Forma

lled

adjust to seasonal changes in food source and availability (Je

an

g the

uspended t al. 2003a). Briefly, the hatching container consisted of a 20 l plastic bucket fitted with a lid, a section of the lower portion of

o

presentative samples

Triplicate samples of female broodstock from each dietary treatment were ki and tissue samples (digestive gland, ovary and tail muscle) were taken for lipid and fatty acid analysis. Samples were taken prior to the commencement of the study, after 3 weeks starvation and 1, 3 and 5 months after the commencement of feeding. A period of starvation would deplete lipid fatty and acid stores and hence provide an insight into the mobilization and utilization of these products. Due to the long lead-time between starvation and egg extrusion (5 months) and the inherent ability of spiny lobsters to

rnakoff et al., 1993; Barkai et al., 1996) it was considered that there would be no undue effects on the processes of maturation.Prior to dissection, broodstock were placed in an ice slurry for 1 h to induce a chill coma. Ovarian stage of development was recorded, with 7 recognized macroscopic stages differentiated primarily on size and colour (Fielder, 1964). Two indices were calculated for broodstock: the org lipid index (organ lipid dw/total body ww x 100) and the gonadal somatic index (GSI, ovary ww/total body ww x 100).

Broodstock tanks were inspected daily for mortalities, molts and ovigerous females, while closer inspection of animals occurred during weekly tank cleaning. Broodstock from both dietary treatments experienced one death each durin

trial. Animals of similar weight replaced mortalities but the replacement animals did not extrude eggs. Prior to hatching (2-7 days), females were removed from the dietary treatment tank and placed into individual 20 l hatching containers s

the bucket has been removed and replaced with a 500 µm screen, phyllosoma larvae are retained within the container by the screen while water is exchanged at 1 l min-

1

.

med from the surface of the hatching

container and allocat ing eatm

ture of la e for 42 d w th length d su vival easured at molt Stages I

Lethal Dose 50 (LD-50), the time (d) for 50% of unfed Stage I larvae to die;

ival o in 1

ple for analysis of lipids and fatty acids.

e applicatio nity tes t ea is

m to test q is th a ex imals to

a se condi th e to t b thei ency

( rt et al., 1 llaló ; C 9 2; 99 nd

L 92; a e ). T l tio en nd any

particular variable will be dependant upon how mu h able between t nd it ce u petency, it will not be a condition that they w lly be exposed to in the wild (Racotta et al., 2003). It is suggested that

s stress tes atio o r rom als

nutritional status to their ability to m

s rgy f pu ing u a , co expected

d salinity c (R al

tive viab ity sur n iv ales at hatch. This is

6.3.4. Phyllosoma assessments

At hatch, phyllosoma larvae were skim

ed randomly to the follow tr ents:

1. cul rva i an r m

- IV; 2.

3. surv f larvae h temperature and salinity challenges (activity test); 4. sam

Th n of sali activity ts to crus ac n larvae a common ethod uality and based on e premise th t you are posing an n adver tion that ey are abl respond o ased on r compet Tackae 989; Vi n,, 1991 lifford, 1 9 Fegan, 1 2; Bray a awrence, 19 Samoch t al., 1998 he corre a n betwe survival a c that vari altered reatments a s influen pon com

ould norma

alinity ts measure a combin n of fact rs anging f an anim

obilize nutrients under duress, in particular, to upply ene or ATPase mps dur osmoreg l tory stress nditions

uring hallenges acotta et ., 2003).

Rela le fecund was mea ed from i d idual fem

a measure of the total number of phyll soma hatc in off a female, dividedo h g by female carapace length (mm) (units – number of phyllosoma per mm of carapace length). It does not include non-viable eggs or animals that do not progress beyond the naupliosoma stage (a brief 0.5 – 1 h post-hatch stage).

The cross-sectional diameter of twenty randomly chosen eggs was measured on e first day that phyllosoma began to hatch from females (a typical hatch period lasts 4-5 days per female). Additionally, total body length (anterior tip of the

cephalo ) was measured in twenty

ne

) and wild broodstock (n=

to

s II low stant

llowing daily total water exchange in the beakers, flushing away uneaten Artemia

Artemia

ere disinfected in a form n bath mg l r 10 mi nd rinsed on a 250 µm wate

th

thorax to the posterior point of the abdomen

wly-hatched Stage I phyllosoma from each sample. Measures of total body length were again taken on days 14, 28 and 42 at Stages II, III and IV, respectively, as described by Lesser (1978). Measurements were obtained using an overhead

projection microscope (Nikon profile projector, model 6C, 20 times magnification).

6.3.6. Larval rearing

Phyllosoma resulting from the Diet SP (n=6), Diet BP (n=9

6) were cultured to Stage IV (42 d) in lightly aerated 1 l glass beakers maintained in water baths at 18ºC. Additional triplicate groups of phyllosoma congenetics remained unfed in the same system and under the same conditions assess their level of endogenous reserves and physiological condition, using a LD- 50.

During Stage I, larval densities in both fed and unfed treatments were 100 larvae l-1. Larval numbers were subsequently reduced to 50 and 30 larvae l-1 at Stage and III, respectively. Larval rearing was conducted in 1 l of water except when survival in previous stages resulted in volumes being reduced to maintain con rearing densities.

Phyllosoma were fed 1.5mm juvenile Artemia at a rate of 3 ml-1 every day, fo

and application of antibiotics (oxytetracycline hydrochloride 25 mg l-1, Intervet Engemycin 100, Melbourne, Australia) to the culture water. Prior to use,

w ali (100 -1 fo n) a

6.3.7. Cumulative activity test

The activity test developed to ascertain larval competency (Smith et al. 2003b) was examined further using the stress parameters of 8, 10, 12‰ and 21, 23, 25°C. Briefly, phyllosoma from individual females (triplicates n=20 larvae) were placed in different combinations of salinity and temperature, and monitored for activity at 3-min intervals. Prostrate larvae were counted as ‘inactive’ and cumulative inactive totals (stress index) were tallied over 1 h. A large stress index indicated that

animals had succumbed sooner to the stress parameters. Results were validated by relating stress indices with survival in culture or an LD-50, a low stress index or high LD-50 is indicative of higher survival of congenetics in culture.