Invasive investigation: uptake and transport of l-leucine in the gill epithelium of crustaceans

Branchial amino acid uptake is evaluated in the invasive Carcinus maenas and compared to native Canadian crustaceans. l-leucine transport and accumulation throughout the body of the green crab is also evaluated in various gill and organ tissues in fed and fasted states.


Introduction
Native to northeast Atlantic waters and the Baltic Sea, the European green crab (Carcinus maenas) is now found in coastal waters worldwide, including the Pacific and Atlantic coasts of Canada.Primary observations of the C. maenas' arrival on Pacific North American coasts were in 1989 in San Francisco Bay, CA, spreading 300 km north to Coos Bay, OR, by 1997, and quickly spreading to Vancouver Island, BC, by 1998 (Jamieson et al., 1998).The expansion of this invasive population has since resulted in severe ecological impacts on Canadian native species and the surrounding ecosystem (Grosholz et al., 2000;Matheson et al., 2016).Despite their more recent introduction on the Canadian Pacific coast, juvenile C. maenas outcompete juvenile Dungeness crab (Metacarcinus magister) for habitat and food resulting in population decline and over $6 million in losses per year to the British Columbia decapod fishery (Colautti et al., 2006).Comparatively, the well-established Atlantic population of C. maenas displaces the American lobster (Homarus americanus), resulting in a commercial loss of $44 to 114 million per year (Colautti et al., 2006).Predation and hunting practices of C. maenas are also particularly destructive to the native ecosystem.Canadian bivalve populations experience reduced recruitment and survival rates due to significant predation, with bivalve populations exhibiting 5-to 10-fold decrease in the presence of C. maenas (Grosholz et al., 2000;Tan and Beal, 2015;Ens et al., 2022).While scavenging for bivalves, C. maenas also causes significant damage to Eelgrass (Zostera spp.) meadows that many estuarine organisms rely on for food and shelter.In Atlantic Canada, eelgrass meadows have experienced biomass declines between 50% and 100% when C. maenas invades their ecosystem (Ens et al., 2022).In an attempt to preserve native species in the Canadian Pacific, researchers are often focused on slowing the growth of existing C. maenas populations to mitigate spread.
While some mitigation efforts have focused on the utilization of chemical agents, such as carbaryl and lindane to target invasive C. maenas populations, these agents are nonspecific and inadvertently target native arthropods (Hanks, 1961;Iliff et al., 2019;Ens et al., 2022).As a result, current mitigation efforts largely rely on manual trapping, with focus on early detection and preemptive capture.Monitoring programs often attempt to pinpoint trapping areas of particular risk to C. maenas invasion, evaluating their habitat preference and physiological parameters to native organisms.In order to accurately predict the spread of C. maenas, it is vital to understand all factors that may influence the survival of both C. maenas and native organisms.However, when evaluating the interactions between native and invasive crustacean species, the role of waterborne amino acids on survival has been largely overlooked.Total amino acid concentrations generally range from 0.1 to 1 μM in the open ocean and increase to 2 μM or greater in estuaries, surrounding marine organisms in free dissolved nutrients (Stephens, 1968;Stephens, 1988).l-leucine in particular has been measured at approximately 8% of the total combined amino acid concentrations in the Pacific surface waters (Bada et al., 1982).Notably, many marine invertebrates are capable of absorbing amino acids from the environment at concentrations as low as 50 nM, which can represent a significant source of their total nutrients (Wright, 1982;Preston, 1993).However, arthropods were historically excluded from this group due to the perceived impermeability of their exoskeleton (Preston, 1993;Chan et al., 2019).Indeed, it was not until recently that the gills of arthropods were found to be able to extract free amino acids (i.e.l-leucine) from the aqueous environment similarly to other marine invertebrates (Blewett and Goss, 2017).
The gills are an ideal transport organ given their direct exposure to the environment, large surface area, and relatively thin membrane for ion transport, in conjunction with high rates of haemolymph perfusion (Henry et al., 2012).The physiological roles of marine crustacean gills are separated into anterior (i.e.gills 2-5) and posterior gills (i.e.pairs 6-9), functioning primarily for respiration or ionoregulation, respectively (Towle et al., 1997;Freire et al., 2008).Research suggests that there are likely two transport pathways for l-leucine across the gill epithelium of C. maenas that are concentration-dependent and display both high and low affinity for this amino acid (Blewett and Goss, 2017).However, it is currently unknown if this ability extends to other species of crustaceans, how environmental stimuli can impact branchial transport, and the destination of transported amino acids within the crustacean body.
The green crab readily acclimates to a variety of environments, including fluctuating estuarine environments (Havel et al., 2015), making it a globally successful invasive crustacean.These crabs can tightly regulate osmolytes and in fact, amino acids are known to play an important role in salinity tolerance and survival (Abe et al., 1999).Thus, we sought to determine if the ability of C. maenas to transport nutrients across the gill epithelium was unique to this species and in turn a contributing factor to their survival and invasive success.To this end, we determined the extent to which other native crustaceans (e.g.Metacarcinus gracilis (graceful crab), Cancer productus (red rock crab), and Metacarcinus magister (Dungeness crab)), of the Pacific can transport amino acids, such as l-leucine across the gill epithelium in comparison to C. maenas.We hypothesized that arthropod species native to the Pacific coast of Canada would be able to transport lleucine across the gill epithelium alongside C. maenas due to their shared gill structures, but with a lower capacity to do so, given C. maenas' increased ability to regulate internal osmolytes (Leignel et al., 2014).To address this hypothesis, we characterized waterborne amino acid transport using gill perfusion techniques to elucidate the transport rate of lleucine in four crustaceans' species, evaluated the influence of feeding events on branchial amino acid transport using both recently fed and fasted crustaceans, and performed whole body bioaccumulation exposures in C. maenas to evaluate waterborne amino acid transport and dispersion throughout crustacean gills and organs.

