Long and Van Sant: Embryo development in golden king crab ( Lithodes aequispinus ) 
69 
guish the different stages. To visualize stage differences, 
the morphometric data were normalized and then ana- 
lyzed with principal component analysis (PCA). To deter- 
mine whether morphometric measurements would allow 
us to distinguish the stages, the data were fourth-root 
transformed and an analysis of similarity (ANOSIM) 
was performed on a Bray-Curtis similarity matrix with 
female and stage as factors. All morphometric analyses 
were performed in Primer, vers. 6.1.15 (PRIMER-E Ltd., 
Ivybridge, U.K.; Clarke and Warwick, 2001). 
When females approached time of hatching, they 
were isolated in individual containers with flow- 
through water. Larvae released from each female were 
collected in nets that were checked daily. The first and 
last day of hatching were noted. 
Results 
Females extruded their eggs between 25 September 
2006, and 6 May 2007, and they began hatching be- 
tween 3 December 2007, and 23 July 2008. The mean 
duration of brooding (from extrusion to the first day 
of hatching) was 437.6 days (standard deviation [SD] 
6.7). We identified 13 stages of embryo development 
(12 of them are shown in Fig. 1) by visual examination 
of the embryos and their morphometric features. The 
MT model was an excellent fit to the data for stage 
transitions (Fig. 2) and provided estimates for the aver- 
age day of transition (£ 50 ) for each stage (Table 1). The 
mean duration of stages was between 10 and 95 days. 
There were some differences among the females (Fig. 
2), as demonstrated by the increased SD in the average 
embryo stage among females, particularly during some 
of the shorter stages (i.e. , stages 3, 4, and 5) and dur- 
ing stages 9, 10, and 11. The following sections describe 
the 13 stages identified. 
Stage 0 (precleavage) 
At this stage, the eggs were newly extruded and firm- 
ly attached to the pleopods; however, no division had 
yet occurred. There was no visible separation between 
the yolk, which was a creamy yellow, and the egg 
membrane. 
Stage 1 (cleavage and blastula) 
This stage began with the first cleavage to the 2-cell 
stage; this cleavage occurred between days 10 and 11 , 
and development progressed rapidly (Fig. 1A). By day 
13, most yolks were at the 8 -to 16-cell stage and by day 
15 they were at the 32-to 64-cell stage. During these 
stages of early division, the yolk often took on a lumpy 
shape and was frequently separated from the egg mem- 
brane, and a nucleus was visible in each cell as a light- 
colored circle when stained with Bouin’s solution. By 
about day 50, the blastula formed, and individual cells 
were no longer discernible. 
Stage 2 (gastrula) 
The beginning of this stage was marked by the blasto- 
pore (Fig. IB) becoming visible in embryos stained with 
Bouin’s solution. This stage began on average on day 
60 and lasted for about 32 days (Table 1). 
Stage 3 (v-shaped embryo) 
This stage began with the appearance of the embryo as 
a yellow v-shape when it was stained in Bouin’s solu- 
tion (Fig. 1C). This stage was short, beginning on about 
day 91 and lasting for about 24 days (Table 1). 
Stage 4 (prenauplius) 
During this stage, the embryos began to rapidly form 
differentiated parts. This stage began when the optical 
lobe, antennules, antennas, mandibles, and abdomen 
were visible as separate entities as opposed to the un- 
differentiated v-shape of the previous stage in embryos 
stained with Bouin’s solution (Fig. ID). The individual 
parts were still somewhat indistinct and widely sepa- 
rated from each other; in particular, the abdomen was 
well-separated from the optical lobe, mandibles, and 
antennas. This stage lasted for approximately 27 days, 
starting on day 115 (Table 1). 
Stage 5 (nauplius) 
This stage was brief, starting at around day 142 and 
lasting only about 18 days (Table 1). During this stage, 
the optical lobes, antennules, antennas, mandibles, and 
abdomen were more distinct, and they lengthened and 
coalesced so that the abdomen was situated between 
the antennas and mandibles and immediately below 
the optical lobes (Fig. IE). 
Stage 6 (maxilliped formation) 
This stage was another brief one, starting at around 
day 160 and lasting only about 15 days (Table 1). It 
began when the maxillipeds became distinct from the 
mandibles. During this stage, the abdomen became 
more elongate (Fig. IF). Before this stage, there were 
almost no changes in the morphological features of the 
eggs; the egg size and dimensions remained constant, 
and the embryo was not visible in unstained embryos 
(Fig. 3). But, during this stage, the embryo became vis- 
ible in unstained embryos, and size and percent area of 
yolk began to decrease (Fig. 4). 
Stage 7 (metanauplius) 
The metanauplius stage began when the abdomen 
was fully bifurcated and the carapace became visible. 
This stage was characterized by the lengthening of 
the antennules, antennas, mandibles, and maxillipeds 
(Fig. 1G). Late in this stage, the telson became visible. 
Growth of the embryo and reduction in the yolk hap- 
