Comparative Biochemistry and Physiology, Part C 151 (2010) 222–228

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Comparative Biochemistry and Physiology, Part C

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Survival, osmoregulation and ammonia-N excretion of blue swimmer crab, Portunuspelagicus, juveniles exposed to different ammonia-N and salinity combinations

Nicholas Romano ⁎, Chaoshu ZengSchool of Marine and Tropical Biology, James Cook University, Townsville, Qld 4811, Australia

⁎ Corresponding author. Tel.: +61 07 47256482; fax:E-mail address: Nicholas.Romano@jcu.edu.au (N. Ro

1532-0456/$ – see front matter © 2009 Elsevier Inc. Aldoi:10.1016/j.cbpc.2009.10.011

a b s t r a c t

a r t i c l e i n f o

Article history:Received 30 August 2009Received in revised form 28 October 2009Accepted 28 October 2009Available online 2 November 2009

Keywords:Portunus pelagicusAmmoniaSalinityNa+/K+-ATPaseHaemolymph osmolalityAmmonia-N excretion

Ammonia-N toxicity to early Portunus pelagicus juveniles at different salinities was investigated alongwith changes to haemolymph osmolality, Na+, K+, Ca2+ and ammonia-N levels, ammonia-N excretion andgill Na+/K+-ATPase activity. Experimental crabs were acclimated to salinities 15, 30 and 45‰ for one weekand 25 replicate crabs were subsequently exposed to 0, 20, 40, 60, 80, 100 and 120 mg L−1 ammonia-N for96-h, respectively. High ammonia-N concentrations were used to determine LC50 values while physiologicalmeasurements were conducted at lower concentrations. When crabs were exposed to ammonia-N, anteriorgill Na+/K+-ATPase activity significantly increased (pb0.05) at all salinities, while this only occurred on theposterior gills at 30‰. For crabs exposed to 20 and 40 mg L−1 ammonia-N, both posterior gill Na+/K+-ATPase activity and ammonia-N excretion were significantly higher at 15‰ than those at 45‰. Despitethis trend, the 96-h LC50 value at 15‰ (43.4 mg L−1) was significantly lower (pb0.05) than at both 30‰ and45‰ (65.8 and 75.2 mg L−1, respectively). This may be due to significantly higher (pb0.05) haemolymphammonia-N levels of crabs at low salinities and may similarly explain the general ammonia-N toxicitypattern to other crustacean species.

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1. Introduction

Ammonia is often an ecologically relevant nutrient in aquatic eco-systems receiving excessive anthropogenic discharges from varioussources, including agricultural run-off, sewage and untreated landfillleachate (Dave and Nilsson, 2005; Sigleo and Frick, 2007). Among thethree major nitrogenous compounds of ammonia, nitrite and nitrate,ammonia-N is generally the most toxic (Meade and Watts, 1995;Romano and Zeng, 2007a,b) with ammonia-N levels of 7 mg L−1

potentially influencing the abundance and distribution of crabs (Rebeloet al., 1999). Even in pristine aquatic systems crustaceans that bury forprolonged periods may experience elevated levels of localized ammo-nia-N due to their continual excretion of metabolic by-products(Weihrauch et al., 1999). Furthermore, within ocean sediments thatserve as habitats for benthic crustaceans, ammonia-N levels as high as39.2 mg L−1 have been reported (Weihrauch et al., 1999).

Since benthic crustaceans may encounter ammonia-N in nature,active ammonia-N excretion against a gradientwas developed to counterexcessive ammonia-N influx to the haemolymph (Weihrauch et al.,2004). Such a mechanism, which is closely linked to osmoregulation, issuggested to be a two-step process (Weihrauch et al., 1999). Based onpast experiments, the currentmodel suggests thatNH4

+ can substitute forK+ via basolaterally located Na+/K+-ATPase activity, and NH4

+ is then

excreted to the environment via an apically located Na+/NH4+ transpor-

ters (reviewed by Weihrauch et al., 2004). This likely explains theincreased gill Na+/K+-ATPase activity of crustaceans exposed to elevatedammonia-N levels in vivo (Chen andNan, 1992;Wang et al., 2003) and invitro (Furriel et al., 2004; Masui et al., 2002, 2005; Garçon et al., 2007).However, excessively high ammonia-N levels may disrupt this process(Chen and Nan, 1992;Wang et al., 2003), which was suggested to be thecause of significantly reduced haemolymph osmolality and/or haemo-lymph Na+ ions of crustaceans (Young-Lai et al., 1991; Chen and Chia,1996; Harris et al., 2001; Romano and Zeng, 2007c).