Animal collection
Invasive adult male C. maenas weighing on average 101 ± 16.5 g and adult male native crab species (M.gracilis  M. magister 528 ± 116 g) were collected in accordance with Fisheries and Oceans Canada license XR 1352020, off the western side of Vancouver Island in Barkley Sound (N49802.274-W125820.710and N49801.749-W125821.515).Captured animals were transported to the Bamfield Marine Science Centre (Bamfield, BC).Animals were held in various meshcovered outdoor 2000 L tanks, on a flow through system of filtered seawater pumped from the ocean (10.6 ± 0.4 • C, 31.5 ± 1.3 ppt).Animals were subjected to natural summer lighting (∼15 h light, ∼ 9 h dark) and fed salmon to satiety every 3 days.A minimum of 7 days prior to experimentation, animals were transported into indoor carbon filtered tanks, fasted, and held at 31.7 ± 0.2 ppt at 17.7 ± 0.8 • C. All collection and holding was performed in accordance with University of Alberta, Bamfield Marine Sciences Centre, and Canadian Council on Animal Care guidelines.

Gill perfusions
Gill perfusion experiments were performed in accordance with the methods described by Siebers et al. (1985) and Blewett and Goss (2017).Briefly, crabs were euthanized via a single spike to the ventral ganglion following 15 minutes on ice, allowing for anesthetization.Posterior gills (7-9), or anterior gill 5 were dissected for perfusion; however, only one gill per individual was used per treatment to avoid pseudoreplication.A range of PE tubing 80 to 210 (Intramedic, Clay Adams) was used based upon the gill size of each crab, and inserted into the afferent and efferent vessels of the gill, and the basal end of the gill was tightly clipped, creating a closed system.In addition to previous work with inulin perfusions (Blewett and Goss, 2017), red dye was perfused through preliminary test gills to ensure no leaks were found within gill clamps, indicating a closed system was being achieved prior to experimental perfusions.Gill perfusion was performed using an artificial hemolymph at 100% SW (saltwater) osmolarity (Table 1), (composition in mM: 470 NaCl, 12 CaCl 2 , 12 MgCl 2 , 11 KCl, 9 NaHCO 3 , 0.1 NH 4 Cl, 0.3 glucose, 0.1 glutathione, 0.5 glutamine, mOsm = 954 ± 2.6) at a rate of approximately 127 ± 1.7 μL/min using a 12-channel peristaltic pump (Fisherbrand FH100M; Fisher Scientific, USA).Gills were then placed in 50 mL of bathing solution comprised of seawater (32 ± 0.2 ppt, 17.7 ± 0.8 • C) spiked with 14 C radiolabelled l-leucine (Perkin Elmer, Boston, MA), at concentrations of 0, 1, 10, 100, and 1000 μM and perfused for 1 h, based on previous work by Blewett and Goss (2017).Each treatment was replicated between three and seven times, depending on individual species gill availability.
In order to compare the influence of feeding on the branchial uptake of l-leucine, C. maenas, M. gracilis and M. magister, were fed salmon tissue to satiety in the hour leading up to euthanization.Posterior gills were then dissected and perfused in 2 μM radiolabelled l-leucine following the procedure above.The influence of feeding on the branchial uptake of l-leucine was not evaluated in C. productus as a result of limited animal availability.Each treatment was replicated between four and six times, depending on individual species gill availability.
A 1-mL subsample of perfusate was collected at the end of each treatment for radioisotope analysis and mixed with 4 mL of Optiphase (Perkin Elmer, MA) liquid scintillant.Individual beta emissions were measured via counts per minute (CPM) of each sample using a TriCarb 29 000 TR liquid Scintillation Analyzer.Gills were weighed and amino acid transport rates were then calculated for each gill (see Calculations and Data Analysis).All "zeros" were considered true zeros as gills were perfused without radioisotope and individual beta emissions were recorded at background levels.

Whole-body exposures
l-leucine was made from a concentrated l-leucine stock of 10 mM (Sigma Aldrich), and added to the seawater 24 h before experimentation to equilibrate.Thirty minutes before the start of the experiment, 1 μCi −1 of 14 C radiolabelled l-leucine (Perkin Elmer, Boston, MA) was added to each exposure chamber for a final concentration in the chambers of 300 μM l-leucine.Water samples of 1 mL were taken at t = 0 and t = 24 to monitor radioisotope concentration.Carcinus maenas were starved for 4 days prior to exposure to whole animal experiments.Five individual crabs were then randomly placed into the containers and allowed to rest for 24 h in the aerated mixture.Upon termination of the exposure, crabs were washed (1 min) in a highly concentrated nonradiolabelled l-leucine wash (10 mM) for displacement of any radiolabelled leucine that was loosely bound to the outside of the animal, they were then washed in 100% seawater for 1 min, and immediately following that, crabs were placed on ice for 15 mins and euthanized as stated above.Once crabs were euthanized, they were dissected and the following organs were weighed and digested as described below for radioisotope analysis: gill 5 (G5), gill 8 (G8), stomach (ST), hepatopancreas (HP), eyestalk (ES), muscle (MU), carapace (CA) and haemolymph (HL).
In order to compare the impacts of feeding on l-leucine accumulation in the gills of C. maenas, the procedure above  A one-way ANOVA was conducted to compare the branchial uptake rate (nmol g-1 h-1) of species at each environmental concentration.p ≤ 0.05 was considered significant.Bars within concentration groups that share lettering are not considered statistically different, while bars that do not share lettering are considered statistically significant.
was also conducted using C. maenas that had been fed to satiety 1 h prior to experimental exposure.Five individual fasted crabs and five individual fed crabs were then randomly placed into the containers and allowed to rest for 24 h in the aerated mixture.Once crabs were euthanized, they were dissected and gills 2 to 9 (G2-G9) were weighed and digested as described below for radioisotope analysis.

