposed to -15°C w ithin the intervals sampled. Lactate concentrations were unchanged at intervals follow ing em ersion at 8°C, while they were elevated at 120 m in following em ersion at -15°C. Rates o f oxygen consum ption (V O2) increased im m ediately follow ing 15, 30, and 45 m in em ersion at 8°C, whereas 30 and 45 m in emersion at -15°C resulted in depressed V O2. All crabs survived handling and emersion at 8°C, w hile exposure to -15°C resulted in increased m ortality. Thus, differences am ong physiological param eters corresponded w ith differences in percentage survival betw een the two tem perature treatm ents. W hile not providing a causal relationship betw een survival and physiology, the m etabolic responses o f Tanner crabs following a sim ulated capture protocol provide a predictive index o f subsequent survival.

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Signature P a g e ...i Title P a g e ...ii A b s tra c t... iii Table o f C o n te n ts ...iv List o f F ig u re s ...vi List o f T a b le s ...vii List o f A p p e n d ic e s...ix A ck n o w led g m en ts...x C h a p te r 1 : G e n e ra l I n tr o d u c tio n ... 1

Tanner Crab Life H istory... 1

Tanner Crab Fisheries B ac k g ro u n d ... 4

Tanner Crab Biology...8

M etabolic p h ysio lo g y...8

Stress physiology...I I Research O bjectives and Chapter O rg an izatio n ... 14

R eferences... 15

C h a p te r 2 : E ffects o f E m e rsio n , T e m p e ra tu re a n d H a n d lin g on H em o ly m p h M e ta b o lites o f T a n n e r C ra b s (C hionoecetes b a ird i)...21

A b stract... 21

Introduction... 22

M aterials and M ethods... 24

Anim als an d husbandry...24

Sam pling o f hem olym ph...24

Glucose assay...25

Lactate assay...27

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Physical responses...28

Glucose concentrations...28

Lactate concentrations...29

D iscussion ...30

A cknow ledgem ents...35

R eferences...36

C h a p te r 3 : E ffects o f E m e rsio n , T e m p e ra tu re a n d H a n d lin g on th e O xygen C o n su m p tio n o f T a n n e r C ra b s (C hionoecetes b a i r d i ) ... 42

A b stract... 42

Introduction... 43

M aterials and M ethods... 45

Anim als a nd husbandry...45

R espirom etry c h a m b e rs...46

M easurem ent o f rates o f oxygen consum ption...46

Changes in VO2 fo llo w in g the emersion trea tm en ts...47

Correction fo r p robe d r ift...48

Statistical analyses...48

R esults... 49

Percentage survival o f Tanner cra b s...49

VO2 o f Tanner crabs between treatm ent g roups...50

VO2 o f Tanner crabs between sam pling periods (S P )...50

D iscussion...51

A cknow ledgem ents...57

R eferences...58

C h a p te r 4 : G e n e ra l C o n c lu s io n ...6 6

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Figure 1.1 Tanner crab fishing sections in the K odiak D istrict...20 Figure 2.1 Concentrations o f (A) glucose and (B) lactate in hem olym ph o f

Tanner crabs... 41 Figure 3.1 Tim eline for m easurem ent o f rates o f oxygen consum ption before

and after Tanner crabs were exposed to subsequent em ersion

treatm ents... 62 Figure 3.2 Rates o f oxygen consum ption o f Tanner crabs at different sampling

p eriods...63 Figure 3.3 Effects o f subsequent stressors on rates o f oxygen consum ption o f

Tanner crabs previously em ersed at 8°C and -15°C for (A) 15 min,

(B) 30 min, and (C) 45 m in... 64 Figure 3.4 Percentage change in rates o f oxygen consum ption o f Tanner crabs 65 Figure A .l A verage daily seaw ater tem peratures in crab holding tanks in 2002

and 2003... 69 Figure C .l Leg and core tem peratures o f Tanner crabs exposed to air at -15°C ...71 Figure D .l Percent dissolved oxygen m easured for 5 hours in aerated-seaw ater 72

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Page Table 2.1 Percentage o f Tanner crabs that survived (S), righted (R), and lost

limbs (L )... 39 Tabic 2.2 A verage concentrations ± SE o f glucose (mg dl"1) and lactate

(mg dl"1) in the hem olym ph o f Tanner crabs... 40 Table 3.1 Percentage survival (S) o f Tanner crabs for all sam pling periods...61 Table B. 1 Tw o-w ay AN OV A (F-test) for levels o f hem olym ph glucose

betw een crabs em ersed at 8°C or -15°C and sam pled at various

tim es post-treatm ent, on Day 0 ... 70 Table B.2 Tw o-w ay AN OV A (F-test) for levels o f hem olym ph lactate betw een

crabs em ersed at 8°C or -15°C and sam pled at various tim es post­

treatm ent, on Day 0 ... 70 Table B.3 T-test for glucose and lactate levels in hem olym ph betw een crabs

handled and undisturbed, on Day 0 ... 70 Table B.4 K ruskal-W allis A N O V A on ranks (H-test) for levels o f glucose and

lactate in hem olym ph betw een crabs em ersed at 8°C and -15°C,

handled and undisturbed, 7 days post-treatm ent... 70 Table E. 1 Standard m etabolic rates (SM R) o f Tanner crabs in the em ersion

treatm ents... 74 Table E.2 V O2 in SPI betw een crabs em ersed at 8°C or -15°C for various

durations... 74 Table E.3 V 02 in SPII betw een crabs em ersed at 8°C or -15°C for various

durations... 74 Table E.4 V O2 in SPIII betw een crabs em ersed at 8°C or -15°C for various

durations... 74 Table E.5 V O2 in SPIV betw een crabs emersed at 8°C or -15°C for various

durations... 74 Table E.6 RM A N O V A for V 02 o f crabs previously em ersed at 8°C for 15 m in

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Page Tabic E.7 RM ANOVA for V O2 o f crabs previously em ersed at 8°C for 30 min

and subjected to a subsequent stressor... 75 Table E.8 RM A N O V A for VO2 o f crabs previously em ersed at 8°C for 45 m in

and subjected to a subsequent stressor... 75 Table E.9 RM AN O V A for V O2 o f crabs previously em ersed at 8°C for all

durations o f exposure and subjected to a subsequent stressor... 75 Table E.10 RM A N O V A for V 02 o f crabs previously em ersed at -15°C for 15

m in and subjected to a subsequent stressor...75 Table E. 11 Friedm an RM A N O V A on ranks for V O2 o f crabs previously

em ersed at -15°C for 30 m in and subjected to a subsequent stressor... 75 Table E.12 RM A N O V A for V O2 o f crabs previously em ersed at -15°C for 45

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Page A ppendix A A verage daily seaw ater tem peratures (°C) in crab holding tanks at

the K odiak Fisheries Research Center in Kodiak, Alaska, in 2002

and 2 0 0 3 ... 69 A ppendix B Sum mary o f A N O V A tables for concentrations o f glucose and

lactate in hem olym ph o f Tanner c ra b s ... 70 A ppendix C Im pact o f the com bined effects o f subfreezing tem perature and

duration on leg and core tem peratures o f Tanner c ra b s ...71 A ppendix D Linear drift o f the oxygen p ro b es...72 A ppendix E Sum mary o f AN OV A tables for rates o f oxygen consum ption (V O2)

o f Tanner c rab s... 74 LIST OF APPENDICES

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I w ould like to thank my m ajor advisor, Dr. C. Loren Buck, for his perseverance, patience and guidance throughout this research project. He gave me unique opportunities to work in fields I was not fam iliar with, and helped me learn and grow from my m istakes. I am forever grateful to my co-advisor, Dr. A lexandra C.M. Oliveira, for her unfailing support and b elief in my abilities. She shared valuable advice and knowledge in every aspect o f this project and was a great source o f positive energy.

