Limulus polyphemus,

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Breeding Season

Fig. 1

To define the length of the breeding season, we monitored a known spawning area (Fig. 1) on the eastern shore of Pleasant Bay on Cape Cod, Massachusetts (James-Pirri et al., 2002, 2005; Carmichael et al., 2003) during daytime high tides from early March to early August, a time period that spans the spawning season described in Pleasant Bay and other sites (Rudloe and Herrnkind, 1976; Barlow et al., 1986, Carmichael et al. 2003, James-Pirri et al. 2005). We also visited the site on one night time high tide during this period, but safety concerns prevented further nocturnal visits. We visited the site, which spanned a 0.5 km section of beach, every two weeks until spawning began, and then on six occasions throughout the season while spawning activity was high. On one of these occasions, we were there for 6 days in a row. To study egg development in the off-season, we returned to the area in August and collected females from the shallows using snorkels. In November and January we obtained crabs that had been recently collected offshore by the Marine Biological Laboratory in Woods Hole.

On six of the visits during the breeding season, we collected crabs in each of three size categories: small (≤210 mm prosomal width (PW) measured across the widest part of the prosoma, or anterior part of the carapace), medium (211-240 mm) and large (>240 mm). Approximately five crabs from each size category were collected each time (15 crabs visit^-1), for a total of approximately 90 crabs. We euthanized females in an anesthetic clove oil bath (Keene et al., 1998; Peake, 1998), removed the carapace from the prosoma and took out the entire volume of eggs.  Other tissue was separated from the eggs by stirring the mixture in seawater; the heavier eggs settled to the bottom, allowing other tissue to be decanted off. In the off-season, we collected 6-10 crabs in each of the three months. Females were euthanized and dissected as above, but eggs were examined only for size, not number.

Egg maturation

To estimate net fecundity, we first assessed whether eggs mature before or continuously during the spawning season, and then used this information to determine whether to count immature eggs in the size-specific fecundity estimates (m_xi) (mean contribution of eggs by a single female in a given size class). We measured egg diameter to the nearest 0.1 mm under a dissecting microscope in samples removed from the total volume of eggs collected from 6-10 crabs from all size categories on each of 4 occasions, during the spawning season (May), and after the season in August, November, and January. Samples of one hundred eggs from each crab were measured. Gardiner (1927) and Dumont and Anderson (1967) described mature horseshoe crab eggs (approximately 1.7 mm diameter) and immature eggs (<0.5 mm). Using these values of egg sizes throughout the spawning season and beyond, we were able to establish in the following way whether horseshoe crabs are determinate or indeterminate spawners and therefore whether we should count immature eggs as well as mature ones. If determinate, crabs would contain a range of developing egg sizes over the winter, but by the onset of spawning only mature and immature eggs would appear, indicating the crabs matured all of their eggs for the current season before breeding began; remaining immature eggs would therefore not be laid until a subsequent year and should not be counted in the current year’s fecundity estimates. If indeterminate, females would contain a range of egg sizes during the season, indicating that eggs were continually maturing as the breeding season progressed; immature eggs should be counted in this case.

Size-specific fecundity

To relate female size to fecundity, we determined the total number of eggs in each female and related that to her prosomal width. Egg number was determined as follows: the entire volume of eggs from each female was dried in an oven (66ºC) for approximately one week and weighed (Turra and Leite, 2001). To convert egg weight to egg number, 5 aliquots of 200 eggs from different crabs were dried and weighed to derive a conversion factor. Size-specific fecundity (m_xi) was calculated by subtracting the number of eggs retained at the end of the breeding season by females of various sizes from the ‘potential’ fecundity (number of eggs in a female before spawning begins), to obtain ‘realized’ fecundity, defined here as the number of eggs actually laid (Wallace and Selman, 1981; Hunter et al., 1985).

Though population ecologists generally use number of female eggs (eggs destined to become females) in their calculations, we did not do that here. Since horseshoe crab eggs have ecological uses beyond reproduction where sex is irrelevant, such as food for shorebirds, it was more useful to consider the total number of eggs. Female eggs can be assumed to be 50% of the total eggs (R.H. Carmichael, unpublished data), so number of female eggs can simply be obtained by dividing total number of eggs by 2, should the reader have need for this information.

  

Breeding behavior

We needed to define certain features of local breeding behavior to interpret our fecundity data. The needed behavioral information included number of eggs deposited per spawning, number of spawning episodes likely to take place per breeding season, and the proportion of eggs laid during the entire season. The number of eggs carried by a female at the start of breeding should correlate with the number of eggs she deposits during a spawning event and the number of times she spawns; we confirmed this correlation from three lines of evidence. First, we directly measured the number of eggs deposited in a spawning episode on two daytime high tides by marking nests where crabs were spawning. We estimated size of spawning females by holding a ruler over them without interrupting the spawning process (Cohen and Brockmann, 1983). After the tide receded, we gently excavated the eggs and collected the discrete clutches (Cohen and Brockmann, 1983; Shuster and Botton, 1985; Brockmann, 1990).

Second, to determine whether females returned to spawn more than once, either during a tidal cycle or later, we tagged 315 females over 4 days (12-15 June), including one nighttime visit during the marking period (2 A.M. 15 June). Female crabs were marked with durable and long-lasting numbered thumbtacks inserted into the prosoma postero-lateral to the right compound eye (Sokoloff, 1978; Cohen and Brockmann, 1983). Sokoloff (1978) determined that tacks could remain in the carapace for at least two years. We searched for tagged crabs on all daytime high tides on return visits during the tagging period and the following two days (16-17 June), and again two weeks (28-29 June) and four weeks (14 July) later.

Third, to roughly estimate the proportion of eggs released during spawning episodes (one visit to the beach), we measured number of eggs carried by females arriving on and leaving the breeding beach on two daytime high tides. Thirty female crabs in amplexus (15 arriving, 15 leaving) were collected on each of these dates. The number of eggs found in the arriving group was compared to that in the departing group.

Net fecundity

To calculate net fecundity we measured size distribution of the female breeding population. We measured 222 females found in the spawning area over the course of the breeding season. Adult females were recognized by the absence of monodactylus pedipalps (‘boxer claws’ found on mature males) and the structure of genital pores (Shuster, 1982; Sekiguchi, 1988). The measurements were grouped into bins of 20 mm for the calculations.

We used data from R.H. Carmichael on size frequency distribution of the whole Pleasant Bay female horseshoe crab population together with our data for size frequency of the breeding population to extrapolate an estimate of the total breeding population by size (N_xi) in Pleasant Bay. Net fecundity (F_xi) (the total number of female eggs produced by all females of a given size category in a season) was calculated by multiplying (N_xi) by size-specific fecundity (m_xi). We assumed a stable population structure, an assumption supported by Carmichael et al. (2003). Standard errors were provided except where they could not be calculated because at least one variable had an N of 1. SE was propagated according to Meyer (1975).