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Color phase-specific ion regulation of the European green crab carcinus maenas in an oscillating salinity environment.

ABSTRACT The physiology of ion regulation in the highly invasive European green crab Carcinus maenas has been widely studied, but mostly in constant salinity conditions, and not in context of their molt cycle-dependent sternite coloration. The ventral sternites are typically green after molting, and turn red through prolonged intermolt, with a concurrent decrease in stress tolerance. In this study, whole animal and molecular physiology was evaluated at constant low salinity (12), and oscillating salinity (12-32 every 6 h). Performance in three whole animal measures revealed that the green phase is more tolerant than the red phase, and that females are more tolerant than the respective males under both salinity conditions. These differences result from larger increases in expression of the drivers behind ion transport ([Na.sup.+]/[K.sup.+]-ATPase, cytoplasmic carbonic anhydrase) in green phase and female crabs. Low salinity exposure resulted in increased expression of these markers compared with oscillating salinity, demonstrating that low salinity is more strenuous, as more cellular regulation is required. This agrees with the crabs' natural environmental conditions where prolonged low salinity is rarely experienced. These findings are presented in context of a crab population survey conducted from May 2012 through November 2016 in southern Maine, USA. Female and red phase crabs were found at higher proportions in the intertidal than previously reported. In addition, gravid females were found year-round, which is indicative of continuous reproduction. The data demonstrate the necessity to evaluate C. maenas in an ecologically relevant context with respect to color phase within each invasive population to truly understand the invasive capabilities of this species and to better inform management strategies.

KEY WORDS: oscillating salinity stress, invasive crustacean, population survey, gene expression, Carcinus maenas

INTRODUCTION

The European green crab Carcinus maenas is a Brachyuran decapod crustacean native to Europe and North Africa and is well documented as a globally invasive species (e.g., Darling et al. 2008, Compton et al. 2010, Darling 2011). This species spread to the east coast of the United States and to the Australian continent over 100 years ago, but more recently has continued its global dispersion because of increased shipping traffic, with invasive populations now established on the west coast of the United States reaching up to Vancouver Island as well as in Japan, South Africa, and eastern Canada (Geller et al. 1997, Yamada et al. 2005, Roman 2006, Darling et al. 2008, Compton et al. 2010, Darling 2011). Particular interest has been given to C. maenas throughout both its native and invasive ranges because of its impacts on shellfisheries (Tettelbach 1986, Floyd & Williams 2004, Beal 2006, Watt et al. 2011), eelgrass beds (Davis et al. 1998, Malyshev & Quijon 2011, Garbary et al. 2014), and saltmarshes (Minello et al. 2003, Neckles et al. 2015).

In recent years, an increased focus has emerged on the polymorphic ventral coloration of this species. The ventral sternites are mostly green after molting and transition to yellow, then orange, and finally to red with prolonged intermolt. Studies have focused on the two ends of this color scale, comparing individuals in the green phase to those in the red phase, also described as "color morphs" (Reid & Aldrich 1989, Reid et al. 1989, Kaiser et al. 1990, McGaw & Naylor 1992a, 1992b, Wolf 1998, Styrishave et al. 2004, Farrell & Nelson 2013, Abuhagr et al. 2014). This ventral color transition has been correlated with changes in life strategies, where green phase crabs allocate energy more toward growth and red phase crabs allocate energy more to reproduction. The color transition is also correlated with thicker carapaces and increased chelae forces in red phase crabs, which would be beneficial during competitive mating interactions (Crothers 1967, Kaiser et al. 1990, McGaw et al. 1992, Reid et al. 1997, Styrishave et al. 2004, Lee et al. 2005).

Red and green phase crabs reportedly show different zonation patterns throughout the intertidal. Green phase animals are more frequently distributed higher in the intertidal zone to take advantage of the abundant food supply that helps to maximize their growth, which they achieve because of their more robust physiological tolerances to a variety of environmental conditions. Red phase animals remain in the more stable subtidal zone as they are less physiologically tolerant to environmental fluctuations. In addition, differences in distribution were reported across sex, where females, regardless of color phase, were subtidally distributed as opposed to males, which showed the clear zonation pattern described here (Crothers 1968, Reid & Aldrich 1989, McGaw & Naylor 1992a, 1992b, Hunter & Naylor 1993, Warman et al. 1993, Reid et al. 1997, Styrishave et al. 1999, 2004, McGaw et al. 2011). The majority of these population surveys have focused on the native European population, with few studies conducted in the various invasive populations. Tepolt and Somero (2014) sampled across two invasive populations on the east and west coasts of the United States as well as the native population in Europe and found very different color phase compositions across these populations. In addition, they suggested different phenotypes for temperature tolerance across populations as a result of physiological variations they observed. These differences in population composition and physiology demonstrate the need for surveying within the various invasive populations.

In addition to focusing more on the various invasive populations, attention needs to be paid to both female color phases, as they have been understudied in previous physiological assessments. The need to understand the physiology of female Carcinus maenas is of further importance because of the past global invasions known to be the result of the transportation of adults (Lau 1995, Crawford 1999, Cohen et al. 2001, Aronson et al. 2015). This shortage of physiological information prevents the accurate creation and implementation of management strategies necessary to curb the spread of this species. A recent study examined physiological differences between these two color phases across sex in response to a low salinity environment in an invasive population and observed that females of both color phases show more robust physiological tolerance compared with their male counterparts, which could contribute to the invasive capabilities of this species (Pennoyer et al. 2016).