In addition to ammonia potentially altering the gill Na+/K+-ATPaseactivity of crustaceans, salinity is also well known to influence thisresponse. Typically at low salinities, gill Na+/K+-ATPase activity ofestuarine or marine crustaceans increases (Holliday, 1985; Piller et al.,1995; Castilho et al., 2001; López-Mañanes et al., 2002; Genovese et al,.2004; Torres et al,. 2007; Lucu et al., 2008) to enhance the rate ofhaemolymph Na+ and Cl− uptake from the environment. In contrast, gillNa+/K+-ATPase activity at high salinities is more varied, includingincreases (Kamemoto, 1991; Holliday et al., 1990; McLaughlin et al.,1996), no change (Genovese et al., 2004, Chung and Lin, 2006; Lucu et al.,2008) or decreases (Castilho et al., 2001; Torres et al., 2007). Thesedifferent findings at high salinities indicate highly species-specificresponses as well as suggesting that Na+/K+-ATPase activity may beinvolved in hypo-osmoregulation (McLaughlin et al., 1996; Lucu andTowle, 2003).

Estuarine systems may routinely present environmental challengesto crustaceans due to wide salinity fluctuations (de Lestang et al., 2003)

223N. Romano, C. Zeng / Comparative Biochemistry and Physiology, Part C 151 (2010) 222–228

while the potential to encounter elevated ammonia-N levels is alsogreater due to anthropogenic discharges and/or burying in sedimentswith high organic contents (Weihrauch et al., 1999; Dave and Nilsson,2005). This is likely to be relevant to early juvenile blue swimmer crabs,Portunus pelagicus, due to their wide distribution and commercialimportance throughout the Indo-Pacific regionwhere the early juvenilelife stages commonly inhabit estuarine systems for months (de Lestanget al., 2003).

While evidence indicates that the mechanisms to cope withelevated ammonia-N and extreme salinity levels in crustaceans arelinked, the acute ammonia-N tolerance of penaeid shrimps decreasewith decreasing salinities (Chen and Lin, 1991; Chen and Lin, 1992;Lin and Chen, 2001; Kir and Kumlu, 2006; Li et al., 2007). This promptsinteresting questions as to how crabs respond and adapt whensubjected to varied salinity and ammonia-N combinations particularlysince it has been previously demonstrated that early P. pelagicusjuveniles are weak osmoregulators (Romano and Zeng, 2006) with ahigh ammonia-N tolerance (Romano and Zeng, 2007d). However, itappears no investigators have yet attempted to link gill Na+/K+-ATPase activity, ammonia-N excretion and haemolymph ammonia-Nlevels from the same crustacean species when simultaneouslysubjected to various ammonia-N and salinity levels. The aim of thecurrent experiment was to hence compare the acute toxicity ofammonia-N to early P. pelagicus juveniles at different salinities andtheir associated responses, including haemolymph osmolality, hae-molymph Na+, K+, Ca2+ and ammonia-N levels along with ammonia-N excretion rates and gill Na+/K+-ATPase activity.

2. Materials and methods

2.1. Source of experimental crabs and acclimation procedure

The crabs used for the present experiments were cultured fromnewly hatched larvae in the laboratory as described by Romano andZeng (2006). Briefly, mature broodstock crabs were caught using baitedtraps from estuaries in the Townsville region of north Queensland,Australia. The females were kept in outdoor recirculating systems untilspawned. Prior to hatching, the berried female was transferred to anindoor 300-L tank with a salinity of 34±1‰. Upon hatching the larvaewere stocked at approximately 500 individuals L−1 in five indoor 300-Ltanks at a salinity of 25±1‰ and temperature of 29±1 °C. Larvaewereinitially fed rotifers (Branchionus sp.) with daily additions of microalgae(Nannochloropsis sp.). By the zoea II stage, Artemia sp. nauplii (INVE,AAA) were hatched added daily to feed the larvae until they settled tofirst staged crabs.