I w ould also like to thank com m ittee m em bers Drs. A lf H. Haukenes, Bradley G. Stevens, and Scott Sm iley for their encouragem ents and insights in each o f their

respective areas o f expertise. A lf Haukenes spent countless hours and m uch effort in the laboratory, collecting data and beating up “the instrum ents” with me.

Financial support for this research was provided by the U nited States D epartm ent o f A griculture - Cooperative State Research, Education, and Extension Service (USDA- CSREES). I w ould like to recognize the Rasm uson Fisheries Research Center and the School o f Fisheries and Ocean Sciences, UAF, for my scholarships and travel awards.

I m ust acknow ledge everyone who was involved directly or indirectly in m y work. I couldn’t possibly nam e everyone but am grateful for your contributions in this research and keeping me sane throughout the process. M y friends and fellow students valiantly encouraged and advised me. The UAF interlibrary loan was a wonderful resource, and the Fisheries Industrial Technology Center (FITC), SFOS and UAF graduate school staff were alw ays so friendly and helpful. I sincerely thank my dear friends and relatives for their love and support.

M y fam ily was alw ays there for me. Their love, encouragem ents and words o f wisdom gave m e self-confidence. Thank you for always believing in me and telling me so. Avec amour, toujours.

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Ta n n e r Cr a b Li f e Hi s t o r y

Tanner crabs (Chionoecetes bairdi\ Rathbun, 1924) are abundant in the N orth Pacific Ocean from the coast o f Oregon to the G ulf o f A laska and in the Bering Sea w here they inhabit depths ranging from the subtidal to > 400 m eters (Jadam ec et al., 1999). In the sum m er o f 1991, m axim um densities o f Tanner crabs were observed around 140-150 m eters in a southeast Alaska fjord (Zhou and Shirley, 1998). G enerally, juvenile Tanner crabs are found in shallow inshore waters and, as they m olt into adulthood, they m igrate to deeper waters (Stevens et al., 1993b). Because they are exposed to a wide range o f depths throughout their lifecycle, they are also exposed to a relatively wide tem perature range. How ever, the preferred w ater tem peratures and upper therm al limits o f Tanner crabs have not currently been determined. Tanner crabs captured at a depth o f 75 m eters were dw elling in w ater tem peratures betw een 3°C in the w inter and 10°C in the summer- fall season (Paul and Paul, 2001a).

Tanner crabs m ate once per year beginning in w inter and ending in spring. Pubescent fem ales are the first to m ate (January-M ay). They generally rem ain in

shallow er waters (< 30m ) and form isolated m ating pairs w ith the sm aller m ales (Stevens et al., 1993b). M ultiparous fem ales mate later in the year (A pril-June) and, as they gather in deeper waters (over 150 m eters on the continental shelf), they mate in aggregate with the larger m ales (Stevens et al., 1994). M ating aggregations consist o f hundreds o f reproductive fem ales m ounded up to a m eter high and several m eters long and males scattered on the seafloor surrounding the m ounds.

W hen a male detects a receptive female, he grasps her by the legs and she faces him in a pre-m ating embrace (Adams, 1982; D onaldson and Adam s, 1989). A male m ating w ith a pubescent female assists the female throughout her m olt to m aturity and

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transfers sperm atophores into the fem ale’s gonopores w hen she is in a soft-shell state. However, if the female is m ultiparous and has undergone her terminal molt, the male internally fertilizes the female in her hard-shell state. The m ale continues to grasp the fem ale until the fertilized eggs are extruded under the abdom inal flap to ensure successful m ating (Jadam ec et al., 1999). Fem ales can store sperm in their sperm athecae and

produce viable eggs w ith 1 or 2 year-old sperm, thus enabling production o f m ultiple broods w ithout proxim al m ating (Adams and Paul, 1983; Paul, 1984). However, only one clutch is produced per year and eggs are brooded for about one year until they hatch as larvae prior to the next m ating (Paul, 1984; Paul and Adam s, 1984).

Several hundred thousand Tanner crab larvae (0.5 to 1 mm in carapace length) are released from each gravid female betw een late w inter and early sum m er and begin

developm ent as plankton (Jadam ec et al., 1999). The planktonic zoea are distributed in the upper 2 0 m eters o f the w ater colum n w here they rem ain for 2 m onths (Incze et al.,

1987). The m egalop or settling stage (6-7 m m in carapace length) m igrates to the sea floor betw een 1 and 10 m onths o f age and m olts into the first instar (3.5 mm in carapace width), w hich resem bles a small adult (Jadam ec et al., 1999).

Small crabs probably settle in shallow inshore waters and m igrate to deeper waters w ith age. Juvenile Tanner crabs have been noted to m olt in m ass at depths < 20 m eters in southeast A laska during the spring m olting season (Stone, 1999). The authors suggested that by m igrating to shallow water, Tanner crabs avoided deepw ater predators such as groundfish and other crab species, including hard-shelled Tanner crabs, while vulnerable in their soft shell form. A fter m olting to m aturity and m ating at least once, adult m ales and fem ales m ove to deeper waters (> 1 0 0 m) w here they rem ain the rest o f their life (Stevens et al., 1993b).

M olting, or ecdysis, for crustaceans requires shedding o f the hard shell

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shell decalcifies. During the m olting process, the cephalothorax is elevated causing the ecdysial suture to break, thus allow ing the soft shell animal to exit the old exoskeleton (Jadam ec et al., 1999). Soft shell crabs are defenseless and barely capable o f m oving, but as the shell hardens, the crab recovers all its abilities (Paul et al., 1995). Calcification o f the shell is a fairly slow process starting within a few hours post-ecdysis and lasting betw een 16 and 71 days (Adams, 1982).

G rowth is defined as the increase in size gained per m olt and the frequency o f m olts (D onaldson et al., 1981). The rate o f growth on a percentage basis decreases with increased size o f crabs regardless o f sex; that is, small crabs grow relatively m ore in size at a m olt than larger crabs (Paul and Paul, 2001b). The frequency o f m olt is both size and m aturity dependent. The interm olt period, tim e betw een m olts, for male Tanner crabs is shorter for sm aller crabs than larger crabs, indicating that small crabs m olt more

frequently. Also, the interm olt period is shorter for juvenile crabs (not bearing sperm atophores) than adolescent crabs (bearing sperm atophores) o f sim ilar size, indicating that juvenile crabs m olt more frequently (Paul and Paul, 2001b).

A dult fem ales are generally sm aller than adult males. In K odiak waters, the average carapace width (CW ) for adult fem ale Tanner crabs is 94 mm com pared to 150 mm for adult male Tanner crabs (D onaldson et al., 1981). D uring the Bering Sea Tanner crab fishery, the largest fem ales m easured 122 mm in CW, w hile the largest males m easured 182 mm in CW betw een 1991 and 1994 (Tracy, 1995).

Fem ales grow for about 5 years and undergo their final m olt at the 13th instar as they attain m aturity. M ales attain functional m aturity after a period o f 6 years and at the

18th instar (Jadam ec et al., 1999). However, it is still debated w hether male Tanner crabs undergo a term inal m olt, like females do, after they have reached functional m aturity or if they continue to grow (Conan and Comeau, 1986; D onaldson and Johnson, 1988).

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enlarged claws. As m ales m olt to m aturity they grow allom etrically such that carapace width (CW ) and height o f chela (CH) increase disproportionately (Stevens et al., 1993a). Larger claws are thought to provide advantages in grasping and handling females in a m ating embrace. Juvenile male Tanner crabs generally do not participate in m ating and have a small CH relative to CW , while the m ajority o f m ales that exhibit grasping behaviors have large claws. Therefore, m orphom etric m aturity is likely a prerequisite for functional m aturity in male Tanner crabs. Functionally m ature male crabs have a

relatively large chela to body size ratio such that CH /CW > 0 .1 7 (Stevens et al., 1993a). In the present study, m ature m ale Tanner crabs were selected using the same ratio number.