Many previous studies have assessed salinity tolerance in Carcinus maenas by exposing individuals to a prolonged low salinity environment (McGaw & Naylor 1992b, Henry et al. 2002, Burke et al. 2003, Towle et al. 2011). Whereas this approach has been widely used, it is not a close facsimile to that of a natural tidal environment. This species would rarely be subjected to low salinity exposures that span days or weeks, as these previous studies have implemented, but instead would only be exposed to low salinity for one low tide during heavy rain, a temporary expansion of a freshwater river plume, or other sudden and time-limited freshwater events. The invasive population studied here from southern Maine, is subjected to semidiurnal tidal cycles, where the maximum period of low salinity exposure is approximately 6 h. Therefore, heavy rain events or freshwater river plumes around estuaries could lower the salinity in the intertidal for no longer than 6 h. Without data that accurately depict how salinity fluctuations affect C. maenas, it is difficult to realistically model potential range expansions, as the effects of varying salinity on this species in its natural environment are not fully understood. Monitoring this species' spread is key to prepare for, and potentially prevent, some of the detrimental effects C. maenas has on the environments it invades.

The goals of this study were therefore 2-fold: firstly, to assess the color phase and sex compositions of an invasive population of Carcinus maenas in southern Maine, each month over years; secondly, to evaluate exposure to constant low salinity and oscillating salinity to determine if the previously observed differential tolerance persists under more realistic salinity conditions. This analysis focused on the osmoregulatory mechanisms involving [Na.sup.+]/[K.sup.+]-ATPase (NKA), two isoforms of carbonic anhydrase [cytoplasmic carbonic anhydrase (CAc) and membrane-bound carbonic anhydrase (CAg)], as well as the ion transporters [Na.sup.+]/[K.sup.+]/2CP cotransporter (NK2C1) and the [Na.sup.+]/[H.sup.+] antiporter (NHE) (reviews in Mantel & Farmer 1983, Geck & Heinz 1986, Pequeux 1995, Krarup et al. 1998, Henry et al. 2012, McNamara & Faria 2012). This multifaceted approach allows for the examination of the population structure and intertidal distribution of this invasive population compared with the native European population, and to evaluate if the widely used constant low salinity exposure is truly representative of the natural intertidal environment that this species inhabits, allowing for its continued use in future studies.

MATERIALS AND METHODS

Population Survey

A monthly survey was conducted along a transect through the rocky intertidal in Biddeford Pool, ME, USA (43[degrees] 26' 31.3" N, 70[degrees] 20' 29.4" W). This transect spanned from the high- to the low-water mark and measured 1.80 m wide and 22.5 m long for a total area of 40.5 [m.sup.2]. This size was chosen to be large enough to obtain a representative sample, while not depleting the transect of all animals, as all individuals were removed for measuring. A similar size (40 [m.sup.2]) is recommended by the Massachusetts Aquatic Invasive Species Program and the Marine Invader Monitoring and Information Collaborative and is implemented in more than 60 different sites throughout New England. The monthly surveys were conducted by turning every rock by hand and collecting all Carcinus maenas that had a carapace width of more than 2 cm. This cutoff for size was chosen because it is difficult to accurately sex individuals below this size class, and capturing all of the smallest individuals for a truly representative sample is challenging. Each individual was sexed, carapace width was recorded, and color was assigned ranging from 1 through 10 based on the color index developed by Lee et al. (2005). Animals were categorized as "green" if they were assigned a color between 1 and 3 (Walmart colors summer field 91,274, lemonade 91,254, and sundrop 91,253, respectively), and were categorized as "red" if assigned a color between 8 and 10 (Walmart colors pumpkin 91,171, jungle orange 91,121, and roasted pepper 91,111, respectively). It was also noted if any individual had a visible egg clutch. Temperature of the air, surface ocean water, and in the large tide pool at the bottom of the transect was recorded. Salinity was recorded in the tide pool at the base of the transect.

Animal Collection for Salinity Trials

Adult Carcinus maenas (carapace width 4-5 cm) were collected both by hand in the intertidal in Biddeford Pool, ME (43[degrees] 26' 39.36" N, 70[degrees] 20' 24.59" W), and by trapping in the Scarborough harbor, ME (43[degrees] 32' 42.78" N, 70[degrees] 20' 2.12" W), in May-October 2013 and 2014 when all experiments were conducted (animal collection permits by the State of Maine, Department of Marine Resources, permit numbers: 2012-41-01, 2013-47-03, 2014-46-04, 2015-46-00, 2016-36-00). Animals were sorted for similar size, sex, and based on the color index as described earlier. Only nongravid females were used for salinity exposures. Animals were held at ambient conditions (salinity of 32 and 15[degrees]C-18[degrees]C) in a flow through seawater system in the Marine Science Center at the University of New England for at least one week before use to diminish activity fluctuations due to circatidal rhythms established in their native environment (McGaw & Naylor 1992a). Animals were fed weekly with herring and mussels ad libitum, but all animals were starved for 48 h before experimentation.

Oscillating Salinity Tank

The oscillating salinity tank design used here expanded upon previous tank designs by Bolt and Naylor (1985), and McGaw and Naylor (1992a). This new tank design used a flow through seawater system as a source of natural unfiltered seawater. Freshwater was pumped into a holding tank that was partially submerged in a larger tank containing flow-through seawater to allow for temperature acclimation (15[degrees]C-18[degrees]C) before mixing. Freshwater flow into the holding tank was regulated by an electric water timer (Vigoro, Atlanta, GA), set to fill the tank immediately after drainage to allow for maximum acclimation time. Electric actuated ball valves (Hayward, Elizabeth, NJ) were connected to digital timers (General Electric, Fairfield, CT) to control the flow of seawater and freshwater into the incubation tank beneath the seawater and freshwater tanks, where animals were kept for the duration of the oscillating salinity trials. Water within the incubation tank was mixed using a small aquarium pump and kept aerated throughout each phase of the cycle. This incubation tank was partially submerged in a larger tank filled with flow-through seawater to minimize temperature variations. An actuated ball valve was also used to regulate the drainage of water from the incubation tank to transition from normal salinity to low salinity and vice versa (Fig. 1). To switch from a salinity of 32 to 12 or from 12 to 32, the ball valve beneath the incubation tank opened and drained about 75% of the water without exposing the animals to air. Then, the valves of the holding tanks opened and filled the incubation tank with either 32 salinity seawater or a mix of seawater and freshwater to reach a salinity of 12, respectively. All timers were synchronized to replicate 6 h cycles with salinity ranging from 32 to 12. Salinity in the incubation tank was recorded throughout the experiments.

