Kilada et al.: An age-deternnination nnethod for 3 commerciaiiy important crustaceans 
43 
Table 1 
Collection location, date, sex, and number of individual red king crab {Paralithodes camtschaticus), southern Tanner crab 
{Chionoecetes bairdi), and spot shrimp {Pandalus platyceros) collected in Alaska during 2013-2014 for investigation of pres- 
ence of growth bands in thin sections of the mesocardiac ossicle of the gastric mill of red king and southern Tanner crabs 
and in eyestalks of spot shrimp. 
Species 
Collection location 
Collection date 
Sex 
n 
Red king crab 
Bristol Bay, Alaska 
June 2013 
Female 
30 
Southern Tanner crab 
Marmot Bay, Kodiak, Alaska 
June 2014 
Male 
34 
Spot shrimp 
Seymour Canal, southeast Alaska 
February and June 2014 
Male and female 
30 
hensive size- and abundance-at-age data (Quinn and 
Deriso, 1999). 
A direct method for determining age based on count- 
ing bands in the endocuticle layer has been presented 
for decapod crustaceans (Leland et al., 2011; Kilada et 
al., 2012). Bands were initially described in the ossicles 
of the gastric mill for 6 species (Leland et al., 2011; 
Kilada et al., 2012) and in the eyestalks of 2 additional 
species (Kilada et al., 2012). These observations have 
since been extended to additional crab, lobster, and 
shrimp species and to euphausids (Kilada and Acuna, 
2015; Kilada et al., 2015; Sheridan et al., 2015; Kilada 
and Ibrahim, 2016; Krafft et al., 2016) and have been 
further supported by technical development, corrobora- 
tion, and validation (Sheridan et al., 2015; Leland et 
al., 2015). 
Our objective was to evaluate the potential of this 
method for 3 commercially important species in Alaska: 
the red king crab (Paralithodes camtschaticus), south- 
ern Tanner crab (Chionoecetes bairdi), and spot shrimp 
(Pandalus platyceros). Feasibility was evaluated 1) by 
identifying the endocuticle layer by histological ex- 
amination, 2) by observing the presence or absence of 
growth bands in the endocuticle of the mesocardiac os- 
sicles of crabs and eyestalks of shrimp, and 3) by deter- 
mining whether the region of the mesocardiac ossicle, 
where growth bands have been observed, may be re- 
tained during ecdysis for these crab species. The me- 
socardiac ossicle was selected as the primary structure 
for investigation in the 2 crab species because of the 
presence of clear bands and evidence of possible reten- 
tion through ecdysis (Leland et al. 2011; Kilada et al., 
2012). The eyestalks of shrimp were selected because of 
the dissimilarity between crab and shrimp gastric mill 
ossicles and because of the evidence of the presence of 
bands in the eyestalk of shrimp (Kilada et al., 2012). 
Materials and methods 
Histological examination 
Red king crab and spot shrimp were collected in south- 
east Alaska in 2014, by using pot gear, and south- 
ern Tanner crab were collected near Kodiak, Alaska, 
in 2014 by using trawl gear (n=3 for each species). 
Gastric mills of red king and southern Tanner crabs 
and paired eyestalks of spot shrimp were dissected 
and preserved in Bouin’s fixative for approximately 1 
month and then transported to the Fish Pathology Lab- 
oratory of the Alaska Department of Fish and Game 
in Anchorage, Alaska. Before processing, mesocardiac 
ossicles of some red king crabs were trimmed to fit 
within histological cassettes (1 cm^), but most ossicles 
and eyestalks did not require trimming. Structures 
were transferred to 70% ethanol and decalcified with 
Evans and Krajian fluid (Evans and Krajian, 1930). 
Tissues were dehydrated, infiltrated, and embedded in 
paraffin with an automatic tissue processor. Histologi- 
cal cassettes were cut longitudinally into 6-pm sections 
with a rotary microtome, and sections were mounted 
onto glass slides. Sections of mesocardiac ossicles of 
crabs and eyestalks of spot shrimp were prepared 
and stained with Masson’s trichrome (Thermo Fisher 
Scientific^, Waltham, MA) and permanently mounted 
with Permount mounting medium (Thermo Fisher Sci- 
entific). Masson’s trichrome was expected to stain the 
endocuticle and exocuticle layers blue and the mem- 
branous layer (hypodermis) and epicuticle red. The 
cuticle layers (for detailed description, see Vatcher et 
al., 2015) were then examined with a Zeiss microscope 
(Carl Zeiss Microscopy, Jena, Germany) and photo- 
graphed with a Jenoptik digital camera and ProgRes 
CapturePro software (JENOPTIK Optical Systems Inc., 
Jupiter, FL). 
Presence of growth bands 
Red king crab, southern Tanner crab, and spot 
shrimp (n=30, 34, and 30, respectively) of a range of 
body sizes were collected across Alaska in 2013 and 
2014 (Table 1). Carapace length (CL) and shell con- 
dition of red king crabs, carapace width (CW) and 
shell condition of southern Tanner crabs, CL of spot 
shrimp, and sex (identified from pleopod morphologi- 
cal features for shrimp) of all specimens were record- 
1 Mention of trade names or commercial companies is for iden- 
tification purposes only and does not imply endorsement by 
the National Marine Fisheries Service, NOAA. 