Tissue and water analysis
Tissues were weighed and collected in 20 mL scintillation vials and digested with 2N HNO 3 (trace metal grade nitric acid, Sigma Aldrich) at the three to five times (exact volume recorded) the weight of the tissue.All vials were sealed and placed in an incubator at 65 • C for 48 h, with vortexing after 24 h.Scintillation fluor was then added to the scintillation vials (Ultima Gold, Perkin Elmer, Waltham, MA) in a 5:1 ratio of volume to tissue and assayed for radioactive beta-emission counts on a TriCarb 29000 TR liquid Scintillation Analyzer.Samples were standardized to a common counting efficiency (quench corrected), using a quench curve constructed from tissue digests.Water samples (1 mL) were treated as above, where Optiphase (Perkin Elmer, MA) liquid fluor was added at a volume of 2:1 ratio of volume to water and run for emission counts on the Liquid Scintillation Analyzer above.T-tests were conducted to compare the branchial uptake rate (nmol g-1 h-1) of each species in their fed and fasted states.p ≤ 0.05 was considered significant.Bars that do not share lettering display statistically significant fed and fasted values.Bars without lettering are considered to show no significant differences between fed and fasted uptake.

Calculations and data analysis
To calculate amino acid uptake, measured perfusate or tissue radioactivity (CPM) was divided by the specific activity (SA) of the exposure solution.To account for the variable gill sizes among species, amino acid concentration was converted into nmol g −1 based on individual gill weights.Finally, amino acid uptake (nmol g −1 h −1 ) or tissue accumulation (nmol g −1 h −1 ) was determined by accounting for total perfusion time in hours (t): Outliers were identified and removed using Grubbs' test, and l-leucine and concentration-dependent uptake was evaluated and plotted using SigmaPlot Version 11.0 (Systat Software, San Jose, CA).Uptake at each given amino acid concentration (μM) versus branchial transport rate (nmol g −1 h −1 ) for the four arthropod species at high and low environment concentrations were plotted on raw data using Sigmaplot Version 11.0.
All statistical analyses were performed in Sigmaplot Version 11.0.ANOVA and Holm-Sidak post hoc tests were conducted to identify any significant differences in transport rate between species at four environmental concentrations (Figure 1), and to evaluate differences in 24-h l-leucine accumulation in various C. maenas organs (Figure 3).Normality was ensured and t tests were performed to evaluate differences between fed and fasted l-leucine transport rates in each species (Figure 2).Whole body feeding state and gill number data were transformed in Sigmaplot 11.0.using the log transform function to achieve normality, a two-way ANOVA was then conducted to evaluate the influence of feeding, gill number, and the interaction between feeding and gill number on 24-h gill accumulation of l-leucine in C. maenas (Figure 4).

Metacarcinus gracilis
higher branchial uptake of l-leucine immediately after feeding, increasing from a fasted value of 1.19 ± 0.278 nmol•g −1 •h −1 to 3.40 ± 0.853 nmol•g −1 •h −1 , and 0.48 ± 0.23 nmol•g −1 •h −1 to 2.00 ± 0.28 nmol•g −1 •h −1 , respectively (Figure 2).While C. maenas did not exhibit a significant difference (p = 0.054) between fed and fasted gills, a general trend can be seen increasing an order of magnitude from fasted gills with a transport rate of 0.132 ± 0.025 nmol•g −1 •h −1 to fed gills with a transport rate of 1.37 ± 0.85 nmol Notably, throughout C. maenas whole-body exposures gill 5 and gill 8 showed significantly higher (p < 0.001, df = 43, f = 19.5)uptake of l-leucine when compared with all other organs, with total accumulation rate at 4.15 ± 0.78 nmol•g −1 •h −1 and 2.90 ± 0.47 nmol•g −1 •h −1 , respectively (Figure 3).No other significant differences (p > 0.05) are noted between the stomach (7.92 When evaluating how feeding state and gill number influence branchial l-leucine accumulation in C. maenas, no significant interaction was found between the fed state and gill number (p = 0.4).Likewise, no significant differences were seen between gill number and l-leucine accumulation in either fed or fasted gills (p > 0.05).

