857 
Age determination of larval strombid 
gastropods by means of growth 
increment counts in statoliths 
Felix A. Grana-Raffucci* 
Richard S. Appeldoorn** 
Department of Marine Sciences 
University of Puerto Rico 
Mayaguez, Puerto Rico 00681-5000 
E-mail address (for R.S. Appeldoorn): R_Appeldoorn@rumac.upr.clu.edu 
‘Present Address: Coastal Zone Division 
Department of Natural and Environmental Resources 
PO Box 5887, San Juan, Puerto Rico 00906 
The queen conch, Strombus gigas 
Linnaeus, and the milk conch, S. 
costatus Gmelin, are important gas- 
tropods of the Caribbean region 
(Appeldoorn and Rodriguez, 1994). 
To age strombid larvae by means 
of their statoliths would be useful 
in order to study aspects of their 
larval life histories and ecology. 
Statoliths, like fish otoliths, are 
formed of aragonitic calcium car- 
bonate deposited on a protein ma- 
trix and exhibit periodic growth 
increments (Radtke, 1983). Re- 
search on statolith microstructure 
has been limited primarily to age 
determination of commercially im- 
portant cephalopods (e.g. Jackson, 
1994). D’Asaro (1965) observed sta- 
tocysts that appeared in four-day- 
old embryos of S. gigas and that 
were fully functional by day six; he 
also noted growth increments or 
rings on these structures. These 
increments were confirmed by 
Salley (1986). Our objective is to 
validate the use of statolith micro- 
structure in S. gigas and S. costatus 
to provide information on age, 
growth, and length of larval life. 
Materials and methods 
Egg masses from eight Strombus 
costatus and one S. gigas were col- 
lected from ovidepositing females 
at a site 7 km south of La Parguera, 
southwest coast of Puerto Rico 
(17.92°N, 67.05°W). Egg masses 
were held in 75-L aquaria subjected 
to the natural light-dark cycle. Cul- 
ture methods of Ballantine and 
Appeldoorn (1983) were used. 
Aquaria were cleaned daily, after 
which one liter of Tahitian Iso- 
chrysis ( 10 6 cells/mL) was added. A 
minimum daily sample of ten indi- 
viduals was removed from each 
aquarium and preserved in 70% 
ethanol. We examined the statolith 
microstructure of larvae from the 
longest surviving cultures. Since no 
veligers reared in our laboratory 
developed through metamorphosis, 
we obtained preserved (5% buffered 
formalin, pH 8.0) S. gigas veligers 
and juveniles of known age from 
the Trade Wind Industries’ hatch- 
ery in the Turks and Caicos Islands. 
Preserved veligers were exam- 
ined with a dissecting microscope, 
their larval shell length (apex to 
siphonal canal) was measured, and 
their shell removed. A drop of 60% 
solution of alizarin red in glycerin 
was added to increase contrast be- 
tween stained soft tissues and the 
unstained statolith. Coverslips 
were added, sealed with Permount, 
and samples were inspected under 
a compound scope at l,000x. Sta- 
tolith diameters were measured 
with an ocular micrometer. Incre- 
ments on statoliths were counted 
by focusing up and down through 
the statolith. For each day of age, 
counts were made from one sta- 
tolith from each of 20 veligers; all 
counts were made by the same 
reader in a blind manner. General 
physical structure was observed 
and described, with emphasis 
placed on the periods preceding and 
following hatching, and, for S. gi- 
gas juveniles, preceding and follow- 
ing metamorphosis to determine 
the presence and nature of any 
transitional marks associated with 
these events. 
Statolith diameter, number of 
growth increments, and shell length 
were averaged for each day and ar- 
ranged with age (in days after 
hatching). Linear least-squares re- 
gressions were calculated to deter- 
mine the relations among these 
three variables. To determine the 
precision (or reproducibility) of 
counts of increments (i.e. verifica- 
tion, Wilson et al., 1983), repeated 
counts for both statoliths were 
made on subsamples. The number 
of increments in these representa- 
tive samples were counted three 
separate times (double-blind), and 
the results were averaged for indi- 
vidual veligers. Standard devia- 
tions (SD) were calculated for each 
individual; standard error of the 
means (SE) was calculated as ap- 
propriate. A nested analysis of vari- 
ance (ANOVA) was done for each 
species to determine if variability 
in incremental counts was due to 
errors in measurement or to natu- 
ral variability in increment depo- 
sition (Sokal and Rohlf, 1981). Data 
were grouped at four levels: 1) all 
** Author to whom correspondence should 
be addressed. 
Manuscript accepted 11 April 1997. 
Fishery Bulletin 95:857-862 (1997) 