Upon settlement, the juvenile crabs were transferred to 4 outdoor1000-L recirculating tanks and the salinity, temperature and totalammonia-N of the water was 34±2‰, 27±2 °C and 0.01 mg L−1,respectively. To reduced cannibalism, numerous coral rock, PVC pipesand nets were provided to serve as shelters. The crabs were fed frozenArtemia for the first two days, while formulated pellet feeds (Ridley)designed for the penaeid shrimp, Penaeus monodon, were alsointroduced for weaning onto pelleted feeds from day 3 onwards.The crabs were fed daily to satiation and each day the tanks weresiphoned to remove uneaten food and faeces. Three weeks aftersettlement, the crabs were randomly selected and individuallytransferred into black plastic containers (diameter 16 cm×height19 cm). Each container had numerous 3.75 mm holes to facilitatewater exchange and all containers were placedwithin 6 static outdoor1000-L oval tanks (two tanks for each salinity level). The salinity wasthen stepwise changed at a rate of 3‰ h−1 until the desired levels of15‰, 30‰ and 45‰ were reached. The salinities of 15‰ and 30‰were created via additions of de-chlorinated freshwater to the sourceseawater of 32‰. The salinity of 45‰ was made by additions of brineto the source seawater. Brine was previously prepared by filling

indoor tanks with seawater and evaporation was accelerated throughstrong aeration and submersible heaters.

During the acclimation period, the crabs were daily fed to satiationusing the pellet feeds (Ridley). For the first 2 days a 10% waterexchangewas performed, however, when the feeding of early juvenileP. pelagicus increased substantially after the first 2 days of acclimationthe water exchange was then increased to 80% for the following5 days to ensure that ammonia-N levels never exceeded 1 mg L−1.After the one week acclimation period, the crabs were transferredindoors for the commencement of the experiment.

2.2. Test solution preparation

A stock solution of 10,000 mg L−1 nitrogen, in the form ofammonia-N, was created by dissolving 38.2 g of NH4Cl (analyticalgrade) to 1-L of distilled water. This stock solution was then dilutedwith pre-adjusted seawater at salinities of 15‰, 30‰ and 45‰, tocreate 7 ammonia-N concentrations of 0 (control with no ammonia-Nadded), 20, 40, 60, 80, 100 and 120 mg L−1.

The seawater used for the experiments had a salinity level of 36‰.To create the desired salinity levels of 15‰ and 30‰, de-chlorinatedfreshwater was used, while brine was added to create a salinity levelof 45‰. The source seawater had an ammonia, nitrite and nitrate levelof b0.01 mg L−1. The pH of all test solutions was maintained at 8.1through the addition of sodium hydroxide (NaOH) pellets.

2.3. Experimental design and set-up

A total of 21 treatments were set up (3 salinities×7 ammonia-Nlevels). Each treatment consisted of 25 replicate crabs (525 crabs intotal) with an initial wet body weight of 2.51±0.21 g. Each crab wasindividually keptwithin a 5-L capacity plastic containerfilledwith 4-L ofthe desired test solution. All containers were organized in a randomblock design and kept within a 1000-L freshwater bath. The freshwaterbath was kept at 28±0.5 °C through air-conditioning and the use ofsubmersible heaters.

Each day a 100% water exchange was performed according to the“static renewal method” described by American Public Health Associ-ation (1985).Mortality observationsweremadeat 12-h intervals for 96-h. Death was assumed when the crab exhibited no movement orresponse after mechanical stimulation.