Experimental Protocol

Two salinity exposures were performed for both color phases and each sex. All salinity exposures were conducted in the summer months to avoid seasonal variability in activity levels for this species. Trials were conducted for each sex separately, consisting of six red and six green phase animals at a time. Constant low and oscillating salinity trials were conducted simultaneously to reduce potential variability between the beginning and the end of summer. All animals were allowed to acclimate to conditions within flow-through holding tanks for at least one week before experimentation. Before the initiation of any experiment, baseline measurements were recorded for each animal and these pooled measurements are indicated in each figure as time point 0 h.

[FIGURE 1 OMITTED]

Oscillating salinity trials were conducted at 15[degrees]C-18[degrees]C in the tank described earlier for 72 h. Whole animal data were collected at 6 h intervals at the end of each normal and low salinity period for the duration of the exposure. To minimize handling stress, multiple oscillating trials were conducted with sampling at different time points to complete this sampling regime and then pooled for analysis. No animal was measured repeatedly within 24 h to match the 24-h sampling regimen of the constant low salinity experiment.

Constant low salinity (salinity of 12) trials were performed in temperature-controlled 100-L incubation tanks at 15[degrees]C-18[degrees]C. The low salinity water in these incubators was pumped from the oscillating salinity tank the day before experimentation began to match the low salinity events during the oscillating salinity trials. Whole animal data were collected at 24-h intervals over the 72-h trial.

At the end of each experiment, the animals were sacrificed by cutting the cerebral ganglion before extraction of the posterior most gill pair for molecular analysis. These gills were used as they are known centers of ion regulation (Pequeux 1995, Krarup et al. 1998, Henry et al. 2012, McNamara & Faria 2012). The gills taken for qPCR were placed in RNAlater solution (Promega, Madison, WI) and stored at 4[degrees]C until processed. Gills for western blots were flash frozen with clamps precooled in liquid nitrogen before storage at -80[degrees]C. For the oscillating salinity trials, gills were collected at the end of the last low salinity event so that conditions are comparable to the constant low salinity samples (66 h). Gills were collected at 72 h for molecular analysis as per the findings of Pennoyer et al. (2016), who observed the largest increase in expression occurring at this time point in low salinity exposures for this invasive population.

Organismal Techniques

Three whole animal parameters were measured at each sampling point during the duration of the 72-h salinity trials. First, a 50 [micro]L hemolymph sample was collected and stored at -20[degrees]C for later analysis with a Wescor Vapro 5520 vapor pressure osmometer (Wescor, Logan, UT) to determine hemolymph osmolality. Next, the animals were tested for righting response where each individual was inverted under water on a flat surface, and the time required for them to right themselves was recorded. Finally, each individual was run on a treadmill underwater, and the time until exhaustion was recorded as a measure of salinity tolerance. Temperature-controlled aerated water was circulated through the treadmill tank during trials and salinity was matched to the two salinity regimes. The treadmill consisted of a motor-driven belt contained within a 19-L acrylic tank with a smaller perforated acrylic box placed around the belt to keep animals centered on the treadmill (see detailed description of the treadmill in Pennoyer et al. 2016). The individuals were run at 20 cm/sec, which is 50% of their maximum speed, determined before the experimentation began. All treadmill trials were ended after 480 sec where any individual still running at that point was counted as 480 sec for calculating means. This maximum was chosen because of the limited time available to perform all whole animal assessments while still placing animals back into the incubation tank before the transition from normal salinity to low salinity, or vice versa, was initiated. Therefore, to prevent salinity shock, a maximum time limit that was not easily accomplished under stressed conditions, but was more easily measured in the time allotted by the oscillating salinity cycle was selected.

Western Blotting

The samples were homogenized in a bullet blender with zirconium oxide beads in ice-cold buffer (in mmol [L.sup.-1]: Tris Base 100, Sucrose 500, NaF 200, NaCl 100, [Na.sub.4][O.sub.7][P.sub.2] 10, sodium orthovanadate 200, [beta]-glycerophosphate 300, with 5 mL/L Sigma protease inhibitor cocktail P8340 containing AEBSF 104, aprotinin 0.08, bestatin hydrochloride 4, E-64 1.4, leupeptin hemisulfate salt 2, pepstatin A 1.5) and protein concentrations were determined spectrophotometrically using Bradford protein assays (Bradford 1976). Protein abundance was quantified by western blotting as described by Frederich et al. (2009) and Jost et al. (2012). Gels were loaded with 45 ng of protein and separated on a 10% SDS gel at 120 V for 60 min. The proteins were transferred to a nitrocellulose membrane for 2 h at 70 V. The membranes were blocked with 3% dry milk in TBS solution and then tested for the respective target proteins using the antibodies primary mouse monoclonal anti-actin IgM, mouse monoclonal anti-creatine kinase IgGl, mouse monoclonal anti-[Na.sup.+]-[K.sup.+]-2[Cl.sup.-]-cotransporter IgGl (all DSHB, Iowa City, IA), rabbit polyclonal anti-AMPK[alpha]1/2 H-300 IgG (Santa Cruz Biotechnology, Inc., Dallas, TX), rabbit polyclonal anti-phospho-AMPK [alpha] Thr-172 IgG (EMD Millipore, Billerica, MA), mouse anti-HSP70 IgGl (SIGMA, St. Louis, MO), and secondary goat anti-mouse IgG or donkey anti-rabbit IgG (LI-COR Biosciences, Lincoln, NE) to visualize the proteins. Actin antibodies were used as a loading control and pre-stained standards (BIO-RAD Kaleidoscope, Hercules, CA) were used as the size standard. All blots were imaged with a LI-COR Odyssey imager (LI-COR, Lincoln, NE) before quantification with ImageJ software (NIH, Bethesda, MD). All western blots were run with control samples and samples from each experimental group to allow for comparisons across blots. The samples were normalized to the red control groups for each sex in addition to the actin normalization.

