Northeastern Naturalist Vol. 22, No. 2 K.T. Lee and J.L. Vespoli 2015 413 2015 NORTHEASTERN NATURALIST 22(2):413–423 Tracking Color Change in Individual Green Crabs, Carcinus maenas (L.) Karen T. Lee1,* and Jessica L. Vespoli1 Abstract - Carcinus maenas (Green Crab) exhibits a color range from green through yellow and orange to red. The change from green to red is hypothesized to signal a change in resource allocation from growth to reproduction. In order to understand color change in individuals, C. maenas were held in captivity or tagged and recaptured in summer 2007 and 2008. Over the course of the study, crabs fell into 4 categories: those which were green and did not change color, those which were red and did not change color, those which became redder, and those which molted, changing from red to green. Crabs did not change from red to green unless they had molted. Our data show that some individual crabs turn progressively redder when they have not molted, and turn green again after molting. Introduction Carcinus maenas (L.) (Green Crab or Shore Crab) is perhaps the most widely introduced shore crab in the world, invading and/or becoming established in shallow marine ecosystems in eastern and western North America, Japan, Australia, and South Africa (Geller et al. 1997) and invading some areas multiple times (Roman 2006). The crab’s importance in shaping the intertidal zone is well documented (Seeley 1986, Trussel 1996). It is a voracious predator, is euryhaline, and is capable of withstanding hypoxia, temperature changes, and desiccation (Crothers 1967, Lee et al. 2003, Reid et al. 1997). Given the crab’s ongoing invasions into new territories and threats to commercial species, it is important to understand the population dynamics and physiology of C. maenas. Carcinus maenas exhibits a range of colors, particularly on the ventral carapace, from green through orange to red, which is correlated with the physiology, reproductive success, and distribution on the shoreline of individual crabs (for reviews see Reid et al. 1997, Styrishave et al. 2004). For the rest of this paper, “green phase” and “red phase” will refer to the morphs, respectively, while the crabs will be referred to as C. maenas since their common name changes with geography. The change from green to red is thought to be evidence of increased inter-molt duration as part of an evolutionarily stable strategy in which some crabs allocate resources to growth while others allocate resources to mating. A tradeoff exists in so far as male crabs that delay molting, turning progressively redder, become physiologically more fragile but are more successful in obtaining and retaining mates. Whether a population is in reproductive or growth mode can be determined by studying color distribution (Rewitz et al. 2004). 1Biology Department, University of Pittsburgh Johnstown, 450 Schoolhouse Road, Johnstown, PA 15904. *Corresponding author - ktlee@pitt.edu. Manuscript Editor: David J. Yozzo Northeastern Naturalist 414 K.T. Lee and J.L. Vespoli 2015 Vol. 22, No. 2 Female C. maenas also exhibit distinct green/red color morphology (McKnight et al. 2000) that mirrors male patterns in osmoregulatory physiology (Lee et al. 2003), size differences (Rewitz et al. 2004), and distribution on the shoreline (McKnight et al. 2000). Both male and female green-phase crabs appear to be better at withstanding anthropogenic stress than red-phase crabs (Costa et al. 2011, Styrishave et al. 2000), and are in better condition as measured by hepatopancreas fatty acid profiles (Styrishave and Andersen 2000). Griffen et al. (2011) used female color as a proxy for time since last molt. There is strong evidence that the change from green to red is associated with increased inter-molt duration (McGaw et al. 1992, Styrishave et al. 2004). Red-phase males are more likely to be fouled by organisms, exhibit wear and tear of their carapace and claws, have missing claws, and have thicker carapaces than green-phase males. All male crabs in early post-molt are green phase and most red-phase males are in later inter-molt stages. Red-phase males are larger on average than those in green phase, and red-phase males are stronger than green-phase males of the same size (Kaiser et al. 1990, Taylor et al. 2009), not because of increases in claw size, but because their closer muscle mass is larger, suggesting they have been adding tissue longer than green-phase crabs of the same size. All of this evidence suggests that redness in male crabs is correlated with intermolt duration. However, no study has tracked individual crabs to monitor color change. We took 2 approaches to tracking individual color change in C. maenas. We marked, recaptured, and recorded the colors of crabs during 2 summers. In addition, we tracked the color of crabs kept in the lab. Several individuals molted in the lab, allowing us to report pre- and post-molt color. If the hypothesis that crabs turn red during prolonged inter-molt and back to green after molting is correct, then crabs would turn progressively redder if they did not molt during the study, and crabs that molted would be green