Discussion
Marine invertebrates utilize waterborne amino acids in various physiological purposes, such as osmoregulation and nutrient uptake, increasing their survival and optimal habitat range (Wright, 1982).Of the four arthropod species tested, all exhibited the ability to not only acquire amino acids over their gills, but to do so at rates akin to that of wellstudied filter feeders (Stephens and Schinske, 1961;Rice et al., 1980).This study compared the ability of three native Pacific arthropod species, Metacarcinus gracilis, Cancer productus, and Metacarcinus magister, to transport l-leucine across their gill epithelia, to that of the invasive green crab (Carcinus maenas).It was hypothesized that, given C. maenas' ability to acquire l-leucine from the environment, native Pacific arthropods would also share this ability and transport l-leucine across their gill epithelium but at a lower rate of transport than C. maenas.Our hypothesis was partially supported in that l-leucine was transported across both the anterior and posterior gill membranes of all four species.The noted uptake suggests that M. gracilis, C. productus, M. magister and C. maenas have the ability to actively transport l-leucine from the environment through their gills.However, C. maenas was only noted to have an increased capacity for transport over M. magister at high environmental concentrations of 100 and 1000 μM, showing a lower transport rate than M. magister at 10 μM and was otherwise comparable to the Pacific native species.Notably, after being transported from the environment, amino acids were shown to remain largely in the gill, with lower concentrations noted in the haemolymph and throughout the organs.Branchial amino acid transport rates were also shown to increase in C. maenas, M. gracilis and M. magister immediately following a feeding event.

Branchial transport among species
Epithelial nutrient uptake in arthropods was previously thought to be an impossible process due to their hard and relatively impermeable exoskeleton (Preston, 1993;Chan et al., 2019).However, Blewett and Goss (2017) provided evidence for branchial uptake of l-leucine in the invasive green crab at environmentally relevant amino acid concentrations.Notably, our results show that branchial amino acid uptake is not only displayed in a single arthropod species, but likely a shared trait among all marine crustaceans.M. gracilis, C. productus, M. magister, as well as C. maenas all display an active transport pathway for the uptake of free It was hypothesized that C. maenas would exhibit a higher capacity for transport than native species given the green crabs' increased ability to regulate internal osmolarity; however, we did not observe this.At high concentrations of lleucine (100 μM-1000 μM), C. maenas exhibited higher rates of transport compared to two of the three native species (i.e.M. gracilis and M. magister) (Figure 1).Interestingly, the invasive C. maenas shows significantly lower branchial amino acid transport when compared to M. magister at environmental concentrations of 10 μM.This suggests that the branchial uptake of l-leucine from the environment in C. maenas offers little competitive advantage over Canadian native crustaceans at ambient environmental amino acid concentrations.It was also previously noted that a low concentration (0-10 μM) pathway could offer greater physiological significance to arthropods based on normal environmental amino acid concentrations than the higher 100 to 1000 μM treatments (Blewett and Goss, 2017).Given that average estuarine amino acid concentrations are generally in the 2 μM range, a significantly higher rate of amino acid transport in C. maenas at environmental concentrations approaching 100 μM would rarely be utilized under normal environmental conditions (Stephens, 1968;Stephens, 1988).This further suggests that the invasive green crab would likely have no competitive advantage with respect to the branchial uptake rate of waterborne l-leucine when compared to the native Pacific species at common environmental amino acid concentrations.However, in isolated environmental instances that result in amino acids at concentrations approaching 100 μM and above (see below), C. maenas may have a competitive advantage over M. gracilis and M. magister.

