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Fig. 1. Experimental design. (A–E) Animal setup. (A) A juvenile crayfish, 2.0
cm from rostrum to telson. (B) Individual cages in an aquarium. Each cage is
5.5 cm in diameter. (C) Diagrams illustrating setup for animals in the Isolate
and Pair groups. Rectangles and circles represent plastic aquaria and cages,
respectively. Small ovals in each circle in the Isolate groups represent
animals in isolation, while small triangles and small squares in each circle
in the Pair group represent dominant and subordinate animals, respectively.
Each gray object inside a circle represents a shelter. For the Pair group, 20
animals were transferred to new cages, two animals per cage, and only one
shelter was provided in each cage. For the Isolate group, ten animals were
individually transferred to new cages and a shelter was provided in each new
cage. (D) Two animals in the Pair group (arrows) in a cage with one shelter.
(E) One animal in the Isolate group (arrow) in a cage with one shelter. (F,G)
Protocols for Cell Proliferation Test (F) and Cell Survival Test (G).
Following the protocol in F and G, the animals in the Isolate and Pair groups
were treated for cell proliferation and cell survival tests. A dotted line
indicates an isolated condition; a broken line indicates a pairing condition;
gray bars indicate immersion of animals in BrdU solution for 24 h. (F) Cell
Proliferation Test. Animals were isolated for 6 weeks, paired or isolated for
1, 7 or 14 days, immersed in BrdU for 24 h on the last day of the pairing
period, and sacrificed. (G) Cell Survival Test. All animals were isolated
either for 5 or 9 weeks, paired or isolated for 7 or 14 days, immersed in BrdU
for 24 h at the beginning of the pairing period, and sacrificed. A separate
group of animals (age-control groups 1 and 2) was treated similarly (isolated
for 5 or 9 weeks, immersed in BrdU for 24 h, and sacrificed immediately) but
without any pairing. The age-control groups show the level of cell
proliferation at the end of the isolation period, while the animals in the
Isolate group show the level of cell survival after 7 days or 14 days of
isolation. (H) Diagram of the cell division cycle. BrdU, a thymidine analog,
labels cells in the S phase during which DNA replication occurs. H3P, a
mitosis marker, labels cells in the M phase during which histone 3, a protein
mediating the folding of DNA, is phosphorylated.
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