after molting. Materials and Methods Field site description This work took place at the Rutgers University Marine Field Station (RUMFS) in Tuckerton, NJ, in the boat basin in front of the station. The salt marsh surrounding the station, part of the Jacques Cousteau National Estuarine Research Reserve (JCNERR), in the Great Bay–Mullica River Estuary, is one of the least impacted salt marshes on the east coast (Lathrop et al. 2000) and has recently been designated a NERR Sentinel Site (Kennish et al. 2014). The marsh is typical of northeastern salt marshes and has a tidal range of approximately 1 m (Lathrop et al. 2000). The boat basin is surrounded by marsh edge, and there are several docks in the basin itself. The maximum depth of the boat basin is approximately 3 m (Able at al. 2012). Field studies Mark–recapture studies were carried out in summer 2007 and 2008. There were 3 trapping periods—early June, late July, and late August— in 2007 and 4 trapping periods—late May, late June, late July, and mid–August— in 2008 (on the last day Northeastern Naturalist Vol. 22, No. 2 K.T. Lee and J.L. Vespoli 2015 415 of collecting in August 2008, no crabs were tagged and only recaptured crabs were recorded). Each trapping period lasted 2–5 days. Temperature ranged from 10.3 to 27.8 °C and salinity from 20.7 to 32.0 ppt during the trapping periods, according to data from a nearby monitoring station (NERRS 2011). Crabs were captured using homemade PVC pipe traps (50–55 cm L x 15 cm W) with plastic hardware-cloth mesh cones (2-cm mesh with 7–10-cm entrances) on each end. We baited the traps with fish and deployed them along the marsh edge or off the dock near an area with shelter for the crabs. They were set out during an incoming tide and pulled on the next outgoing tide or deployed overnight to catch the incoming tide and pulled the next morning. We placed the traps such that they were at least partially exposed during low tide. Location, carapace width, sex, color, missing limbs or damage, tag number (if one was attached), and presence of a tag hole (indicating tag loss) were recorded for each crab. For crabs larger than 32 mm carapace width, we injected monofilament tags (Floy FF–94 Fine Fabric Anchor Tags with molt cone) between the carapace and abdomen of the crabs using a Denison Mark II Fine Fabric tagging gun. Tagged crabs were returned to the site from which they were trapped. Laboratory studies Laboratory studies were carried out in summer 2007. Crabs for lab studies were either trapped, but not tagged, at RUMFS, or purchased from nearby bait shops. We transported crabs from the field to the laboratory at the University of Pittsburgh Johnstown in a cooler on ice. In the lab, we maintained crabs in individual aquaria of various sizes in artificial seawater at 16–18 °C in a 12–12 light/dark cycle. We fed crabs either fish or mussels twice per week and recorded their color once a week. Data analysis We coded crab color using a 10-category index (see Lee et al. 2005 for details) that facilitated statistical analysis of color and allowed intermediate-color crabs to be included in the analysis rather than ignored or lumped into red phase or green phase. We assigned color index values as follows: 1 = green, 2–5 = various shades of yellow (progressively from greenish to nearly orange), 6–8 = progressively darker orange, and 9–10 = shades of red. Crabs that fell between index values were indicated with a 0.5 as part of their index value. Thus, any crab with an index value 5.5 or higher would generally be considered as orange or red. Since errors of 1.5 index units were not uncommon even with a single scorer (Lee et al. 2005), only crabs which changed 2 or more index values between captures were considered to have changed color, even if one value was clearly orange or red and the other green or yellow. Statistical analysis was carried out using SPSS. All means are expressed as mean ± 1 standard error. We analyzed color change between captures for individual crabs using the related samples Wilcoxon signed-rank test, unadjusted for ties, and the differences between time periods across the population of crabs using Kruskal– Wallis and pairwise multiple comparisons, unadjusted for ties. We plotted graphs using Kaleidograph. Northeastern Naturalist 416 K.T. Lee and J.L. Vespoli 2015 Vol. 22, No. 2 Results Field studies We tagged a total of 878 crabs—331 in 2007, and 547 in 2008 (Table 1)—and recaptured a total of 102, for a recapture efficiency of 11.39%. No crabs tagged in 2007 were recaptured in 2008. Of those crabs recaptured, ~40% were captured within a few days during the same trapping period and thus would not be expected to have changed color. A few crabs (3 in 2007 and 14 in 2008) had tag holes or carapace damage that could have been caused by an attempt to molt around a tag. A total of 59 crabs (57.9% of recaptured crabs) were recaptured in different time periods. Both green-phase and red-phase crabs were marked and recaptured, but none of the red-phase crabs changed color between captures. Of the green-phase crabs, 56% of females and 65% of males changed color between ca ptures. Because