Feeding events and branchial transport
Throughout this study, we also sought to examine the influence of feeding events on branchial uptake of waterborne amino acids in order to understand the nutritional role of branchial amino acid uptake.Often, increased transport rates are indicative of an increased expression of transporters after an external stimulant.Interestingly, M. gracilis and M. magister both exhibited significant increases in branchial l-leucine uptake immediately after a feeding event (Figure 2).Similarly, even in the absence in statistical significance, a nearly 10fold increase in branchial uptake can also be observed in C. maenas immediately after a feeding event (Figure 2).This suggests that the branchial uptake of waterborne amino acids in crustaceans is heavily influenced by a feeding event, displaying uptake rates ranging from nearly 3-to 10-fold that of fasted uptake.Comparatively, in perhaps the most notorious example of postprandial regulation of nutrient transport, the Burmese python shows a 5.7-fold increase in l-leucine uptake to maximize nutrient acquisition during times of feeding, and conserve digestive energy while fasting (Cox and Secor, 2008).In many vertebrates, including the Burmese python (Secor et al., 2001), cholecystokinin/gastriclike peptides (CCK/gastric) increase after ingestion and act as regulatory peptides in the gastrointestinal endocrine system, stimulating pancreatic enzyme secretions (Larson and Vigna, 1983).Similarly, CCK/gastric peptides are found within the gastrointestinal system of C. maenas and other crustaceans, and in turn are shown to stimulate the release of digestive enzymes by the hepatopancreas in the freshwater crustacean O. limosus (Larson and Vigna, 1983;Resch-Sedlmeier and Sedlmeier, 1999).While the exact endocrine response pathway remains unknown, we propose that upon ingestion, the release of CCK/gastric peptides or other similar hormones into the haemolymph not only stimulates the release of digestive enzymes in the hepatopancreas but also induces the increased expression of transport proteins on the gill epithelia.The authors also suggest that during a feeding event, the creation of a localized "feeding cloud" could lead to a significantly higher environmental (>100 μM) concentration of waterborne amino acids, leeching out of the given food source and creating a microclimate in which upregulating branchial nutrient uptake could be extremely advantageous.Notably, postprandial oxygen consumption in crustaceans increases between 1.5-and 3-fold compared to resting levels, resulting in a high rate of energy production and consumption, increasing nutrient transport throughout the body (McGaw and Curtis, 2013).In conjunction with the creation of a feeding cloud, an increase in branchial amino acid transport rate would allow for the increased uptake of nutrients that would otherwise be lost to the surrounding environment; however, further research is required to confirm this theory.