2.4. Osmolality, Na+, K+ and Ca2+ and ammonia-N levels of the culturewater and haemolymph

After 96-h of exposure, the surviving crabs at the intermolt stage(n=9 to 15) were determined according to Chen and Chia (1997).These crabs were sampled for measurements of haemolymph osmolal-ity, haemolymph Na+, K+ and Ca2+ levels as well as haemolymphammonia-N levels (n=4). To obtain the haemolymph, a syringe wasinserted through theproximal anthropodalmembrane at the base of thesecondwalking leg of the crabs towithdraw thehaemolymph. Aportionof the haemolymph (50 μL) was immediately analyzed on a cryoscopicosmometer (Gonotec) for osmolality measurements while 20 μL ofhaemolymph was immediately diluted with distilled water (2 mL) andanalyzed on a flame photometer (Sherwood, Cambridge, UK) for totalNa+, K+ and Ca2+ levels. Meanwhile for the measurements ofhaemolymph ammonia-N, the haemolymph samples were dilutedwith distilled water, immediately frozen at −20 °C and measured forhaemolymph ammonia-N at the Australian Centre for TropicalFreshwater Research within 2 days using the Nesslerization method(4500 NH3-G) according to APHA (1989).

Ammonia-N excretion rates of the crabs were measured over a 24-h interval (started at 72-h and ended at 96-h). At 72-h of theexperiment, immediately after the containers received a 100% waterexchange, newly replaced water was sampled from 3 replicate

Table 1The 96-h LC50 ammonia-N and NH3 values (mg L−1) (and the 95% confidence intervalsin parenthesizes) of early Portunus pelagicus juveniles subjected to 15, 30 and 45‰.

Salinity (‰) 96-h LC50 ammonia-N value(mg L−1)

96-h LC50 NH3 value(mg L−1)

15 43.4 (37.8–48.2) 2.9 (2.5–3.3)30 65.8 (60.1–70.9) 4.3 (4.0–4.7)45 75.2 (69.4–80.7) 4.7 (4.3–5.0)

Table 2The osmolality (mOsm kg−1), Na+, K+ and Ca2+ (mmol L−1) of the seawater atsalinities of 15‰, 30‰ and 45‰ and from the haemolymph (mean±SE) of early Por-tunus pelagicus juveniles after a 7-day acclimation to salinities of 15‰, 30‰ and 45‰followed by a 96-h exposure to different ammonia-N levels (mg L−1) and subjected tosalinities of 15‰, 30‰ and 45‰.

Treatment Osmolality Sodium Potassium Calcium n

15‰ salinity 435 197.5 4.5 4.5

Ammonia-N0 mg L−1 579.8±10.2a 217.7±7.7a 7.6±0.3a 7.2±0.2a 1420 mg L−1 584.0±9.2a 223.7±5.1a 7.4±0.5a 7.3±0.3a 1340 mg L−1 582.3±11.8a 222.1±3.8a 7.2±0.2a 7.3±0.2a 1560 mg L−1 589.5±5.7* 231.3±3.2* 7.5±0.1* 7.4±0.2* 930‰ salinity 882 393.7 8.0 8.0

Ammonia-N0 mg L−1 938.2±10.8b 408.6±8.3b 12.3±0.4b 12.4±0.4b 1020 mg L−1 931.9±6.3b 410.2±8.0b 12.5±0.6b 12.1±0.5b 1240 mg L−1 944.3±9.3b 404.9±6.3b 12.4±0.4b 12.2±0.4b 1360 mg L−1 942.9±11.5b 411.3±5.8b 12.6±0.3b 12.5±0.3b 1145‰ salinity 1332 590.1 12.0 12.0

Ammonia-N0 mg L−1 1321.2±12.5c 575.1±3.2c 15.3±0.3c 15.4±0.3c 1520 mg L−1 1319.6±11.9c 587.6±6.4c 15.2±0.4c 15.3±0.4c 1440 mg L−1 1319.5±8.7c 566.9±5.2c 15.3±0.4c 15.6±0.3c 1560 mg L−1 1325.2±13.5c 578.4±6.6c 15.1±0.2c 15.2±0.5c 11

⁎No statistics performed due to an insufficient number of surviving intermolt crabs(n=2) sampled.No significant ammonia-N effect (pN0.05) was detected. Significant salinity effectswere detected (pb0.05) between each salinity treatment indicated by differentlowercase letters.