of the small number of recaptured crabs that changed color, data from 2007 and 2008 were pooled for statistical analysis. Mean color index for pooled crabs which changed color (n = 13, mean cw = 48.86 ± 1.61 mm) increased significantly between first capture (mean color = 2.08 ± 0.34) and recapture (mean color = 4.88 ± 0.35) (Z = 3.204, P = 0.001; Fig. 1). Only 1 crab was recaptured that had molted between captures: a male with carapace width = 44.1 mm. He increased carapace width by 9.9 mm (22.1%) and showed healing around the tag. Though he was very light orange pre-molt and showed no orange post-molt, the color change was fewer than 2 index values. Color change during the summer across all of the crabs captured (Table 2, Fig. 2) showed similar patterns in summer 2007 and 2008. Females were more red than males in pooled data (2007 females: n = 175, mean color = 6.89 ± 0.199; males: n = 205, mean color = 4.3 ± 0.117; Z = –10.132, P < 0.001; 2008 females: n = 330, mean color = 6.421 ± 0.1501; males: n = 258, mean color = 4.04 ± 0.105; Z = –11. 608, P < 0.001). While male crabs became redder over the course of the summer (2007: n = 205; H = 31.57, P < 0.001; 2008: n = 258; H = 8.078, P = 0.044), female crabs showed a different pattern, with redness peaking in June and August and decreasing in May and July (2007: n = 175; H = 18.757, P < 0.001; 2008: n = 330; H = 23.991, P < 0.001) Though the overall trend was significant, there were significant differences only between some months (Table 2). Laboratory studies Color change during inter-molt. A total of 47 crabs (39 females [F] and 8 males [M]) were transported to the lab. Of those, 6 molted and were analyzed separately. Table 1. Mark–recapture data. Number of crabs marked and recaptured in summer 2007 and 2008. Recapture (different time) indicates the number of crabs recaptured in separate trapping periods. Crabs captured during the same trapping period would not have changed color and so were not part of the tracking data. Year Marked Recaptured (total) Recaptured (different time) Changed Color 2007 331 30 15 4 2008 547 72 44 9 Total 878 102 59 13 Northeastern Naturalist Vol. 22, No. 2 K.T. Lee and J.L. Vespoli 2015 417 Twenty-seven (19 F, 8 M) of the remaining 41 survived at least 4 weeks in captivity. Of these survivors, 9 crabs (6 F, 3 M) changed color more than 2 index units. The group of crabs became significantly redder by week 4 (mean week 0 = 2.94 ±0.57, mean week 4 = 5.7 ± 0.46; Z = –2.680, P = 0.007; Fig. 3). Of the 18 surviving crabs Figure 1. Color change in crabs between captures. Index values for pooled crabs (n = 13; mean cw = 48.86 ± 1.61 mm; mean color change = 2.81 ± 0.28) increased significantly between first capture (mean color = 2.08 ± 0.34) and recapture (mean color = 4.88 ± 0.45) (Z = -3.204; P = 0.001). Table 2. Comparison of crab population color among capture periods (months). * indicates significance at α ≤ 0.05. Months t P 2007 Females June vs July 0.033 0.973 June vs August 3.820 ≤0.001* July vs August 3.732 ≤0.001* 2008 Females May vs June 2.894 0.004* May vs July 0.689 0.491 May vs August 1.383 0.167 June vs July -4.528 ≤0.001* June vs August -1.954 0.051 July vs August 1.230 0.219 2007 Males June vs July 3.871 ≤0.001* June vs August 5.284 ≤0.001* July vs August 2.389 0.017* 2008 Males May vs June 0.849 0.396 May vs July 1.587 0.112 May vs August 2.635 0.008* June vs July 0.939 0.348 June vs August 2.262 0.024* July vs August 1.572 0.116 Northeastern Naturalist 418 K.T. Lee and J.L. Vespoli 2015 Vol. 22, No. 2 Figure 2. Color change in crab populations. Light gray bars are females, dark gray bars are males. A is 2007, B is 2008. Northeastern Naturalist Vol. 22, No. 2 K.T. Lee and J.L. Vespoli 2015 419 that did not change color in captivity, 10 had index values of 5.5 or greater during week 0 and thus were already red phase at the beginning of the study period. Molting and color change. Six female crabs molted in captivity (Fig. 4). Crabs were significantly larger after molting (pre-molt mean carapace width = 40.85 ± 1.85 mm vs. post-molt mean carapace width = 48.97 ± 1.76; Z = 2.201; P = 0.028), with an average increase in size of 19.9%. All 6 crabs were red phase before molting (mean index value = 6.75 ± 0.56), and 5 of the 6 were green phase post-molt (mean index value = 3.33 ± 0.70). The color change from pre–molt to post–molt was statistically significant (Z = –2.201, P = 0.028; Fig. 4). Discussion This work is the first attempt to track color change in individual C. maenas. These data support the hypothesis that red coloration develops in crabs that have not molted (Reid et al. 1997, Styrishave et al. 2004). Crabs that had not molted in late spring or early summer would be green phase at the beginning of the study and would become redder as the study progressed. These crabs would be transitioning from green phase to red phase. This pattern was seen in both tagged individuals in the wild and crabs held in captivity, even over the short period of time (4–12 weeks) in our study. Any crabs molting during the study were expected to be green post-molt, even if they were red prior to molting. In