The two different antibodies for AMP-activated protein kinase (AMPK) used here are to quantify total AMPK protein levels (anti-AMPK [alpha]1/2 H-300) and AMPK. activity (anti-phospho-AMPK [alpha] Thr-172). The anti-phospho-AMPK [alpha] Thr-172 antibody specifically targets phosphorylated AMPK, which is commonly used as a measure of activity levels (Frederich & Balschi 2002, Bartrons et al. 2004, Mulligan et al. 2007, Frederich et al. 2009, Goodchild et al. 2016, Pennoyer et al. 2016).

Quantitative Real-Time PCR

The posterior most pair of gills from each individual were extracted and homogenized in liquid nitrogen for total RNA isolation via a phenol chloroform extraction, including a DNAse treatment step, according to the manufacturer's guidelines (Promega RNAgents, Madison, WI). Purity of the extracted RNA was measured with a 190-840 nm spectrum on a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Newington, NH), specifically focusing on the 260/280 nm ratio and a potential phenol contamination at 230 nm. A total RNA mass of 1.8 [micro]g was then reverse transcribed to cDNA using the Invitrogen Superscript III First-Strand synthesis system for qPCR (Thermo Fisher Scientific, Grand Island, NY). The cDNA was amplified with the appropriate primer pairs from Pennoyer et al. (2016) for each target [Carcinus maenas [Na.sup.+]/[K.sup.+]-ATPase alpha subunit mRNA GenBank: AY035550.1; C. maenas [Na.sup.+]-[K.sup.+]-2CL-cotransporter mRNA GenBank: AY035548.1; C. maenas sodium/hydrogen exchanger mRNA GenBank: U09274.1; C. maenas arginine kinase (AK) mRNA GenBank: AF167313.1; C. maenas partial hsp70 gene for heat shock protein 70 GenBank: AM 116767.1; C. maenas cytoplasmic carbonic anhydrase mRNA GenBank: EU273943.1; C. maenas glycosyl-phosphatidylinositol-linked carbonic anhydrase mRNA GenBank: EU273944.1; Cancer irroratus AMP-activated protein kinase alpha subunit mRNA GenBank: FJ496868.1] using the Brilliant II SYBR Green qPCR Master Mix (Agilent Technologies, Santa Clara, CA). The PCR reaction was carried out on a Stratagene Mx3005p instrument (Agilent Technologies, Santa Clara, CA) and used 35 cycles with 1 min each for denaturation (92[degrees]C), annealing, and amplification (72[degrees]C). The annealing temperatures were 55[degrees]C for AMPK, heat shock protein (HSP), and AK; 54[degrees]C for NKA, NK2C1, and NHE; and 53.5[degrees]C for CAc and CAg. A melting curve analysis after each run confirmed that only one DNA product was amplified. Before the actual analyses, the qPCR products were sequenced and identified by BLAST analysis as the respective target. All data were normalized to the red control group for each sex. Alien qRT-PCR Inhibitor Alert (Agilent Technologies, Santa Clara, CA) was included as a non-endogenous RNA transcript for normalization. The Alien qRT-PCR Inhibitor Alert is a system designed for both detecting inhibitory compounds present in a sample and for overall assay normalization procedures. This system was chosen because of the differential PCR inhibition between the two color phases, where red phase animals show increased inhibition compared with green phase animals (K. Pennoyer, A. Himes, M. Frederich, unpublished observation). The samples where inhibition occurred were excluded and PCR data were normalized to the AlienRNA transcript as reported in previous studies (Huggett et al. 2005, Goodchild et al. 2016, Pennoyer et al. 2016). This method reduces the widely documented intersample variability that can occur by using a conventional reference gene such as actin (for example, Schmittgen & Zakrajsek 2000, Selvey et al. 2001, Bas et al. 2004, Olsvik et al. 2005, Sellars et al. 2007, Tong et al. 2009) and is more reliable than using a reference gene that is known to have at least a several-fold increase as is the case for both actin (Lovett et al. 2003, Henry et al. 2006, Serrano et al. 2007, Serrano & Henry 2008, Mitchell and Henry 2014) and AK (Towle & Weihrauch 2001, Luquet et al. 2005, Serrano et al. 2007. Pennoyer et al. 2016) in response to low salinity.

Statistics

Whole animal, western blot, and qPCR data were analyzed for significant differences with GraphPad Prism version 6 (San Diego, CA) and R version 3.1.1 (Vienna, Austria). Significant interaction terms within the full multifactor ANOVA model were investigated using one-way or two-way ANOVA based on the respective dataset. A Sidak post hoc test was conducted to evaluate orthogonal contrasts of interest while reducing the type I error rate. All tests were run with an [alpha]-level of 0.05. Statistical significance is denoted in all figures using upper case letters for the green phase and lower case letters for the red phase. Different letters indicate significant differences between points, and the same letter indicates no significant difference. Asterisks are used to indicate significant differences between color phases.

RESULTS

Population Survey

Surveys were conducted monthly from May 2012 through November 2016. Average crab density was 1.1 [+ or -]0.76 crabs per square meter but showed seasonal variation from 0.2 crabs/[m.sup.2 ]in winter to 3.8 crabs/[m.sup.2] in summer (Fig. 2A). These densities correlate with high and low water and air temperatures (Fig. 2B). Salinity remained constant between 30 and 32 in the tide pool at the base of the transect. As animals were collected under rocks in small puddles, buried in sand, and running over rocks, measuring salinity within the transect was unfeasible.