Ta n n e r Cr a b Fi s h e r i e s Ba c k g r o u n d

The com m ercial harvest o f Tanner crabs in A laskan waters is a significant industry. The greatest catch o f Tanner crabs was in 1977 in the Bering Sea w hen 78.3 m illion pounds o f crab were landed. To put this in perspective, in the same year in Bristol Bay, 11.9 m illion pounds o f snow crab (Chionoecetes opilio) and 70 m illion pounds o f red king crab (Paralithodes cam tschaticus) were landed (Ruccio, 2003). Econom ically, the red king crab industry yields the greatest ex-vessel price per pound. In 2002, prices reached $6.27/lb for A leutian Islands red king crabs com pared to $2.21/lb for K odiak Tanner crabs and $1.40/lb for Bering Sea snow crabs (ADFG, 2004). Even though the Tanner crab fishery developed later than the red king crab fishery it quickly grew and contributed m illions o f pounds o f crab for m any consecutive years. How ever, stocks o f Tanner crabs have undergone extreme fluctuations in recent years and are currently so low that com m ercial fisheries have been closed in the Bering Sea and A leutians since the mid-

1990s and are only sporadically open in some areas in the G u lf o f Alaska.

Fisheries regulations are im plem ented to m anage stocks o f Tanner crabs to ensure long-term sustainability o f the species. Pot and trawl surveys are conducted by the A laska

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D epartm ent o f Fish and Game (ADFG) to estim ate abundance o f m ature m ales, predict recruitm ent, and determ ine levels o f annual harvest (Cavin et al., 2002). A season closure was established in 1975 as April 30 to protect crabs at the onset o f m ating and m olting in the late springtim e, and in 1976 a m inim um CW o f 140 m m (5.5 inches) w as established to allow crabs to mate at least once in their life before becom ing legally harvestable by the fishery (Cavin et al., 2002).

Com m ercial fishing for Tanner crab m ay occur in Southeastern Alaska, Yakutat, Kodiak, and South Peninsula areas on January 15 once harvest strategies are m et for each district (Iverson, 2001). In the K odiak district, the A laskan B oard o f Fisheries (BOF) established harvest strategies in 1999 such that a threshold abundance o f m ature male crabs m ust be m et or exceeded in order for fisheries to occur and guideline harvest levels (GHL) m ust be greater than 400,000 pounds o f crab for the w hole district (Ruccio, 2003). GH L are based upon abundance estim ates and differ betw een districts and years.

The Tanner crab fisheries began in 1967 in the K odiak District. This district encom passes w aters around the Kodiak A rchipelago from 1 to 200 nm o ff shore and is divided into eight m anagem ent sections - N ortheast, Eastside, Southeast, Southwest, W estside, N orth M ainland, South M ainland, and Semidi Islands (Figure 1.1). Tanner crab fisheries can be open in some sections and closed in others depending on estim ates o f population size and pre-season GH L per section (Iverson, 2001; Ruccio, 2003). Catches, in the Kodiak District, peaked in 1978 w ith over 33 m illion pounds o f crab landed and averaged 20.9 m illion pounds annually betw een 1970 and 1980. How ever, over the next

15 years, num bers o f Tanner crab rapidly declined w ith harvests totaling only 1.2 m illion pounds in 1994. The K odiak Tanner crab fishery closed in 1995 and rem ained so for the next 6 years. In 2001, it was reopened but population levels o f Tanner crab rem ain relatively depressed. The 2003 harvest o f Tanner crabs in the K odiak district totaled 510,000 pounds o f crab for an ex-vessel value o f $1.18 m illion (Cavin et al., 2002; AD FG , 2004; Ruccio, 2003).

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Boats may use pots or traps o f limited dim ensions ( lO’x lO ’x 4 2 ” ) to legally exploit Tanner crabs. N either traw ling nor ring nets are legal types o f gear in the Kodiak District. An escape panel o f at least 7 % inches stretched m esh or a m inim um o f 4 escape rings o f at least 5 inches in diam eter are required to allow undersized crabs to exit. The m axim um num ber o f pots allow ed per vessel can increase as the GH L increases. Pots are baited and dropped to the sea bottom and left for a lim ited duration after w hich pots are retrieved and crabs are sorted on deck by species, sex, and size. Only large adult male crabs are legally harvested. Sub-legal male (< 140 m m CW ) and female Tanner crabs, as well as all other species, are considered bycatch and are returned to the sea (Iverson, 2001; Ruccio, 2003).

The num ber o f Tanner crabs caught and returned to the sea is substantial. During the 1994 Bering Sea Tanner crab fishery, an estim ated 3.8 m illion legal Tanner crabs w ere retained while 5.9 m illion sub-legal m ales and 3.6 m illion fem ale Tanner crabs were caught and released (Tracy, 1995). Other fisheries yield considerable num bers o f Tanner crab bycatch such as the Bering Sea scallop fisheries and snow crab fisheries

(Rosenkranz, 2002; Tracy, 1995). During the 1994 Bering Sea snow crab fishery, sub- legal m ale Tanner crabs constituted the largest portion o f crab bycatch and were estim ated at 6.4 m illion followed by 2.3 m illion females and 0.4 m illion legal m ales (Tracy, 1995). G iven these num bers, any decrease in viability or fitness o f anim als captured and subsequently returned to the sea m ay have m easurable impacts on future recruitm ent. Hence, changes in the m ethods o f harvest that increase survivorship o f bycatch have the potential for trem endous benefit to sustainable fisheries.

The Tanner crab fishery occurs in the w inter m onths and therefore anim als m ay be exposed to extreme w eather and sea conditions as they are brought up on deck. The typical conditions in the Bering Sea betw een January and M arch include average m onthly air tem peratures ranging from -0.6 to -3.6°C and average m onthly w ind speeds ranging from 9 to 11 m s' 1 (W arrenchuk and Shirley, 2002a). During the sorting process, crabs are

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exposed to air for a few m inutes to hours depending on the type o f fishery and the num ber o f crabs caught. Anim als harvested in pots are handled relatively quickly,

w hereas those caught as bycatch during traw ling operations can rem ain on deck for hours (Stevens, 1990). Rough handling on board and contact with heavy gear m ay cause severe injuries. Investigating the effects o f any o f these stressors on survival o f Tanner crabs returned to the sea can be used to improve estim ates o f bycatch m ortality, a param eter o f im portance to fisheries managers.

H andling, air exposure and low tem peratures either alone or in com bination have adverse effects on the health, perform ance, and viability o f crabs. Tanner crabs exposed to air and low tem peratures had reduced righting response, increased limb autotom y (leg dropped in response to limb injury or other stress), and increased m ortality (Carls and O'Clair, 1995). Similarly, the effects o f windchill com bined w ith subfreezing tem perature on the congeneric snow crabs induced high rates o f limb loss, reduced righting response, and resulted in 40-100% m ortality follow ing severe treatm ents (W arrenchuk and Shirley, 2002b). M acintosh et al. (1996) reported that 85% o f Tanner crabs experim entally injured in the legs had autotom ized their limbs w ithin 24 hours. Injuries were m eant to simulate handling conditions on fishing boats and included pinched, bent, and cracked limbs. Such injuries, even if not im m ediately fatal, m ay render an individual m ore vulnerable to predation or less likely to reproduce. During a com m ercial sole trawl, only 22% o f all Tanner crab bycatch (n = 16,498) survived after 48 hours observation (Stevens, 1990). W hile these data dem onstrate a m easurable impact o f environm ental stressors on the vitality o f Tanner crabs, little data describing their physiological responses to various stressors is available.

