FISHERY BULLETIN: VOL. 85, NO. 1 



dividual identification, and alternately assigned to 

 either an exposure or control group. 



After marking, control spiny lobsters were 

 weighed to the nearest 0.1 g and promptly placed 

 inside a shaded, wood-slat fish box two-thirds sub- 

 merged inside the acclimation tank. Weights were 

 also recorded at 1 and 2 h. Excess water clinging 

 to the exoskeleton and inside the branchial cham- 

 bers was removed prior to each weighing by holding 

 the spiny lobster around the carapace in a head down 

 position and gently moving it through a short down- 

 ward arc six times. Exposed spiny lobsters were 

 marked and weighed as above, but were held in a 

 fish box located in a fully shaded outdoor area. 

 Evaporative water loss was indicated by weight 

 decrease over time. 



During the period when desiccation experiments 

 were performed (late March to early May 1984), 

 relative humidity was 61-72%, air temperature 

 22-30 °C, wind speed 10 km/h or less, and cloud cover 

 ranged from clear to lightly scattered or hazy. Ex- 

 periments were not performed on very wet or windy 

 days to avoid excessive variation in desiccation rates 

 between experiments. 



Hemolymph Chemistry 



To assess effects of exposure on hemolymph chem- 

 istry, spiny lobsters were air-exposed in fish boxes 

 for ¥2, 1, or 2 h as previously described. Control 

 spiny lobsters were removed directly from the ac- 

 climation tank. 



Hemolymph sampling was via cardiac puncture. 

 A 1.6 mm (yie-in) hole drilled through the dorsal 

 carapace directly over the heart allowed easy hypo- 

 dermic removal of 8-10 mL of hemolymph. There 

 is no suitable chemical method to prevent hemo- 

 lymph clotting (Young 1972). At ambient tempera- 

 ture, spiny lobster hemolymph forms a tough rub- 

 bery clot within seconds. Prompt cooling of the 

 hemolymph by immersion of the syringe in an ice 

 water bath (4°C, 60 s) inhibited clotting long enough 

 to prepare subsamples for pH, ammonia, and lactic 

 acid analysis. All hemolymph samples were collected 

 between the hours of 10:00 and 16:00 and analyzed 

 the same day. 



Intervals between netting and completion of 

 hemolymph removal were 70 s or less, thus mini- 

 mizing trauma associated with handling and cardiac 

 puncture. Since net confinement reduced struggling, 

 spiny lobsters were not removed from the net for 

 hemolymph sampling unless access to the dorsal 

 carapace was restricted. In preliminary experi- 

 ments, repetitive handling and sampling of controls 



depressed hemolymph pH values. Consequently, 

 each spiny lobster was sampled only once in ex- 

 periments reported here. Hemolymph pH was deter- 

 mined by a digital pH meter with a calomel micro- 

 electrode. Hemolymph subsamples (2 mL) and a 7.0 

 buffer solution were chilled to 4 °C in a second ice 

 water bath before recording pH. Blood pH at 4°C 

 probably varies from in vivo pH at ambient tem- 

 perature, but this was an essential concession to 

 retard clot formation. Anaerobic, radiometer-type 

 pH measurements were also impossible due to clot- 

 ting. However, care was taken to minimize hemo- 

 lymph air contact since changes in CO9 equilibrium 

 can alter pH values. Truchot (1975) reported the pH 

 of crustacean blood exposed to air without mixing 

 varies little from anaerobically obtained samples. 



Serum was prepared by injecting the remaining 

 6-8 mL of chilled hemolymph into a 15 mL tissue 

 grinder, then gently grinding for 1-2 min until the 

 clotting hemolymph was liquified. The still cool 

 serum was then refrigerated in capped test tubes 

 for subsequent ammonia analysis. 



Ammonia was measured using the Conway micro- 

 diffusion method (Conway and Byrne 1933) with 

 modifications suggested by Seligson and Seligson 

 (1951). With this method, ammonia from a 0.5 mL 

 blood sample was diffused onto an acidified glass 

 rod inserted inside a microdif fusion cell. Microdif- 

 fusion cells were rotated for 50 min to facilitate 

 diffusion, then the rods were washed off with 5 mL 

 of Nessler's reagent. Intensity of color developed 

 in Nessler's reagent, corresponding to ammonia con- 

 centration, was measured in a colorimeter at 420 

 nm. All samples were done in duplicate as were 

 blanks and two concentration standards (10 ;.<g/mL 

 and 20 /ug/mL) accompanying each group of un- 

 knowns. 



Blood serum lactic acid concentrations were deter- 

 mined with a Sigma Chemical Company^ lactic acid 

 analysis kit (826UV). With this kit, blood lactic acid 

 is converted to pyruvic acid by lactate dehydroge- 

 nase, resulting in reduction of an equivalent amount 

 of NAD. Reduction of NAD causes an increase in 

 sample absorbance at 340 nm proportional to the 

 initial lactic acid concentration. The same 2 mL 

 blood sample used to measure pH was subsequent- 

 ly deproteinated with 4 mL of 8% perchloric acid 

 and used in the lactic acid analysis. After 90-min 

 incubation of the reaction mixture at 25°C, absorb- 

 ance readings were stable, indicating the end point 

 of the reaction. A chemical modification of the 



'Reference to trade names does not imply endorsement by the 

 National Marine Fisheries Service, NOAA. 



46 