Overall, green phase animals were more abundant than red phase individuals for 72% of the collections (Fig. 2C), and females were more abundant than males 74.4% of the time (Fig. 2D). Nearly all months where more males were captured than females occurred in winter when overall abundance was low. The green phase was evenly distributed across sex, 55% of all green phase animals caught were male and 45% were female. Conversely, of all red phase animals caught, 83% were female.

The percentage of red females collected for each month was calculated along with the percentage of gravid females (Fig. 2E). Both metrics follow very similar trends, peaking in summer and decreasing in winter. Gravid females were still found at low levels in winter. From September to March, there were occasional months without any gravid crabs found, but October was the only month in which no gravid females were ever found in the 5 y of surveys. Red phase females were found all year long, except during the months where the total collection was very low.

Organismal Responses

Righting Response

At constant low salinity, there was no significant difference in righting time between red and green color phases for either females (Fig. 3B) or males (Fig. 3E). No differences were detected within sex, but green phase males had a significantly longer overall righting time than green females [3.5 [+ or -] 4.3 versus 1.5 [+ or -] 0.5 sec; ANOVA, F(l,37) = 4.996, P = 0.0315].

During oscillating salinity conditions, there was a significant difference between the two female color phases [ANOVA, F(1123) = 31.28, P < 0.0001; Fig. 4B], In particular, there was a large difference between the two female color phases at 60 h (ANOVA, P = 0.0006) where red phase females showed a large increase in righting time. There was no change within the green phase females over the course of the trial, but red phase females showed a significant increase over time [ANOVA, F(11,59) = 2.422, P = 0.0146]. The male color phases (Fig. 4E) followed similar trends, where a significant difference between the two color phases was detected [ANOVA, F(1129) = 51.77, P< 0.0001]. Green phase males differed from females in that a slight, yet significant, increase was detected by 72 h [ANOVA, F(11,66) = 2.087, P = 0.0334]. Red phase males showed no change over the duration of the trial.

Treadmill Endurance

The effects of low salinity exposure on treadmill endurance were similar for both sexes. Both female color phases showed a decreasing trend in endurance over the course of the trial (Fig. 3C) where green phase females appear to have higher endurance than red phase females, but this was not significant for either phase. Both male phases showed similar decreasing trends that were also not significant (Fig. 3F). A significant difference between the two male color phases was observed [ANOVA, F(l,32) = 11.40, P = 0.0019].

The results from the oscillating salinity condition were more variable and partially fluctuated with the oscillations in water salinity. For females (Fig. 4C), a significant difference between each color phase was detected [ANOVA, F(1121) = 28.96, P < 0.0001]. Specifically, green phase females showed significantly higher endurance than red phase females at 18 h (ANOVA, P = 0.0007) and 42 h (P = 0.0089), which are both low salinity time points in the oscillating cycle. A significant decrease in endurance was observed in green phase females [ANOVA, F(11,58) = 2.639, P = 0.0083], but green phase females never decreased to the same levels as their red counterparts, which showed a decreasing trend over the course of the trial as well. Similar trends were observed in both male color phases (Fig. 4F), where green phase males showed significantly higher endurance than red phase males over the duration of the salinity exposure [ANOVA, F(1124) = 55.97, P < 0.0001]. Like green females, green phase males showed a significant decrease in performance over the course of the trial [ANOVA, F(11,59) = 2.126, P = 0.032], which was also observed in red phase males [ANOVA, F( 11,65) = 2.478, P = 0.0116]. Of particular interest here is that green phase male treadmill performance patterns fall in line with the oscillations in salinity. These morphs showed higher mean endurance levels at 12, 24, 36, 48, 60, and 72 h, which are normal salinity time points, with decreases in endurance observed at the low salinity time points between each of these respective points.

Hemolymph Osmolality

Hemolymph osmolality results for the low salinity treatment were similar across sex. Both female phases (Fig. 3D) showed a significant decrease at 24 h [ANOVA, green: F(3,20) = 104.1, P < 0.0001, red: F(3,20) = 57.63, P < 0.0001], which then stabilized and remained constant for the duration of the experiment. A significant difference between the color phases was detected after this initial decrease at each time point (ANOVA, 24 h: P < 0.0001, 48 h: P < 0.0001, 72 h: P < 0.0001). These same trends were observed in males (Fig. 3G), where a significant decrease in osmolality was detected in both phases at 24 h [ANOVA, green: F(3,19) = 138.9, P< 0.0001, red: F(3,13) = 183.5, P < 0.0001]. This decrease resulted in significantly higher osmolality in green phase males at 24 h (ANOVA, P < 0.0001), 48 h (P = 0.0006), and 72 h (P = 0.001) compared with red phase males. Interestingly, green phase females had significantly higher osmolality than green phase males at both the initial time point (ANOVA, P = 0.0002) and all three points after the low salinity transfer (ANOVA 24 h:, P < 0.0001, 48 h: P < 0.0001, 72 h: P< 0.0001). Red phase females were also found to have significantly higher osmolality levels than red phase males at the three time points post transfer (ANOVA, 24 h: P = 0.0162, 48 h: P = 0.0339, 72 h: P = 0.0188), but no difference was found at 0 h.

