All hermit crabs for the following experiments were collected on a flat rocky intertidal shore at Kattoshi, Hakodate Bay, Hokkaido, Japan (41°44′34″N, 140°36′08″E). The breeding season of Pagurus filholi ranges from February to August, but was mainly from March to July at our study site (Goshima et al. 1998). Sampling of guarding pairs was performed during low tides from March to July in 2005–2009.
Maturity condition of guarded females
Guarding pairs were collected haphazardly 8 times at Kattoshi and were brought to the laboratory. Each pair was separated and the maturity condition of the guarded female was ascertained as non-ovigerous or ovigerous (egg carrying). For the ovigerous females, we determined the egg developmental stages microscopically and classed the females into one of three categories as follows: stage I ovigerous female had newly deposited eggs and more than 70% of original yolk volume; stage II ovigerous female had eggs of less than 70% of original yolk volume and some embryos having incomplete pigmented eyes visible; stage III ovigerous female had pigmented embryo with well-developed oval eyes visible. Percentages of the guarded females were compared among four different maturity conditions using repeated measure ANOVA to clarify whether males in the field frequently guarded any particular maturity stage of the females. As the sphericity assumption did not hold (Mauchly test, W = 0.00026; P < 0.0001), the P value of the F test was corrected based on the degrees of freedom multiplied by Greenhouse–Geisser epsilon (0.3403). Multiple comparisons were performed by paired t-tests with separate error terms depending on the two levels being compared. The type I error rate was corrected by sequential Bonferroni.
“Female conditioned” water sample collection
Guarding pairs were collected at Kattoshi and brought to the laboratory. Thirty females that had been guarded by males in the field were separated from the males and placed in the same plastic cup (5 cm diameter, 9 cm high) with 100 ml artificial seawater (Perfect Marine, Nisso Corporation) and kept for 24 h at 12 °C. The resulting “female conditioned” water samples were tested to confirm the presence of sex pheromones triggering male assessment response (Imafuku 1986; Goshima et al. 1998; Minouchi and Goshima 1998; Okamura and Goshima 2010). Only females sampled within 2 days from the field were used to collect “female conditioned” water samples.
Solitary males of the hermit crab often show assessment behavior toward mature females, while no response behavior was observed toward immature females or shells with no females. The assessment behavior is preliminary to guarding behavior, and it is elicited by female sex pheromones (Imafuku 1986; Goshima et al. 1998; Minouchi and Goshima 1998; Okamura and Goshima 2010). The male embraces the female with its ambulatory legs, and rotates the female into a face-to-face position. The male then brings the anterior parts of his cephalothorax close to the shell aperture of the female, and examines it by touch with antennae, walking legs, and chelae on a part of the female’s body just protruding from the shell. We determined whether this assessment behavior occurred or not as well as duration of the interaction when observed in experiments to detect sex pheromonal activity in the samples of “female-conditioned” water as follows.
Guarding males from the field were separated off the females, and each male was placed in a plastic case (10.5 cm diameter, 2 cm high) full of natural seawater. Most guarded females in Kattoshi were in Batillaria cumingi shells (Yoshino et al. 2002). Two groups of empty Batillaria shells which were similar in size were plugged with cotton containing either artificial seawater (n = 17) or “female-conditioned” water (n = 17) and then presented, in that order, to the males in the plastic cases. The behavior of the male was observed initially for 30 s. If assessment was initiated during this time period, male behavior was observed for 30 s more. The difference in assessment duration between artificial seawater and “female-conditioned” water shell groups was analyzed with the Wilcoxon signed-rank test.
To verify the assay protocol, the above procedure was repeated, but this time, shells plugged with cotton containing “female conditioned” water (n = 20) were presented first, followed by the shells containing artificial seawater (n = 20). This was done specifically to test sample presentation order effect on male assessment behavior. The behavior of males presented with plugged shells was observed initially for 30 s. If assessment behavior was initiated during this interval, observation continued for over 30 s. The difference in assessment duration between “female-conditioned” water and artificial seawater shell groups was analyzed with the Wilcoxon signed-rank test.
Only males that performed assessment behavior to the “female-conditioned” water samples (positive control) were used in experiments 2 and 3. In the later experiments, we tested seawater first and then samples because the order of the treatments had no effects on experimental results (see Results Section for details).
Experiment 1: “pheromone” molecular size
We performed ultrafiltration of the “female-conditioned” water samples using YM-1 filters (Amicon ultra, Mollipore) to separate each water sample into two fractions of different molecular size; <1 or >1 kDa. The pheromonal activity of the remaining fraction on the filter (>1 kDa) and filtrate (<1 kDa) was tested for each sample (n = 30) by the following procedure.
Guarding males from the field were separated off the females, and each male was placed in a plastic case (10.5 cm diameter, 2 cm high) full of natural seawater to test “female conditioned” water fractions (<1 or >1 kDa) separately, both against seawater. First, one group of Batillaria shells was plugged with cotton containing artificial seawater (n = 30) while another group of shells was plugged with cotton soaked in unfiltered fraction sample (n = 30) or filtrate fraction sample (n = 30). The unfiltered fraction (>1 kDa) was obtained by rinsing the YM-1 filter membrane with 100 ml distilled water. Artificial seawater and fraction samples were presented sequentially (one at a time) to the males. The behavior of males was observed initially for 30 s. If assessment was initiated during this period, we observed male behavior for 30 s more. Difference in assessment duration between artificial seawater and fraction shell groups was analyzed with the Wilcoxon signed-rank test.
Experiment 2: “pheromone” polarity
To reveal the polarity of the compounds with sex pheromone properties, and in order to remove large amounts of salts from “female conditioned” water samples, ODS resin fractioning was employed as follows. “Female conditioned” water was passed through an ODS open column (3.5 cm diameter, 4 cm high, Fuji Silysia Chemical). The column was washed with distilled water and successively eluted with 30 or 50% MeOH. Each extract was evaporated in vacuum and dissolved in 10 ml distilled water and used as a sample. The behavior of males presented sequentially with cotton plugged Batillaria shells, containing column samples eluted with 30% (n = 38) and 50% (n = 28) MeOH, was examined separately, both against plugged shells with artificial seawater for each sample in a bioassay as noted under Bioassay procedures Section.
Experiment 3: stability
One of the major concerns during handling of natural chemical products including sex pheromones is their stability. Herein, we present handling tests and storing procedures of the pheromone potentially contained within 100 ml of “female conditioned” water and its effect on male assessment behavior.
“Female conditioned” water was freeze-dried and taken up in 10 ml distilled water. The freeze-drying effect on pheromonal activity was evaluated with the behavioral assay for n = 14 samples, described under Bioassay procedures Section.
The effect of high temperature on pheromonal activity of “female conditioned” water was tested by increasing the sample temperature to 90 °C for 15 min. The behavioral assay described under Bioassay procedures Section was performed for n = 14 samples.
Stability at ambient temperature
The objective of the bioassay for this item was to test the stability of effect of the compound involved in triggering male assessment behavior at ambient temperature. “Female conditioned” water samples (made using natural seawater in this experiment) were kept in plastic bottles for 6 days at 12 °C before presentation to the males in an assay as described in the Bioassay procedures Section for n = 22 samples.