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containers (containers sterilized prior to the water exchange) for eachammonia-N/salinity treatment. Each sampledcontainerwas then sealedfor the following 24-h and at 96-h (i.e. 24-h later) the water from thesame containers was sampled again. The water was measured forammonia-N using the same procedure mentioned above for haemo-lymph ammonia-N levels. The rate of ammonia-N excretion wascalculated using the following formula:

ððCfinal− CinitialÞ × VÞ= ðWt= hÞ

where Cfinal is the final ammonia-N concentration of the water, Cinitial

is the initial ammonia-N concentration of the water, V is the watervolume of the container, Wt is the wet mass (g) of the crab and h isthe hour interval between ammonia-N sampling.

Measurements of ammonia-N levels for the blank controls werealso conducted in containers without crabs which were used to adjustthe readings of ammonia-N excretion rates.

To confirm the actual ammonia-N concentrations used in theexperiment, 3 samples were taken from each treatment at 0-h and 72-h (immediately after new test solutions were added to the containers)and measured using the above mentioned procedure. The meanvalues of the actual ammonia-N concentrations at 0-h and 72-h werewithin the 5% of the stated ammonia-N concentrations.

2.5. Gill Na+/K+-ATPase activity

To determine the gill Na+/K+-ATPase activity following the 96-h exposure to various ammonia-N/salinity levels, the anterior (1st–3rdpair) and posterior (5th–8th pair) gills of the surviving crabs werequickly dissectedout on iceand thegills of the crabs (n=9–15) fromthesame treatment were pooled to yield 5 replicates for each treatment.These samples were then snap frozen in liquid nitrogen and then storedin a −80 °C freezer until processing (within one month). Gill Na+/K+-ATPase activity wasmeasured according to Holliday (1985). Briefly, thegills were homogenized in an ice-cold buffer (1:9 w/v) containing0.25 M sucrose, 6 mM EDTA, 50 mM Tris and 0.1 % (w/v) sodiumdeoxycholate with a pH of 7.2. The homogenatewas then centrifuged at4 °C at 16000g. The supernatant was obtained and centrifuged again at4 °C at 30,700 rpm (author: replace with g-force). No pellets wereobserved and only the supernatant was obtained which was kept in a−80 °C freezer until the Na+/K+-ATPase activity assay was conducted.

To conduct the assay the supernatant was thawed on ice and 67 µLsupernatant was then added to two separate mediums (200 µL). Oneconsistedof 167 mMNaCl, 50 mMKCl and33mMimidazole-HCl at apHof 7.2 while the other medium consisted of 217 mMNaCl, 1.67 ouabainand 33 mM imidazole-HCl at a pH of 7.2. These were pre-incubated in awater bath at 30 °C for 10 min., followed by the addition of 67 µL of asolution containing 25 mM Na2ATP and 50 mMMgCl2 at a pH of 7.2 toeach of the twomedia. The twomediawere then allowed to incubate for30 min at 30 °C. After 30 min., the reactionwas stopped by adding 2 mLof “Bonting's” reagent containing 560 mM H2SO4, 8.1 mM ammoniummolybdate and170 mMFeSO4. After adding “Bonting's” reagent another20 minwas allowed for color formation and the absorbancewas read at720 nm on a spectrometer after prior readings with phosphatestandards. Liberated phosphate was determined by subtracting thereadings between the two media and the Na+/K+-ATPase activity wasexpressed in µmol Pi h−1mg protein −1. Duplicates from each salinity/ammonia-N treatment were performed.

2.6. Data analysis

The LC50 values and 95% confidence intervals at each salinitycondition were determined by the Spearman-Karber method usingSPSS version 16.0. The response frequency was the observedmortality, the total observed were the sample population, the factorwas the hours during which mortality observations were made and

the co-variate was the ammonia-N concentration used. The Pearsongoodness-of-fit test confirmed the data adequately fit the model.Significant differences (pb0.05) were assumed when the 95%confidence intervals did not overlap.

The NH3 (un-ionized form) values of ammonia-N was calculatedaccording to the tables or equations from Spotte and Adams (1983)based on a temperature of 28 ºC, pH of 8.1 and salinities of 15‰, 30‰and 45‰, respectively.