[FIGURE 2 OMITTED]

Hemolymph osmolality results from the oscillating salinity trials closely follow the salinity fluctuations for each sex. Both female color phases (Fig. 4D) had similar osmolalities at 0 h and showed significant decreases in osmolality from 0 to 6 h (ANOVA, green: P < 0.0001, red: P < 0.0001). At 6 h, however, green phase females had significantly higher osmolality than red phase females (P < 0.0001). Both phases then showed increases in hemolymph osmolality at 12 h, back to the same levels as those observed at 0 h. This trend continued for the remainder of the trial where both phases showed decreases at low salinity time points, but red phase females showed significantly larger decreases, before both phases returned to similar osmolality levels at the normal salinity time points. Male color phases (Fig. 4G) showed the same significant trends as females. Both color phases began the trial with similar hemolymph osmolalities that significantly decreased at each successive low salinity event before returning to previous levels at the next normal salinity time point. At each of these low salinity periods, red phase males exhibited significantly larger decreases in osmolality compared with green phase males. When comparing across sex, similar differences were found in osmolality as those for the low salinity exposure where both female phases showed significantly higher osmolalities than male phases [ANOVA, green: F(1151) = 61.91, P< 0.0001, red: F(1156) = 28.97, P< 0.0001].

Protein Abundance in Gill

Significant differences were detected in females for AK and HSP70, two proteins involved in general cellular stress responses (Fig. 5). In particular, green phase females exposed to constant low salinity showed increased expression levels of AK compared with either the control group (ANOVA, P = 0.0038) or the oscillating salinity group (ANOVA, P = 0.0295), with no difference detected between the control and the oscillating groups. A similar trend was observed in green phase females for HSP70, where exposure to low salinity resulted in significantly higher expression levels compared with the control (ANOVA, P = 0.0405), but no differences were found compared with the oscillating salinity group or between the control and oscillating groups. No significant changes were detected in NK2C1 or AMPK activity for either salinity condition.

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

[FIGURE 7 OMITTED]

No significant changes were detected between male color phases (Fig. 6) exposed to each salinity condition for any of the proteins evaluated here. More variability was observed among the male groups, but few increasing or decreasing trends were seen. No significant differences were found across sex.

Gene Expression in Gill

Females of both color phases in each salinity condition showed a large increase in gene expression for the cytoplasmic form of CAc (between 5.5 and 90.3-fold; Fig. 7). Green phase females subjected to low salinity showed significantly higher gene expression of CAc than the control group (ANOVA, P = 0.0348). No significant differences were detected between the green low salinity group and the green oscillating salinity group or between the oscillating group and the control. Red phase females mirrored these trends in upregulation. Although they were not significant, the observed increases in expression were smaller compared with green phase females across each treatment. Whereas the upregulation was not as large, similar trends across the groups were detected for CAg and NKA, although these were not significant. No significant changes were found in AMPK, HSP70, NK2C1, NHE, or AK between the two color phases or salinity conditions.

More significant differences were found within the male color phases (Fig. 8). Like their female counterparts, males showed the largest increase in gene expression in CAc, where green phase males under low salinity conditions showed higher expression levels than both the control (ANOVA, P = 0.0004) and the oscillating salinity group (ANOVA, P = 0.003). Whereas no significant difference was found between the control and oscillating groups, the oscillating group did show an apparent increase in CAc expression over the control. Green phase males exposed to low salinity were found to upregulate CAc significantly more than red phase males under the same condition (ANOVA, P = 0.0013). Green phase males showed a larger increase in gene expression than red morphs for CAg (ANOVA, P = 0.0302), similar to the trends observed in females. Unique to the male color phases is the significant difference in AMPK where green phase males showed more upregulation than red phase males under the oscillating salinity condition (ANOVA, P = 0.0364). The only difference detected across sex was that red phase females exposed to low salinity show significantly larger expression of NKA than red phase males (ANOVA, P = 0.0461). The observed lack of significant differences between the sexes as well as the lack of overall significant trends within each sex may be because of the elevated variation present within both the male and female datasets.

[FIGURE 8 OMITTED]

DISCUSSION

Population Survey

Previous population surveys conducted throughout the native population of Carcinus maenas have observed clear trends in the distribution of the two different color phases studied here as well as across sex. Specifically, it is widely stated that green phase individuals dominate the intertidal, and that the majority of these intertidal green phase crabs are male, whereas red phase individuals of either sex are subtidally distributed. In addition, males were described to be more abundant in the intertidal, with females more frequently observed in the subtidal regardless of color phase (Crothers 1967, Reid & Aldrich 1989, McGaw & Naylor 1992a, Hunter & Naylor 1993, Warman et al. 1993, Reid et al. 1997, Styrishave et al. 1999, McKnight et al. 2000, Styrishave & Andersen 2000, McGaw et al. 2011). In contrast to these previous findings, the results reported here show an even distribution of green phase animals across sex in the intertidal with red phase crabs of both sexes present in the intertidal throughout the year and at nearly the same abundances as green phase crabs during summer months. It is important to note that the majority of the red phase crabs collected in this study were females, and that all of the gravid females collected here were red, supporting the interpretation that red phase crabs are in a reproductive phase. The abundance of females collected in the intertidal zone in this study, regardless of color phase, is in stark contrast to previous studies conducted throughout the native range of this species (McGaw & Naylor 1992a, Hunter & Naylor 1993, Warman et al. 1993, Styrishave & Andersen 2000). The results of this study show that females were more abundant than males 74.4% of the time, and that males were only more abundant than females during winter when overall abundances were low. The supposed subtidal distribution of females has been tied to the reproductive strategies of this species where gravid females remain in the subtidal because the adult in this weaker physiological state or the egg clutch carried by the female cannot withstand the environmental variation in the intertidal (Crothers 1967, Warman et al. 1993, Pequeux 1995, Reid et al. 1997, Bravo et al. 2007). Based on the findings here, however, there must be more factors involved as to why gravid females in the native population avoid the intertidal, potentially because of native predators, but are prevalent throughout the intertidal in the invasive population along the coast of the eastern United States (Hunter & Naylor 1993, Reid et al. 1997).