Understanding m etabolism o f crabs following various stressors can provide a quantitative m easure o f the response o f crabs to more subtle stressors. These stressors do not necessarily kill crabs, but they m ay cause animals to incur a m ctabolic cost or im pair their ability to escape a subsequent stressor. Q uantifying physiological responses to environm ental and physical insults m ight be helpful in predicting perform ance and

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predicting health and subsequent survivorship o f individual Tanner crabs selected for live delivery to markets.

Ta n n e r Cr a b Bi o l o g y

M etabolic physiology

M etabolism integrates all chemical processes and energy transform ation that sustain a living organism, and can be sum m arized by two m ain m echanism s - energy extraction and energy use (Schm idt-Nielson, 1997). Anim als extract energy by breaking down food com ponents such as carbohydrates, amino acids, and fatty acids through processes known as catabolism . This energy is stored in the form o f adenosine triphosphate (ATP) and cells use it to biosynthesize new m acrom olecules needed by the organism through processes know n as anabolism. ATP is also used for other physiological activities such active transport and m ovem ent while some energy is lost in the form o f heat. It appears that in crustaceans, the principal biochem ical pathw ays that produce ATP are sim ilar to vertebrates (Chang and O 'Connor, 1983).

Glucose is found to be the m ajor com ponent o f circulating carbohydrates in crustacean hem olym ph and is a m ajor source o f potential energy (Chang and O 'Connor,

1983; Schm idt-N ielson, 1997). In the cytosol o f the cell, glucose undergoes glycolysis to form pyruvate. This pathw ay yields 2 m olecules o f ATP and does not require oxygen. Pyruvate can either be used in oxidative respiration or be reduced to lactate depending on the levels o f oxygen present. In the m itochondrial m atrix w here oxidative m etabolism takes place, the acetyl group o f pyruvate com bines w ith coenzym e A to form acetyl coenzym e A (acetylCoA ), w hich is then fed into the citric acid cycle. High-energy electrons that are released during this process are fed into the electron transport chain to synthesize m olecules o f ATP through a reaction know n as oxidative phosphorylation.

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The com plete oxidation o f one m olecule o f glucose generates H2O, CO2, and 38 ATP (Schm idt-Nielson, 1997). W hen oxygen is in short supply, pyruvate is converted to lactic acid. This final product cannot be further catabolised and accum ulates in the tissues. W hen oxygen becomes available, lactic acid is converted back to pyruvate, a process that, in crabs, norm ally takes place in the hepatopancreas (Chang and O'Connor, 1983).

In crustacean tissues like in vertebrates, when oxygen supply is low, anim als m ust acquire their m etabolic energy from anaerobic reactions, nam ely glycolysis. Glycolysis contributes only 2 ATP per glucose m olecule ferm ented to lactate com pared to 38 ATP by oxidative respiration. Therefore, glucose stores m ust be m obilized to increase the rate o f glycolysis to m eet energy dem ands that are not m et by oxidative m etabolism alone. Prolonged anaerobic m etabolism has been evidenced in m any crustacean species by increased concentrations o f lactate and increased concentrations o f glucose in hem olym ph (W ebster, 1996; M orris and Oliver, 1999; Bergm ann et al., 2001). Thus, changes in hem olym ph concentrations o f glucose and lactate can serve as indicators o f m etabolic status o f crustaceans.

Gas exchange in decapod crustaceans occurs alm ost exclusively across the surface o f the gills (Schm idt-Nielson, 1997). Gills o f Tanner crabs are enclosed in the bronchial cavities located dorsally on each side o f the anim al underneath the carapace. Gill lam ellae are small filam entous structures o f vascular m em branes that serve to facilitate dissolved gas exchange by increasing surface area per gill filam ent (M cM ahon and W ilkens, 1983). Crabs m aintain a gradient in gas tension betw een their hem olym ph and the m edia by flushing their gills w ith w ater in a unidirectional way. Incurrent w ater enters through openings at the base o f the legs, fills the bronchial cham bers, and exits through the mouth. Due to the density o f the m edium and low oxygen content in water, extra ventilation is often required, such as m echanical pum ping w ith the scaphognathites, exopodite o f the second m axilla, to enhance w ater circulation. The beats generate force

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sufficient to m ove w ater through the bronchial cavity and across the gills (M cM ahon and W ilkens, 1983; Schm idt-Nielson, 1997).

As water passes through the gills, dissolved gases diffuse across the gill

m em brane separating the hem olym ph and am bient w ater (Schm idt-N ielson, 1997). Both O2 and C O2 diffuse down their pressure gradient. Thus, O2 diffuses from the relatively O2

rich am bient w ater to the relatively O2 replete hem olym ph, and C O2 diffuses from hem olym ph to water. A countercurrent flow system betw een hem olym ph and w ater optim izes gradients and efficiency o f gas exchange (M cM ahon and W ilkens, 1983).

Like m ost advanced crustaceans, Tanner crabs have an open circulatory system and a heart that flushes hem olym ph throughout the body. The heart is a single cham bered organ with pairs o f valved openings (ostias) that lie dorsally w ithin a pericardial cavity, above the gut and betw een the thoracic gills (Brusca and Brusca, 1990). Oxygen-rich hem olym ph at the gills flows to the heart where it is pum ped to all body parts. From the heart, m ajor arteries facilitate circulation to vital organs, and em pty into an open

hem ocoel w here hem olym ph flows freely betw een tissues. A t the tissue and m uscle level, oxygen is used in oxidative phosphorylation. O xygen-poor hem olym ph gathers at the gills w here it is replenished w ith 02 and freed o f CO2 (M cLaughlin, 1983; B rusca and Brusca, 1990).

O xygen carrying capacity o f the hem olym ph is facilitated by a copper-based respiratory pigm ent, hem ocyanin. Flemocyanin serves to bind, store, and transport oxygen from the site o f uptake to the rest o f the body (M angum , 1983). The hem ocyanin com plex is m ade o f proteins containing copper ions as the site for 02-binding. One subunit consists o f a large protein w ith 2 Cu2+ ions that reversibly bind one m olecule o f O2. Groups o f 2 or 3 subunits constitute one m onom er and 6 m onom ers constitute one hexamer. Polym eric hem ocyanin in crustaceans typically consists o f 1, 2 or 4 connected hexam ers. This respiratory pigm ent is free in solution, pale blue when oxygenated and colorless w hen deoxygenated (M angum , 1983; W ithers, 1992).

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Anim als break down food to produce most o f their energy needs through oxidative respiration. Thus, quantifying the amount o f oxygen an anim al consumes is a m easure o f its energy m etabolism and can be used to express the energy cost o f a state such as exercising, stress, or resting (Schm idt-Nielson, 1997). Respirom etry is a m ethod to determ ine rates o f oxygen consum ption (Cech, 1990; Handy and Depledge, 1999) and has been used in other species to determ ine changes in m etabolic rate associated with tem perature, exposure to air, handling and oxygen tension (M orris and Oliver, 1999; Crear and Forteath, 2001; Y aikin et al., 2002). During the course o f this study we wanted to evaluate survivorship and perform ance o f Tanner crabs exposed to stressors associated with com m ercial crab fisheries while m onitoring m etabolic indices. W e m easured

hem olym ph concentrations o f glucose and lactate, and oxygen consum ption o f crabs subjected to air at cold and subfreezing tem peratures for different durations to define the range o f m etabolic responses. These data were used to evaluate perform ance o f crabs following treatments.

Stress physiology

Stress is defined as the response o f an organism to a stim ulus (stressor) that causes the disruption o f the resting physiological state to the extent that chances o f survival m ay be reduced (Barton, 1997). Or put simply, stress is a “threat to or disturbance o f

hom eostasis” . The stress response involves physiological and behavioral changes by an organism to m aintain hom eostasis in face o f a threat. This does not necessarily im ply that stress is detrim ental to the individual. The response to a stressor is considered an adaptive m echanism that prom otes survival. How ever, if the intensity o f the stressor is severe or long lasting and the organism m ay not escape, the threat becom es detrim ental to the individual’s health and w ell-being. This is also know n as a m aladaptive or distressed state (Barton, 1997; Barton et al., 2002).