our study, crabs that molted, including captive Figure 3. Color change in captive crabs. The crabs that changed color in lab got significantly darker by week 4 (mean week 0 = 2.94 ± 0.57, mean week 4 = 5.7 ± 0.46; Z = -2.680, P = 0.007). Northeastern Naturalist 420 K.T. Lee and J.L. Vespoli 2015 Vol. 22, No. 2 individuals and one individual that molted while tagged, were all red phase premolt and green phase post-molt, with the exception of one female which stayed red phase post-molt. Crabs did not decrease index value (from redder to greener) unless they had molted. Other crabs did not fit the hypotheses. Some crabs were green and stayed green, while other crabs were red and stayed red during the course of the study. It is possible that the green crabs that did not turn red during the study have recently molted, and were unlikely to molt again during the 3-4 month trapping period (Crothers 1967). Given that prolonged inter-molt is thought to cause color change, crabs which had recently molted would not likely turn red. The crabs that were already red either had not molted in a very long time or were in terminal anecdysis. When the mean color of all the population of crabs captured over the course of the summer is examined, the pattern is more complicated. Male crabs became redder from early to late summer. Females, however, showed a different pattern, with a decrease in color index in July compared to June and August. Clearly color change was present in females, but was different than the pattern in males. Even given the differences in the pattern of overall color change in the population, female color index is higher than that of males throughout the summer, confirming the pattern seen in previous studies (McKnight et al. 2000). Figure 4. Molting in captivity. Light gray bars are pre-molt, dark gray are post-molt. Six female crabs molted in captivity. Crabs were significantly larger after molting (Z = -2.201; P = 0.028). The color change from pre-molt (red phase) to post-molt (mostly green phase) was statistically significant (Z = -2.201; P = 0.028). Northeastern Naturalist Vol. 22, No. 2 K.T. Lee and J.L. Vespoli 2015 421 The differences in the pattern of color change in males and females over the course of the summer may be due to the pattern of egg–bearing in females. Females bearing eggs do not molt, but females must molt in order to mate. In the Gulf of Maine, eggs are extruded in late spring (April–May), and peak mating occurs in July–August (Berrill 1982). Hatching occurs from May to July in Europe (Crothers 1967). At our field site with its warmer water, extrusion and peak mating would occur a bit earlier than in the Gulf of Maine, but might be similar to Europe. Egg– bearing females were previously observed at RUMFS in June and July but not in August or September, (K.T. Lee, unpubl. data). The decrease in color in females in July could be explained by increased molting and mating in females. Neither mating females nor molting females would be expected to come to traps (Crothers 1968), so they, mainly red phase, would be under-represented in the captured crabs. The decrease in red coloration seen in captured females in May 2008 might be explained by the time of egg extrusion if some females molt just prior to extrusion or if females do not feed in the days surrounding extrusion. These data are only the beginning of understanding color change in C. maenas. The time in captivity was shorter than planned because of crab mortality. Recapture rates in this study are comparable, albeit on the low side, to other mark– recapture studies in crabs (rates ranging from 2% to 25%; Brousseau et al. 2002, Corgos at al. 2007, Hyland et al. 1984, Yamada et al. 2005), however, the small number of captured crabs makes generalizing the data difficult. None of the crabs captured in 2007 were recaptured in 2008. Where did the crabs go? It is tempting to conclude that the crabs were eaten in increased numbers because of the tags, but casual examination of crab remains left by foraging gulls did not reveal the presence of tags (K.T. Lee, pers. observ.). Perhaps crabs leave the trapping area on a regular basis and are replaced by other crabs. Without long-term studies, these questions cannot be answered. Baited traps, by their nature, sample only actively foraging crabs. Location and density of traps influence capture rates. These data were collected at a single field site and only in summer. What about other types of shorelines or other regions? Only with multi-site, intensive trapping efforts can color change in individual crabs be fully understood. Acknowledgments This study was funded by the Department of Biology, The Natural Sciences Division, and the Office of the Vice President of Academic Affairs at the University of Pittsburgh at Johnstown and by a University of Pittsburgh at Johnstown College Research Council Small Grant. We thank the staff of Rutgers University Marine Field Station for the use of their laboratory facilities and field sites. Literature Cited Able, K.W., D.N. Vivian, G. Petruzzelli, and S.M. Hagan. 2012. 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