The survey results presented here question the assumption that females mate only once a year after molting, generally between May and October (Crothers 1968, Reid et al. 1997), because gravid females were consistently collected from December through August, with the highest number of gravid females collected in the summer months. The only month where gravid females were never observed was October. With such a large time window in which gravid females are present in the intertidal, it seems unlikely that females only mate once per year in a synchronized mating season, but in fact reproduce year-round. These findings for females are in contrast to the published native population survey data, but follow the nutritional results of Styrishave and Andersen (2000), who found that female Carcinus maenas have increased fatty acid requirements compared with males to facilitate egg development. Females would be able to maximize their fatty acid intake by exploiting the more abundant food supply of the intertidal zone.

The survey results from this study are in agreement with the findings for the European population in regard to the seasonal migration out of the intertidal. Carcinus maenas is reported to move offshore when water temperatures drop below 8[degrees]C in winter and then return as the water warms in spring (Crothers 1968, McGaw & Naylor 1992a, Hunter & Naylor 1993, Warman et al. 1993, Reid et al. 1997). A similar drop in abundance was observed here in the fall as temperatures declined, and an increase in abundance was observed in late spring as water temperatures begin to warm.

Many previous surveys of Carcinus maenas relied either partially or entirely on baited traps for collecting animals, but this method has been shown to bias collections toward larger individuals in crustaceans (Miller 1990, Ihde et al. 2006). In addition to this potential size bias, several studies have shown that red morph males outcompete similarly sized green morph males for food, which could bias baited trap collections with respect to color phase (Kaiser et al. 1990, Reid et al. 1997, Styrishave et al. 2004). Therefore, the collection method selected for this study was to turn rocks by hand within the intertidal and collect all C. maenas within the specified transect to get the most accurate depiction of the sex and color phase composition for this invasive population. This collection method also alleviated any issue with the previously observed seasonal variations in activity for C. maenas, as decreased activity levels during winter months would reduce interest in baited traps (Crothers 1968, Aagaard et al. 1995, Styrishave et al. 1999).

As a whole, these similarities and differences between the native population and the well-established invasive population along the coast of the eastern United States demonstrate the need for specific surveys in each invasive population to truly understand the dynamics within these different regions. Recently, differences across the populations were illustrated by Tepolt and Somero (2014), where sampling across the native European populations as well as the invasive populations on the east and west coast of the United States yielded very different color phase ratios. These differences could help explain the narrower reproductive period in the European population as it was found to have a larger percentage of green phase crabs, known to be in a growth phase, compared with the population studied here.

Salinity Exposures

The results from the whole animal assessments support previous observations that green phase crabs are physiologically more resilient than red phase crabs when exposed to low salinity (Reid et al. 1989, McGaw & Naylor 1992b, Lee et al. 2003). This difference was observed in all whole animal parameters where righting response showed that red phase crabs were consistently slower than green phase crabs, treadmill endurance showed green phase crabs had higher endurance than red phase crabs, and hemolymph osmolality showed that green phase crabs maintained higher osmolality than red phase crabs regardless of sex or salinity condition for any of these parameters. Nevertheless, fluctuations in treadmill endurance and hemolymph osmolality in relation to the oscillations in water salinity were observed for both color phases across sex, with green phase crabs exhibiting less fluctuation than red phase crabs. Although these fluctuations occurred, osmolality levels in both color phases never dropped to the levels observed in the low salinity exposure, contributing to the conclusion that low salinity is the more taxing of the two salinity conditions.

This difference between the salinity conditions is supported by the molecular results reported in this study that revealed significantly increased gene expression in both sexes as a result of low salinity exposure compared with either the oscillating salinity condition or the normal salinity control. Whereas oscillating salinity exposure resulted in increasing trends in upregulation for osmoregulatory genes, they were not significant and were never as large as those observed in the low salinity exposure. In particular, the observed increases were largest for CAc, CAg, and NKA, which are the enzymes involved in the generation and maintenance of the ion gradient that drives ion transport within the cell. This upregulation supports previous studies that have also observed increased gene expression in these key ion-regulating enzymes (Henry et al. 2006, Serrano & Henry 2008, Stillman et al. 2008, Towle et al. 2011, Havird et al. 2013, Pennoyer et al. 2016). Only slight changes in the gene expression of the ion transporters NHE and the NK2CI were observed, which is in agreement with previous reports (Pequeux 1995, Henry et al. 2006, 2012, McNamara & Faria 2012). No changes were observed in the regulation of HSP70 or A MPK, which supports the hypothesis that this common stress response mechanism does not play a major role in salinity tolerance in this species (Stillman et al. 2008, Towle et al. 2011).

Interestingly, green phase crabs also showed increased gene expression of the routinely used reference gene AK (Kotlyar et al. 2000, Towle & Weihrauch 2001, Henry et al. 2003, 2006) compared with red phase crabs across all salinity conditions tested, although the results were not significant. This could potentially skew expression results if used for normalization purposes in future color phase studies. This increase in gene expression was followed by a significant increase in protein levels in green phase females, revealing that AK may actively contribute to the low salinity response in this euryhaline crustacean, similarly to how it functions in other osmoregulators (Kotlyar et al. 2000). Through its function as a catalyst for the hydrolysis of phosphoarginine to yield ATP, AK could contribute to the energetic requirement for the active transport of ions during osmoregulation (Towle & Weihrauch 2001, Holt & Kinsey 2002). This additional energy pathway in the cell could be beneficial to organisms in a taxing environment as energy must be allocated for the maintenance of cellular functions and the respective stress response, while also reducing energy allocations to growth and reproduction (Sokolova et al. 2012). The ability of green phase crabs to more easily activate this additional energy-producing pathway compared with red phase crabs could aid in the increased whole animal performance observed in this study, as they would be able to osmoregulate more efficiently. Aside from this increase in AK abundance, the only other significant change detected in protein abundance was an increase in green phase females for HSP70, which was not accompanied by a significant increase in gene expression and is contrary to previous findings that HSP70 does not function in osmoregulation (Stillman et al. 2008, Towle et al. 2011).