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The response o f fish to a m ultitude o f stressors such as handling, crowding, disease, w ater quality, and tem perature has been especially well studied, not only for theoretical interests but also for aquaculture and m anagem ent applications. W edem eyer et al. (1990) described com pensatory responses in terms o f prim ary (neuronal and endocrine pathw ays), secondary (physiological com pensations) and tertiary (whole animal

perform ance) changes. In brief, the prim ary response involves the stim ulation o f the central nervous system culm inating in the secretion o f horm ones into the blood stream. This includes stim ulation o f chrom affin cells in the kidney by the sym pathetic nervous system to release catecholam ines (epinephrine or adrenaline), and stim ulation o f the hypothalam us and pituitary that regulate the production and secretion o f corticosteroids (m ainly cortisol) into circulation. The secondary response refers to alterations o f ion concentrations and m etabolite levels in plasm a and tissue that is caused by physiological adjustm ents o f m etabolism , respiration, acid-base balance, hydrom ineral balance and imm une function. The tertiary response concerns w hole-anim al perform ance such as growth, reproduction, resistance to diseases, activity and overall survival, w hich if sufficiently reduced m ay have effects at the population and com m unity levels (W edem eyer et al., 1990; Sumpter, 1997; Barton et al., 2002).

These responses to stress are interrelated such that prim ary responses m ay directly or indirectly influence secondary and tertiary responses. For example, the release o f epinephrine and cortisol serve to m obilize energy stores to cope with the increased energy dem ands from m etabolic activity associated w ith stress. The m ain effects o f these horm ones are to optim ize cardiovascular and respiratory functions and regulate energy substrate m obilization and utilization (W edem eyer et al., 1990). The latter includes the conversion o f glycogen into glucose for the m ore im m ediate response and replenishm ent o f glycogen stores in the liver via gluconeogenesis for the longer-term m aintenance o f m etabolic substrate. Thus, following an acute stressor, such as handling or b rief disturbance, concentrations o f plasm a glucose increase to provide energy to the animal for the ‘fight-or-flight’ reaction. In some cases, concentrations o f plasm a lactate m ay also

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increase as a result o f m uscular activity associated with stress. Overall, non-essential processes (growth, reproduction) are tem porarily halted and resources are redirected tow ards m etabolic processes that w ould favor survival (Barton et al., 2002).

In invertebrates, the basic characteristics o f a stress response are analogous to those found in fish and higher vertebrates. The m ajor glands and production sites

involved in stress m ay be different in such a w ay that in invertebrates, glands are few er in num ber and hold m ultifunctional roles. However, the same nervous and endocrine

responses are im plicated, but in a m ore sim plistic way, and the order o f the m olecular response is analogous (Ottaviani and Franceschi, 1996). Data rem ains sparse and extensive inform ation on the effects o f stressors, responses and m olecules concerned in stress are still needed for a broad num ber o f species. In crustaceans, the sinus gland located in the eyestalk stores and releases neuroendocrine horm ones, o f m ain interest the crustacean hyperglycem ic horm one (CHH; Borst, 2003). CH H and CH H-like peptides secreted from the sinus gland regulate a wide variety o f physiological processes such as glucose levels, osmosis, reproduction and m olting. The control o f hyperglycem ic activity has received some attention and recent work has shown that stress such as hypoxia induced the release o f CHH and elevated levels o f glucose in hem olym ph o f the crab Cancer pagarus (W ebster, 1996).

Changes in physiological param eters are characterized by com m on features w hether the stress results from fishing and rearing practices (capture, handling, transport, diseases), environm ental alterations (pollution, hypoxia and w ater quality), or behavioral factors (social hierarchies, biological interactions). How ever, the type and m agnitude o f the response and the tim e it takes an anim al to recover depend on the nature, duration and severity o f the stressors, the species, and individuals within a species, each w ith different capacities to respond to stress (Barton, 1997). A dditionally, the degree to w hich an anim al responds to m ultiple stressors is influenced in part by the cumulative

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m ultiple stressors m ay have additive or antagonistic effects on anim al physiology and behavior, it is important to clearly define the biological system and consider all factors that m ay affect the stress response.

Re s e a r c h Ob j e c t i v e s a n d Ch a p t e r Or g a n i z a t i o n

This research project was initiated in the fall o f 2002 to characterize the physiological responses o f Tanner crabs to acute physical stressors m odeled on the types o f events that can occur in the com m ercial crab fisheries. Analytical m ethods used in this project include respirom etry and assays o f hem olym ph constituents to m onitor m etabolic

changes following physical insults such as exposure to air, subfreezing tem peratures, and handling. By characterizing these responses to stress and the tim e to recover, one can better understand the consequences o f the physical insult im posed upon crabs captured. The objectives o f this research project were to 1) evaluate the im pact o f various stressors through m onitoring physiological changes, and 2) predict the relative vitality and

recovery o f crabs following stressors by em ploying physiological indices.

In chapter one, we present background on Tanner crabs and com m ercial fisheries in A laska and give a general review on energy m etabolism and stress physiology. In chapter two, concentrations o f glucose and lactate in the hem olym ph o f Tanner crabs are determ ined follow ing various treatm ents o f tem perature and air exposure. In chapter three, rates o f oxygen consum ption are determ ined before and after Tanner crabs are subjected to subsequent periods o f exposure to air at different tem peratures. Rates o f oxygen consum ption were m easured with a static respirom etry chamber.

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Incze, L. S., D. A. A rm strong, and S. L. Smith. 1987. Abundance o f larval Tanner crabs {Chionoecetes spp.) in relation to adult fem ales and regional oceanography o f the southeastern Bering Sea. Canadian Journal o f Fisheries and Aquatic Sciences 44(6): 1143-1156.

Iverson, K. 2001. The K odiak district Tanner crab (Chionoecetes bairdi) fishery, 1985 to 2001. Com m ercial Fisheries Entry Com m ission, Report No. 01-4N, Juneau, Alaska. 22 pp.

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M acintosh, R. A., B. G. Stevens, J. A. Haaga, and B. A. Johnson. 1996. Effects o f

handling and discarding on m ortality o f Tanner crabs (Chionoecetes bairdi). High Latitude Crabs: Biology, M anagem ent, and Econom ics. Pp. 577-590. Vol. 13. Low ell W akefield Fisheries Sym posium Series. U niversity o f A laska Fairbanks, Alaska, Alaska Sea Grant Report 96-02.

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M cM ahon, B. R., and J. L. W ilkens. 1983. V entilation, perfusion, and oxygen uptake, pp. 289-372. In, D. E. Bliss, and L. H. M antel, eds. The Biology o f Crustacea:

Internal A natom y and Physiological Regulation. Vol. 5. A cadem ic Press, New York.

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122A(3): 299-308.

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Paul, A. J., and J. M. Paul. 2001a. Effects o f tem perature on length o f interm olt periods in juvenile male Chionoecetes bairdi. A laska Fishery Research B ulletin 8(2): 132-

134.

Paul, A. J., and J. M. Paul. 2001b. Interm olt durations o f captive juvenile and adolescent male Tanner crabs Chionoecetes bairdi. Journal o f Shellfish Research 20(1): 373- 376.

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Rosenkranz, G. E. 2002. M ortality o f Chionoecetes crabs incidentally caught in Alaska's weathervane scallop fishery. In, A. J. Paul, E. G. Dawe, R. Elner, G. S. Jamieson, G. H. Kruse, R. S. Otto, B. Sainte-M arie, T. C. Shirley, and D. W oodby, eds. Crabs in Cold W ater Regions: Biology, M anagem ent, and Econom ics. Pp. 717- 737. Vol. 19. Lowell W akefield Fisheries Sym posium Series. University o f Alaska Fairbanks, Alaska, A laska Sea Grant Report 02-01.