Uptake specificity and accumulation
While waterborne amino acid uptake has been studied in 10 different marine invertebrate taxa, now including four arthropod species, the identification of amino acid transporters in marine invertebrates relies heavily on mammalian models (Stephens, 1988;Glover et al., 2011;Katayama et al., 2016;Blewett and Goss, 2017).As such, little is known about the exact transporters present on invertebrate gill epithelia; however, most mechanistic evidence suggests a combination of SLC6 family Na + /AA cotransporters on the basolateral and apical membrane, allowing amino acid transport across the gill epithelia (Wright, 1987;Applebaum et al., 2013;Blewett and Goss, 2017).It has been widely described that the epithelia of the posterior (i.e.6-9) and anterior gills (i.e.2-5) of euryhaline crabs are specialized for unique primary purposes (Towle and Weihrauch, 2001;Freire et al., 2008;Blewett and Goss, 2017).While the anterior gills are believed to serve a mainly respiratory function owing to their relatively thin epithelia, the posterior gills are characterized by a thicker, mitochondrially rich epithelium (Freire et al., 2008).The increased expression of mitochondria provides energy in the form of ATP to power the Na + /K + ATPase, driving an increased electrochemical gradient in the cell, thus creating the Na + /AA co-transport capacity of the posterior gills (Towle and Weihrauch, 2001).However, contrary to existing  (Blewett and Goss, 2017), no significant differences were exhibited between the anterior and posterior transport of l-leucine in C. maenas (Figure 3), suggesting that regardless of the noted physiological differences, amino acid transport pathways appear uniform across all gills.This lack of gill specificity across physiologically different tissues could be explained in part by the presence of multiple unique amino acid pathways across epithelial surfaces, similar to marine echinoderms which exhibit 11 specific amino acid SLC6 family cotransporters throughout their epithelium and tube feet regardless of tissue specificity (Applebaum et al., 2013).It is, therefore, challenging to draw a conclusive statement regarding the specificity of anterior and posterior C. maenas gills and their role in the transport of waterborne amino acids without further investigation.
Understanding the ability of individual gills to transport amino acids from the environment into the epithelia cells offers valuable information regarding the accumulation of waterborne amino acids in the body of marine crustaceans.However, gill transport rates offer little evidence regarding the dispersion and utilization of amino acids throughout the crustacean body.The tissue-specific distribution of transported l-leucine throughout the body of C. maenas was therefore also evaluated to understand the whole-body distribution of accumulated amino acids.Generally, marine organisms maintain their internal amino acid concentration at 10 3 to 10 6 fold higher than their external environment (Clark, 1968;Stevens and Preston, 1980).As suggested above, amino acids are transported across the gill epithelia into the haemolymph via Na + /AA cotransporters on the apical and basolateral membrane, before being transported throughout the body via the circulatory system.Therefore, as expected, waterborne amino acids were transported through gills 5 and 8 into the haemolymph of C. maenas, and further dispersed into