Any significant differences among treatments for the haemolymphosmolality, haemolymph Na+, K+, Ca2+, haemolymph ammonia-Nlevels, ammonia-N excretion rates and gill Na+/K+-ATPase activity aswell as any potential interaction of ammonia-N and salinity on thesemeasured variables were analyzed using a two-way ANOVA. Signif-icant differences among treatments (pb0.05) were determined usingDuncan's multiple range test (Duncan, 1955).

3. Results

3.1. Survival

No crabs died in any of the control treatments (no addedammonia-N). The 96-h LC50 values of ammonia-N (with the 95 %confidence intervals in parenthesis) at salinities 15‰, 30‰ and 45‰were 43.4 (37.8–48.2), 65.8 (60.1–70.9) and 75.2 (69.4–80.7) mg L−1,respectively (Table 1) while the 96-h LC50 values of NH3 were 2.9(2.5–3.3), 4.3 (4.0–4.7) and 4.7 (4.3–5.0), respectively (Table 1). The96-h LC50 value of both ammonia-N and NH3 at a salinity of 15‰ wassignificantly lower (pb0.05) than those from both 30‰ and 45‰

Table 3The mean haemolymph ammonia-N concentration (mg L−1) (±SE) of early Portunuspelagicus juveniles after a 7-day acclimation to salinities of 15‰, 30‰ and 45‰followed by a 96-h exposure to different ammonia-N levels (mg L−1) at salinities of15‰, 30‰ and 45‰.

Ammonia-N (mg L−1) Salinity

15‰ 30‰ 45‰

0 A 0.03±0.01a A 0.01±0.01a A 0.01±0.01a

20 B 16.3±1.9a B 11.1±1.5b B 10.2±1.2b

40 C 34.4±3.1a C 28.8±2.4a,b C 27.3±2.9b

60 D 58.7±5.6a D 51.3±8.2a,b D 47.5±4.2b

Different upper and lower case letters indicate significant differences (pb0.05) withineach column and row, respectively.In all treatments n=4.

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salinity treatments while no significant difference (pN0.05) wasdetected between the 30‰ and 45‰ treatments.

3.2. Haemolymph osmolality, Na+, K+, Ca2+ and ammonia-N levels

Themeanhaemolymphosmolality of the control crabs at salinities of15‰, 30‰ and 45‰, were 579.8, 938.2 and 1321.2 mOsm kg−1,respectively. Meanwhile, for the control crabs subjected to salinities of15‰, 30‰ and 45‰, themeanhaemolymphNa+, K+andCa2+was217,408 and 575 mmol L−1, 7.6, 12.3 and 15.3 mmol L−1, and 7.2, 12.3 and15.3 mmol L−1, respectively (Table 2).

A significant salinity effect (pb0.01) was detected on the haemo-lymphosmolality andhaemolymphNa+, K+andCa2+ levels of the crabssince these values decreased as salinities decreased (Table 2). Atsalinities of 15‰ and 30‰ the haemolymph osmolality of the crabswere hyper-osmotic to the experimental test solutions while thehaemolymph osmolality at a salinity of 45‰ was near iso-osmotic.Meanwhile the haemolymphNa+ levels of the crabs at salinities of 15‰,30‰ and 45‰ were hyper-ionic, near iso-ionic and hypo-ionic,respectively (Table 2).

The mean haemolymph ammonia-N levels of the control crabs sub-jected to salinities 15‰, 30‰ and 45‰were 0.03, 0.01 and 0.01 mg L−1

ammonia-N, respectively andwere not significantly different from eachother (pN0.05) (Table 3). However, at 20 mg L−1 ammonia-N, the

Fig. 1. The mean gill Na+/K+-ATPase activity (µmol Pi mg protein−1h−1) (±SE) of the adifferent ammonia-N levels (mg L−1) from salinities 15‰, 30‰ and 45‰. Different letters

haemolymph ammonia-N of the crabs subjected to a low salinity of 15‰was significantly higher (pb0.05) than those at 30‰ and 45‰.Meanwhile, when exposed to 40 and 60 mg L−1 ammonia-N, thehaemolymph ammonia-N levels of the crabs at 15‰ were significantlyhigher than those at 45‰ only (Table 3). Furthermore, within the samesalinity treatments, the haemolymph ammonia-N levels of the crabssignificantly increased (pb0.01) as the ammonia-N levels increased(Table 3).