Ruccio, M. P. 2003. Fishery m anagem ent plan for the com m ercial Tanner crab fishery in the K odiak D istrict o f registration area J, 2004. A laska D epartm ent o f Fish and Gam e, Report No. 4K 03-57, Kodiak, Alaska. 30 pp.

Schm idt-N ielson, K. 1997. Anim al Physiology, Adaptation and Environm ent. Cam bridge U niversity Press, Cam bridge, U nited Kingdom. 612 pp.

Stevens, B. G. 1990. Survival o f king and Tanner crabs captured by com m ercial sole trawls. Fishery Bulletin 88(4): 731-744.

Stevens, B. G., W. E. Donaldson, J. A. Haaga, and J. E. M unk. 1993a. M orphom etry and m aturity o f paired Tanner crabs, Chionoecetes bairdi, from shallow and

deepw ater environm ents. Canadian Journal o f Fisheries and Aquatic Sciences 50(7): 1504-1516.

Stevens, B. G., J. Haaga, J. E. M unk, and W. E. Donaldson. 1993b. M orphom etric m aturity and aggregative m ating behavior o f Tanner crab, Chionoecetes bairdi (Decapoda: M ajidae), sam pled by scuba and submersible. Journal o f Shellfish Research 12(1): 132.

Stevens, B. G., J. A. Haaga, and W. E. Donaldson. 1994. A ggregative m ating o f Tanner crabs, Chionoecetes bairdi. Canadian Journal o f Fisheries and Aquatic Sciences 51(6): 1273-1280.

Stone, R. P. 1999. M ass m olting o f Tanner crabs Chionoecetes bairdi in a southeast A laska-estuary. A laska Fishery Research Bulletin 6(1): 19-28.

Sum pter, J. P. 1997. The endocrinology o f stress, pp. 95-118. In, G. K. Iwama, A. D. Pickering, J. P. Sumpter, and C. B. Schreck, eds. Fish Stress and H ealth in A quaculture. Cam bridge U niversity Press, Cambridge.

Tracy, D. A. 1995. A laska departm ent o f fish and gam e sum m ary o f the 1994 m andatory shellfish observer program database. A laska D epartm ent o f Fish and Game, Com m ercial Fisheries M anagem ent and D evelopm ent D ivision, Report No. 4K 95-32, Kodiak, Alaska. 96 pp.

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W arrenchuk, J. J., and T. C. Shirley. 2002a. Effects o f w indchill on the snow crab

( Chionoecetes opilio). In, A. J. Paul, E. G. Dawe, R. Elner, G. S. Jamieson, G. H. Kruse, R. S. Otto, B. Sainte-M arie, T. C. Shirley, and D. W oodby, eds. Crabs in Cold W ater Regions: Biology, M anagem ent, and Econom ics. Pp. 81-96. Vol. 19. Lowell W akefield Fisheries Sym posium Series. U niversity o f Alaska Fairbanks, Alaska, Alaska Sea Grant Report 02-01.

W arrenchuk, J. J., and T. C. Shirley. 2002b. Estim ated m ortality o f snow crabs Chionoecetes opilio discarded during the Bering Sea fishery in 1998. Alaska Fishery Research Bulletin 9(1): 44-52.

W ebster, S. G. 1996. M easurem ent o f crustacean hyperglycaem ic horm one levels in the edible crab Cancer pagurus during em ersion stress. Journal o f Experim ental B iology 199: 1579-1585.

W edem eyer, G. A., B. A. Barton, and D. J. M cLeay. 1990. Stress and acclim ation, pp. 335-356. In, C. B. Schreck, and P. B. M oyle, eds. M ethods for Fish Biology. A m erican Fisheries Society, M aryland.

W ithers, P. C. 1992. Com parative Anim al Physiology. Saunders College Publishing, Florida. 949 pp.

Yaikin, J., R. A. Quinones, and R. R. Gonzalez. 2002. A erobic respiration rate and an anaerobic enzym atic activity o f Petrolisthes laevigatus (Anom ura, Porcellanidae) under laboratory conditions. Journal o f Crustacean Biology 22(2): 345-352. Zhou, S., and T. C. Shirley. 1998. A subm ersible study o f red king crab and Tanner crab

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A L A S K A , «Sk ;i ' ' **4^ % ■ Kodiak Island L A*-V- J>/ J s J & W ; : ./(t/i . ALASKA PENINSUL^ v> Xf j>, ^ " V A £ tev A V H ,;1 w ' " ; / . * \ J . ,''* V ' Northeast V; ' , . * ; S J , 1*1 A * > '.I’SAf’V' ( *«.Sl >. ' ' ^ ll" , A J " . . . r KODIAK is/ ’. ‘•9 1 \ *{■ *s4 \ * i t..J Southwest ** - ' . • ’ K Eastside / Vv"'*' v \ .$ r i \ \ Semidi island \ ,/9 < j- Southeast

Figure 1.1 Tanner crab fishing sections in the K odiak District.

This m ap shows the location o f K odiak Island in relation to A laska and the fishing sections for Tanner crabs around Kodiak. M aps were reproduced from the Alaska D epartm ent o f Fish and Gam e (ADFG).

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CH APTER 2 : EFFECTS OF EM ERSIO N, TEM PERA TU RE AND HANDLING ON HEM OLYM PH M ETABOLITES OF TA NNER CRABS (CH IO N O ECETES BAIRDI)*

Ab s t r a c t

In this study, we characterize m etabolic responses and subsequent recovery o f Tanner crabs following 45 m in o f air exposure (em ersion) at either 8°C or -15°C. Concentrations o f glucose and lactate in hem olym ph were determ ined at 30, 60 and 120 m in following emersion. Physical responses, including mortality, righting response and limb loss were recorded for up to 7 days o f recovery. Concentrations o f glucose were significantly lower 30 to 120 m in follow ing em ersion at -15°C than at 8°C. C oncentrations o f lactate

increased significantly (2 tim es pre-treatm ent levels) by 1 2 0 m in follow ing em ersion at -15°C but rem ained unchanged at intervals following em ersion at 8°C. N inety percent o f crabs survived handling and em ersion at 8°C, while em ersion at -15°C resulted in 71% survival. A m ong surviving crabs, righting response was significantly lower and limb loss significantly higher am ong anim als em ersed at -15°C than those em ersed at 8°C

throughout the 7 days o f recovery. Thus, physiological responses to subfreezing

tem perature were consistent w ith low survival, low righting response and increased limb loss.

*E1 M ejjati S. Y ., A. C. M . O liveira, A. H. H aukenes, and C. L. B uck. 2006. E ffects o f E m ersion, T em perature and H andling on H em olym ph M etabolites o f T an n er C rabs (C hionoecetes bairdi). Prepared for subm ission to the Journal o f C rustacean B iology.

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In t r o d u c t i o n

Com m ercially harvested and incidentally captured Tanner crabs (Chionoecetes bairdi; Rathbun, 1924) in Alaskan waters are subjected to environm ental and physical stressors that m ay im pinge on individual survival and fitness. Crabs captured in pots and trawls are exposed to rough handling during the sorting onboard, exposure to air ranging from a few m inutes to hours and extreme w eather conditions in the w inter m onths with tem peratures generally below 0°C. Handling and exposure to low air tem peratures is know n to induce limb loss, reduce vigor (m easured by righting response) and lower survival o f crabs (Carls and O'Clair, 1995; M acintosh et al., 1996; W arrenchuk and Shirley, 2002).

A lthough crabs m ay survive after capture, physical injuries and stress load m ay render an individual m ore vulnerable to predation, less likely to successfully reproduce or result in delayed m ortality. Stevens (1990) estim ated only 22% survivorship o f Tanner crabs inadvertently captured as bycatch in traw ls during a com m ercial fishery for sole. W hile these data dem onstrate a m easurable impact on crabs captured, there are little data available describing the physiological responses o f Tanner crabs to stressors.