the stomach, hepatopancreas, eyestalk, muscle tissue, carapace, and heart of C. maenas.While both gills 5 and 8 exhibit significantly higher accumulation of l-leucine compared to all other organs and the haemolymph, this could be due in part to l-leucine that was actively being acquired and sequestered in the epithelial cells at the end of the exposure period.Comparably, Kucukgulmez and Celik (2008) found no distinct differences in l-leucine accumulation between various muscle tissues and the hepatopancreas of the blue crab (C.sapidus), indicating little specificity between l-leucine accumulation and unique organ tissues similar to the results of this study.After the gills, the highest rate of accumulation throughout the body can be seen in the haemolymph.This elevated level of free amino acids within the circulatory system of C. maenas after a 24-h exposure period suggests that the primary utilization of l-leucine within the green crab, may not be protein synthesis.In C. maenas, protein synthesis is known to return to fasted levels within 16 h of a single meal, peaking at a rate nearly four times that of fasted crustaceans (Carter and Mente, 2014).Carter and Mente (2014) also note no difference in protein synthesis rates in the gill, heart, leg, and claw of unfed and continually fed C. maenas.Thus, the metabolic fate of branchial acquired amino acids may largely depend on feeding regimes, suggesting that after a feeding event and increase in metabolic rate, l-leucine could serve as secondary nutrient source for protein synthesis.However, given the large retention of l-leucine in the gills and haemolymph in periods of fasting, in combination with research suggesting low protein conversion efficiency, and a maximum of 40% amino acids utilization for protein synthesis (Mente et al., 2010), we suggest a primarily osmoregulatory function of branchial amino acid uptake.
It is therefore possible that these amino acids are being utilized as an osmolyte throughout the body, not unlike the osmoregulatory strategy of the Japanese mitten crab (Eriocheir japonicus) and many marine bivalves (Abe et al., 1999;Toyohara et al., 2005).The Japanese mitten crab exhibits increased retention of amino acids, such as lalanine, in muscle tissue when exposed to increasing salinity (>32 ppt), using free l-alanine as an osmolyte in muscle tissues to regulate osmolarity in an estuarine environment (Abe et al., 1999b).Similarly, the red swamp crayfish (Procambarus clarkia) exhibits a 5.4-fold increase in amino acid accumulation in response to increasing salinity in both muscle tissue and the hepatopancreas (Fujimori and Abe, 2002).Likewise, giant Pacific oysters (Crassostrea gigas) and the blue mussel M. galloprovincialis exhibit increased amino acid transporter expression in the gill epithelia, adductor muscle and kidney in response to both hypoosmotic and hyperosmotic stress (Yamauchi et al., 1992;Toyohara et al., 2005).This could suggest that the utilization of amino acids occurs throughout the body of arthropods and marine invertebrates, not only for protein synthesis but also as an osmolyte and key component in the osmoregulatory capabilities of crustacean species (Abe et al., 1999).Further research is required to conclusively identify the utilization of branchial amino acid uptake in organ tissue and haemolymph of arthropods, as well as arthropods' ability to transport amino acids in varying environmental salinities.