The observed differential expression of these osmoregulatory genes across color phase explains in part how green phase crabs are better able to tolerate low salinity and outperform red phase crabs in the whole animal assessments evaluated here. Also contributing to this variable ion regulatory capability is the differing water permeability in the gills that has been noted across the two color phases as the result of the accumulation of a biofilm over the surface of the gill epithelium as intermolt duration increases (Legeay & Massabuau 2000). The accumulation of this biofilm, however, has been shown to affect the invasive and native populations in different ways. Pennoyer et al. (2016) observed that red phase crabs from the same invasive population as studied here did not exhibit the reduced respiration rates of those reported for the native population (Reid & Aldrich 1989, Reid et al. 1997, Legeay & Massabuau 2000, Rivera-lngraham et al. 2016). Therefore, it is not fully understood what significance this accumulation plays in the variation noted between the two color phases. It seems unlikely that this biofilm is the only factor contributing to the described variations between the color phases as there are no known differences in gill morphology between the sexes that would account for the differences between the male and female color phases observed here.

The differences between the commonly studied low salinity condition and the oscillating salinity condition, more closely resembling the natural fluctuations of an intertidal environment, frame the widely studied osmoregulatory mechanism in a more natural setting. Whereas previous studies employing constant low salinity exposures have clearly demonstrated that significant upregulation occurs in NKA, CAg, and CAc, these studies have consistently found that upregulation of these genes does not occur immediately after low salinity exposure, but in fact occurs after 24 h of exposure. This lag in upregulation is followed by a further lag in the activation of the translated gene product (Luquet et al. 2005, Mitchell & Henry 2014). With the upregulation of the osmoregulatory mechanism requiring such a long time period, it is unlikely that this would be actively involved in the low salinity exposures routinely experienced by this species from daily tidal fluctuations or local weather events. This is supported by the oscillating salinity gene expression results that exhibit lower expression than the constant low salinity results in this study. Some studies suggest that the lack of upregulation observed in the oscillating salinity experiments could be a sign that the routine cellular mechanisms of Carcinus maenas are sufficient enough to cope with the relatively short period of environmental challenge they experience and therefore further upregulation is unnecessary (Henry et al. 2003, Mitchell & Henry 2014). Another possible explanation to these differences is that there is a large constitutively expressed cellular pool of the key osmoregulatory enzymes that make it unnecessary for C. maenas to upregulate these mechanisms unless the exposure is prolonged, as in the constant low salinity condition. These findings and alternative explanations emphasize the need to assess invasive species in a more natural setting to fully understand their physiological capabilities and the implications for future range expansions.

Sex Comparison

Differences between color phases were observed within both sexes, but variations were also found between males and females. Female crabs had significantly higher hemolymph osmolality than their male counterparts under both low and oscillating salinity conditions. Females also demonstrated increased expression compared with males for the key enzymes involved in osmoregulation, potentially explaining their enhanced ability to maintain internal ionic concentrations. The finding that female phase crabs outperform male phase crabs is in agreement with previous low salinity work by Pennoyer et al. (2016), and alters the general understanding of the physiological changes throughout the molt cycle for this species.

It is currently accepted that green phase crabs are in a growth phase that occurs after molting and red phase crabs are in a reproductive phase, leading to a prolonged intermolt period because of increased energy allocation for gamete production (Crothers 1968, Reid & Aldrich 1989, Styrishave et al. 1999). This follows for males as prolonged intermolt results in increased carapace thickness and chelae strength, which allows them to better compete for mates (Kaiser et al. 1990, Reid et al. 1997, Styrishave et al. 2004). Whereas this period provides a clear advantage for males, its purpose in females is less clear. This weaker physiological state in females could contribute to egg survival, as it is known that adults can tolerate more environmental variability compared with their eggs; however, the true purpose of this phase in females is not fully understood (Pequeux 1995, Bravo et al. 2007, Compton et al. 2010, Pennoyer et al. 2016).

CONCLUSIONS

The differential osmoregulatory capabilities observed here across color phase and sex in both the low salinity and the more ecologically relevant oscillating salinity conditions illustrate the necessity for more focused studies to fully understand the physiology of this highly invasive species to therefore better inform management strategies. In particular, the understanding of the osmoregulatory mechanism vital to the low salinity survival of Carcinus maenas may need to be reevaluated as it was determined here that upregulation of the genes necessary for osmoregulation are significantly lower in both color phases exposed to the more realistic oscillating salinity condition. When these differences are framed in context of the observed seasonal variations in the population throughout the year, they provide useful information for future management programs. Whereas C. maenas are robustly tolerant to environmental conditions as a whole, there are times of the year when the lesstolerant red phase crabs are more abundant, in late spring and early summer, indicating that the population as a whole is more vulnerable to external challenges. This variability in the resiliency of the population as a whole could support potential management strategies that seek to curb the spread of this invasive species, as these seasonal fluctuations in physiological tolerance provide a clear window as to when the implementation of management strategies would be most successful.

ACKNOWLEDGMENTS

The authors thank Shaun Gill for his expertise in building and maintaining the treadmill and oscillating salinity tank. This project was funded in part by National Science Foundation grant IOB-0640478 to M. F. and an American Physiological Society summer fellowship (UGSRF) to A. H.

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ANTHONY R. HIMES, ([dagger]) WINGCUE S. BALSCHI, GWENDOLYN PELLETIER AND MARKUS FREDERICH (*)

Department of Marine Sciences, University of New England, Biddeford, ME 04005

(*) Corresponding author. E-mail: mfrederich@une.edu

([dagger]) Current address: Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, PA 19129.
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Author:Himes, Anthony R.; Balschi, Wingcue S.; Pelletier, Gwendolyn; Frederich, Markus
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:1U1ME
Date:Aug 1, 2017
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