D uring exposure to air, the gills o f aquatic crustaceans m ay become im paired (Johnson and Uglow, 1985). As a consequence, the dem and for oxygen becom es greater than the supply and oxygen requirem ents are not met. Thus, as the endogenous oxygen supply is depleted an oxygen debt is established and these anim als rely on increased anaerobic m etabolism to survive (Johnson and Uglow, 1985). In m ost crustacean species glucose is the prim ary m etabolic substrate and lactate is the m ost im portant end product o f anaerobic activity (Chang and O 'Connor, 1983). Thus, a switch from prim arily aerobic to anaerobic m etabolism is typically evidenced by an increase in hem olym ph lactate and often an increase in hem olym ph glucose (Santos and Keller, 1993; W ebster, 1996; M orris and Oliver, 1999a; Bergm ann et al., 2001; Speed et al., 2001). Sw im m ing crabs

(.Liocarcinus depurator) and squat lobsters (M unida rugosa) captured by trawls and then exposed to air for 45 m in increase concentrations o f both glucose and lactate in

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hem olym ph com pared to controls (Bergm an et al., 2001). Similarly, southern rock lobsters (Jasus edwardsii) emersed for prolonged periods o f time have higher

concentrations o f glucose and lactate com pared to controls (M orris and Oliver, 1999a). There is some evidence that hem olym ph levels o f glucose can be depleted during prolonged exposure to a stressor, thus, yielding high concentrations o f lactate and low concentrations o f glucose in hem olym ph (Huang and Chen, 2001). Therefore, changes in hem olym ph concentrations o f glucose and lactate m ay serve as valuable indicators o f m etabolic status o f crustaceans, although it is not know n for Tanner crabs if glucose levels should increase or decrease following exposure to air.

R ecovery involves the reversal o f physiological disturbances incurred during exposure to air and is com plete w hen anaerobic end products are cleared from the tissues upon re-im m ersion. Recovery is typically a slow process that takes a few hours for large decapods. Im m ediately after western rock lobsters (P am dirus cygnus) are re-imm ersed, rates o f oxygen consum ption increase by 2.5 tim es the control rates (Crear and Forteath, 2001). These gradually decrease, as do concentrations o f both glucose and lactate in the hem olym ph, and return to pre-stress levels within 8 hours. R ecovery o f energy stores is extrem ely im portant for animals to cope with further periods o f stress. Therefore, crabs m ight survive stress from capture, but animals returned to the sea or retained in holding tanks m ay rem ain vulnerable and susceptible to predation, including cannibalism.

In the present study, concentrations o f glucose and lactate in hem olym ph o f Tanner crabs were determ ined at various sampling tim es follow ing exposure to 45 m in em ersion at 8°C and -15°C in the laboratory. Physical responses such as survival, righting response and limb loss were recorded for up to 7 days post-treatm ent. W e hypothesized that: 1) m ortality, righting response and limb loss differ betw een anim als exposed to stress treatm ents, 2) concentrations o f hem olym ph glucose and lactate vary among anim als subjected to stress treatm ents and 3) the degree o f changes in these constituents is related to the intensity o f the stressor.

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Ma t e r i a l s a n d Me t h o d s

A nim als a nd husbandry

Tanner crabs (Chionoecetes bairdi) were captured in pots by a com m ercial fishing vessel in N ovem ber 2002 from wild stocks near Kodiak Island, A laska and transported to laboratories at the Kodiak Fisheries Research Center. A pproxim ately 24 crabs were m aintained in each rectangular fiberglass tank (1.75 x 1.00 x 0.30 m). Holding tanks were supplied w ith 100% sand-filtered seaw ater drawn from 25 m eters beneath the surface o f Trident Basin at am bient outdoor w ater tem perature (Appendix A). Trials were carried out in Decem ber 2002 when the average m onthly tem perature o f seaw ater was 6.0 ± 1°C. For identification purposes, uniquely coded tags (Floy Tag & M fg, Inc., Seattle,

W ashington) were attached to a w alking leg o f each crab. Carapace width (CW; range 101.1 to 153.5 m m) and chela height (CH) were m easured to the nearest 0.1 m illim eter at the point o f m axim um dim ension not including the spines, and live w et weights (range 0.32 to 1.32 kg) were recorded to the nearest 0.01 kilogram. O nly m ature males with a ratio o f CH /CW > 0.17 were used in this project (Stevens et al., 1993). Crabs were fed tw ice per w eek ad libidum w ith m ajestic squid (Berryteuthis magister) supplem ented with salm on (Oncorhynchus spp.) and halibut (H ippoglossus stenolepis) as available. Tanks were cleaned the following day to rem ove uneaten food and wastes. Crabs were starved for 3 days prior to testing (Paul and Fuji, 1989).

Sam pling o f hemolymph

Fifty-six crabs were random ly selected and divided into 8 groups {n = 7 per group). In one group, crabs were not subjected to any experim ental treatm ent and hem olym ph was collected (undisturbed treatm ent). In a second group, crabs w ere held out o f the w ater and handled for approxim ately 5 seconds, returned to seaw ater in a holding tank, and 75 min later hem olym ph w as collected (handled treatm ent). In the rem aining 6 groups, crabs

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were exposed to air for 45 min either at 8 ± 0.5°C or -15 ± 2.1°C, returned to seaw ater in a holding tank, and 30, 60 or 120 m in after re-im m ersion hem olym ph was collected (em ersion treatm ents). All trials were conducted on the same day (Day 0) and animals were placed in a com m unal recovery tank. Seven days post-treatm ent (Day 7)

hem olym ph was collected for a second time from all surviving crabs. Hem olym ph samples were taken from crabs by inserting a 22-gauge needle 11/2 inch long with a 5 ml syringe (M ed-V et International, M ettawa, Illinois) through the epimeral line at the rear m argin o f the carapace (Speed et al., 2001).

Anim als were inspected and physical responses were assessed following exposure to a treatm ent im m ediately after crab hem olym ph was collected on Day 0, the following day (Day 1), and on D ay 7. M ortality was recorded and am ong surviving crabs, limb loss and righting response (ability o f a crab to right itself w hen placed on its back underw ater) were measured. Crabs were judged dead when m ovem ents o f the scaphognathites and locom otory appendices ceased; righting response o f a crab was recorded as successful or failure after 5 m in (Carls and O ’Clair, 1995).

H em olym ph was collected from each crab for the determ ination o f concentrations o f glucose and lactate. Prior to analysis, samples o f hem olym ph (ca. 1ml) were

transferred into 1.5 ml polypropylene snap-cap vials and placed in the refrigerator (4°C) overnight to clot. Samples were then centrifuged at 14,000 rpm for 5 min in a

m icrocentrifuge (IEC M icro-M B International Equipm ent Com pany, USA). Supernatants were transferred to new 1.5 ml polypropylene vials with Pasteur pipettes and stored at -80°C until assayed (Bergm ann et al., 2001).

Glucose assay

Before the glucose assay, hem olym ph samples w ere treated w ith perchloric acid (PCA) to precipitate proteins out o f the solution, using a m ethodology adapted from Bergm ann et al. (2001). Briefly, hem olym ph samples were thaw ed at room tem perature and 400 |il o f

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each sample was m ixed with 100 |il o f 8% PCA. The precipitated proteins were separated by centrifugation at 14,000 rpm for 10 min and the supernatants were transferred using Pasteur pipettes to new 1.5 ml polypropylene vials. A 4 M solution o f potassium bicarbonate (KH CO 3) was added to achieve a final pH o f 7.0 as determ ined with pH paper. A fter cooling in a freezer (-20°C) for 5 min, the precipitated potassium perchlorate was separated by centrifugation at 14,000 rpm for 10 min. The supernatants were then transferred w ith Pasteur pipettes to new 1.5 ml polypropylene vials and stored at -80°C overnight.