Environmental conclusions
In order to conserve the diversity and abundance of native species, it is crucial to understand any and all physiological factors that influence species interactions and resultant survival.Throughout this study, we sought to expand our understanding of marine crustacean ion transport processes, and more importantly, gain insight into a potential key factor in the invasive success of the globally dominant C. maenas.The invasive green crab is a known euryhaline species with survivable salinity ranging from 4 to 54 ppt (Leignel et al., 2014).This is particularly interesting when compared to the native Pacific crustaceans Metacarcinus gracilis, Metacarcinus magister, and Cancer productus, which all possess a lower ability to regulate internal ions.M. magister in particular displays a moderate ability for osmoregulation, surviving prolonged periods in salinity ranging from 12 to 35 ppt (Cleaver, 1949;Sugarman et al., 1983).In contrast, M. gracilis and C. productus have weak osmoregulatory abilities, unable to withstand extended periods in salinities lower than approximately 18 to 20 ppt and 13 to 16 ppt, respectively (Selby, 1980;Carroll and Winn, 1989;Curtis et al., 2007).With a high thermotolerance (0-35 • C), aggressive nature, and extensive thermal tolerance, C. maenas leaves no surprise surrounding its ability to thrive in vastly different coastal waters around the world (Leignel et al., 2014).While the invasive green crab displayed the highest capacity for uptake of l-leucine when compared with Canadian native species at high amino acid concentrations, it showed no advantage at environmental levels.However, in the presence of a feeding cloud, or localized area of high amino acid concentrations, the competitive ability of C. maenas could be greatly increased.l-leucine was determined to accumulate throughout all tested tissues within the body of C. maenas, suggesting widespread utilization.While the exact utilization of these amino acids remains unknown, a deeper understanding of branchial transport could improve clarity regarding success of crustacean species in variable habitats and offer insight into the future spread of C. maenas.

Figure 1 :
Figure 1: l-leucine transport in the posterior gills (7-9) of Carcinus maenas, Metacarcinus gracilis, Metacarcinus magister, and Cancer productus at waterborne l-leucine concentrations of (A) 1 μM, (B) 10 μM, (C) 100 μM, and (D) 1000 μM determined via gill perfusion.Values represent mean ± s.e.m (n = 4-7).A one-way ANOVA was conducted to compare the branchial uptake rate (nmol g-1 h-1) of species at each environmental concentration.p ≤ 0.05 was considered significant.Bars within concentration groups that share lettering are not considered statistically different, while bars that do not share lettering are considered statistically significant.

Figure 2 :
Figure 2: Fed vs Fasted branchial uptake of 2 μM l-leucine in the posterior gills (7-9) of Carcinus maenas, Metacarcinus gracilis, and Metacarcinus magister.Bar values represent mean ± s.e.m (n = 4-5).T-tests were conducted to compare the branchial uptake rate (nmol g-1 h-1) of each species in their fed and fasted states.p ≤ 0.05 was considered significant.Bars that do not share lettering display statistically significant fed and fasted values.Bars without lettering are considered to show no significant differences between fed and fasted uptake.

Figure 4 :
Figure 4: Gill accumulation of 2 μM l-leucine in the anterior gills (G2-G5) and posterior gills (G6-G9) of Carcinus maenas in both fasted and fed states.Values represent mean ± s.e.m (n = 4-5).A two-way ANOVA was conducted to compare the accumulation of l-leucine (nmol g-1 h-1) between gill number, and the influence of fed state.p ≤ 0.05 was considered significant.Data was transformed (Log10) in Sigmaplot 11.0 to achieve normality.No significant differences were seen in relation to gill number and l-leucine accumulation.Bars that do not share lettering display statistically significant l-leucine accumulation values between fed and fasted states.Bars without lettering are not considered statistically different between fed and fasted states.