The enzym atic m icroassay for hem olym ph glucose was adapted from the m ethodology described in (W ebster, 1996). Enzym atic chrom ogen reagents were prepared according to instructions provided with Glucose C2 autokits (W ako Chemicals USA, Inc., Richm ond, Virginia). Aqueous solutions o f glucose standards were prepared at concentrations o f 0.0 (blank), 1.0, 2.5, 5.0, 10.0, 20.0, and 40.0 mg dl"1 and digested sim ilarly to the hem olym ph samples. H em olym ph samples and glucose standards (50 jj.1 each) w ere added in triplicate into the wells o f a 96-well m icroplate (Becton Dickinson Labware, Franklin Lakes, N ew Jersey) with 200 fil o f reagent. To account for inter and intra-plate variation, glucose standards were analyzed on every m icroplate in replicate. The m icroplates were placed on an orbital shaker (Troem ner, Inc., Thorofare, New Jersey) at 150 rpm for 30 seconds. Samples and standards were then placed in an incubator (model 1555, Sheldon M anufacturing Inc., Cornelius, Oregon) at 34°C for 5 min. To reduce evaporative loss during incubation, m icroplates were placed on damp paper in a glass container and covered w ith alum inum foil. Absorbance readings were determ ined w ith a m icroplate reader (Universal M icroplate Reader EL800; Bio-Tek Instrum ents, Inc., W inooski, V erm ont) at a w avelength o f 515 nm. The average absorbance o f the blanks was used to correct the readings o f glucose standards and hem olym ph samples. Concentrations o f glucose were derived from the predictive equation and expressed in m g dl' 1 o f hemolymph.

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Lactate assay

The enzym atic m icroassay for hem olym ph lactate was adapted from the m ethodology described in Chung-Yen et al. (1999). H em olym ph samples were thaw ed at room tem perature. Enzym atic chrom ogen reagents were prepared according to the instructions provided with the Lactate Instant kit # 735 (Trinity Biotech Inc., Saint Louis, M issouri) and aqueous solutions o f lactate standards were prepared at concentrations o f 0 (blank), 5, 10, and 20 m g dl"1. Hem olym ph samples and lactate standards (2.5 jj.1 each) were analyzed in triplicate with 250 |xl o f reagent. Samples and standards were hom ogenized and incubated as per the procedures described in the glucose section. Absorbance readings were determ ined at a w avelength o f 540 nm and the average absorbance o f the blank was used to correct the readings o f lactate standards and hem olym ph samples. C oncentrations o f lactate were derived from the predictive equation and expressed in m g dl"1 o f hemolymph.

Statistical analyses

All statistical tests were perform ed with STA TISTICA 6.1 (StatSoft Inc., Tulsa,

Oklahom a). W e used a Fisher exact test to determ ine significant differences in survival, righting response, and limb loss betw een undisturbed, handled and em ersion treatm ents. Significant differences in concentrations o f hem olym ph glucose and lactate were determ ined w ith the following statistical tests: we used a tw o-w ay analysis o f variance (ANO VA ) to com pare crabs exposed to air with tem perature and sampling time as factors (A ppendix B). Specific differences were determ ined with T ukey’s Unequal N H onestly Significant D ifference (HSD). W e used a one-w ay ANOVA to compare all treatm ent groups on Day 0 and Day 7. The D unnett’s post-hoc test was used to determ ine

significant differences betw een crabs exposed to each tem perature at sam pling intervals with undisturbed crabs. W hen data failed to m eet the assum ptions o f norm ality and equal variance, a K ruskal-W allis AN O V A on ranks (H-test) was used. We used a T-test to

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com pare crabs handled with crabs undisturbed. To take into account the variations in sample size, a type III sum o f squares was used to calculate P-values. All differences were considered significant at P < 0.05. W eighted m eans and standard errors (SE) are represented in all graphs and tables for concentrations o f glucose and lactate.

Re s u l t s

Physical responses

The w eight (F(7> 4sj = 1.64; P = 0.15) and size ( F ^ 4gj = 0.50; P = 0.83) o f crabs did not differ significantly betw een experim ental groups prior to any treatm ent. Crabs that were em ersed at -15°C for 45 min displayed dim inished vigor throughout the 7 days o f recovery as dem onstrated by righting response, limb loss and survival data (Table 2.1). A lthough the percentage survival did not differ significantly am ong the treatm ent groups, only 71% o f crabs em ersed at -15°C survived after 7 days post-treatm ent com pared to 90% o f the crabs em ersed at 8°C, 100% o f the crabs undisturbed and 100%) o f the crabs handled for 5 seconds. Am ong the crabs that survived em ersion at -15°C, only 33% could right them selves w ithin 5 m in by Day 7, w hich was significantly lower com pared to the other treatm ent groups (P < 0.05). M oreover, 80%o o f these crabs had lost limbs by Day 7, w hich w as significantly greater than the other treatm ent groups (P < 0.05).

Glucose concentrations

The m ean concentration o f glucose in hem olym ph o f undisturbed Tanner crabs was 11.15 ± 1.45 m g dl' 1 (Table 2.2). Com pared to undisturbed crabs, glucose levels at the intervals sam pled follow ing em ersion at -15°C were not significantly different (F ,i 23) = 2.30; P =

0.10). Likewise, glucose levels at the intervals sam pled follow ing em ersion at 8°C did not differ significantly from pre-treatm ent levels ( F ^ ?:ij = 0.78; P = 0.52), but they tended to be greater (1.4 tim es) at 120 m in post-treatm ent (15.97 ± 3.49 mg d l'1). There

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w as a significant effect o f tem perature on concentrations o f glucose in hem olym ph {Fa ,36) ~ 9.77; P - 0.0035). Concentrations o f glucosc m easured between 30 and 120 min

were significantly lower following the -15°C em ersion treatm ent than the 8°C em ersion treatm ent (Figure 2.1).

H andling had a significant effect on short-term changes in concentrations o f hem olym ph glucose (T = -2.64; d.f. = 11; P = 0.023). Seventy-five m inutes after crabs were handled for 5 seconds, the m ean concentration o f glucose (17.51 ± 1.85 mg d l'1) was significantly greater (1.6 tim es) than pre-treatm ent levels (Table 2.2). Seven days post-treatm ent glucose levels o f crabs undisturbed, crabs handled for 5 seconds, and crabs em ersed within each tem perature were sim ilar (H (ij 8} = 6.16; P = 0.104).

L actate concentrations

The m ean concentration o f lactate in hem olym ph o f undisturbed Tanner crabs was 6.12 ± 1.49 mg dl’1 (Table 2.2). Lactate levels o f crabs em ersed at -15°C were significantly greater (2.1 tim es) at 120 m in post-treatm ent (13.07 ± 2.44 m g d l'1) com pared to undisturbed crabs (F^, 24) = 3.63; P = 0.027). A lthough lactate levels at the intervals

sampled follow ing em ersion at 8°C did not differ significantly from pre-treatm ent levels (F(3,24) = 1.47; P = 0.25), they tended to be greater (1.8 tim es) at 60 m in post-treatm ent

(11.08 ± 2.73 mg dl"1). There was a significant interaction betw een tem perature and sam pling tim e on concentrations o f hem olym ph lactate (F q m > = 4.28; P = 0.022). W hile em ersion at -15°C led to increased concentrations o f lactate upon re-im m ersion, lactate levels were not different betw een 30 and 120 m in following em ersion at 8°C

(Figure 2.1).

Seventy-five m inutes after the 5-second handling treatm ent, concentrations o f lactate in hem olym ph o f crabs (6.97 ± 1.70 mg dl"1) did not differ significantly from pre­ treatm ent levels (T = -0.38; d.f. = 12; P = 0.71; Table 2.2). Seven days post-treatm ent,

References

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