A two-way ANOVA detected no significant interaction (pN0.05)between the salinity and ammonia-N level on the haemolymphosmolality, haemolymph Na+, K+ and Ca2+ levels or haemolymphammonia-N levels.

3.3. Gill Na+/K+-ATPase activity and ammonia-N excretion

The Na+/K+-ATPase activity in response to salinity and ammonia-N were measured from both the anterior and posterior gills. For thecrabs from the control, the Na+/K+-ATPase activity on the anteriorgills showed no significant change (pN0.05) at different salinities andremained significantly lower (pb0.01) than the posterior gills (Fig. 1).In contrast, the Na+/K+-ATPase activity of the posterior gills showed asignificant salinity dependence (pb0.01); the highest activity wasfound for the crabs subjected to the lowest salinity of 15‰, which wassignificantly higher (pb0.05) than those subjected to the highersalinities of 30‰ and 45‰ (Fig. 1). Compared to the control crabs, theNa+/K+-ATPase activity on the anterior gills generally increasedsignificantly (pb0.05) when exposed to increasing ammonia-N levelsat all salinities tested. However, the Na+/K+-ATPase activity on theposterior gills only significantly increased (pb0.05) with increasingammonia-N levels at a salinity of 30‰ (Fig. 1). No data was obtainedfor the crabs subjected to a salinity of 15‰ at 60 mg L−1 ammonia-Nsince the surviving number of crabs were insufficient to conduct gillNa+/K+-ATPase measurements.

Aammonia-N excretion rates generally reduced as the salinitiesand/or the ammonia-N levels increased (Table 4). A two-way ANOVAdetected significant effects of both ammonia-N and salinity (pb0.01)on ammonia-N excretion. The highest ammonia-N excretion rate wasdetected for the crabs subjected to the lowest salinity of 15‰withoutadded ammonia-N. Furthermore, as the ammonia-N levels increased,the crabs subjected to a low salinity of 15‰ consistently had

nterior and posterior gills of early Portunus pelagicus juveniles after 96-h exposure toindicate significant differences (pb0.05) between all treatments.

Table 4Following a 7-day acclimation to salinities of 15‰, 30‰ and 45‰, themean ammonia-Nexcretion rates (µg g−1h−1) (±SE) of early Portunus pelagicus juveniles exposed todifferent ammonia-N levels (mg L−1) and subjected to salinities of 15‰, 30‰ and 45‰over a 24-h period.

Ammonia-N (mg L−1) Salinity

15‰ 30‰ 45‰

0 A 265.1±12.3a A 231.2±13.7a,b A 201.4±12.1b

20 A 232.4±10.2a AB 211.4±12.3a,b A 186.2±20.7b

40 B 168.9±9.6a B 142.3±10.3a,b B 125.4±10.1b

60 C 121.2±10.8a C 104.4±9.2a C 81.2±8.3b

Different upper and lower case letters indicate significant differences (pb0.05) withineach column and row, respectively.In all treatments n=4.

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significantly higher (pb0.05) ammonia-N excretion rates than thoseexposed to the same ammonia-N level but subjected to highersalinities of 30‰ and 45‰ (Table 4).

A two-way ANOVA detected no significant interaction (pN0.05)between the salinity and ammonia-N level on the gill Na+/K+-ATPaseactivity or ammonia-N excretion.

4. Discussion

The current study investigated the survival, osmoregulatoryresponses and ammonia-N excretion of early P. pelagicus juvenilesunder a wide range of ammonia-N and salinity combinations to betterunderstand adaptive responses to osmotic and ammonia-N stress. Fromthe upper end of the ammonia-N concentrations used, the current studydemonstrates that the ammonia-N tolerance of early P. pelagicussignificantly decreases at decreasing salinities. This ammonia-N toxicitypattern has also been reported for a number of penaeid shrimpsincluding Penaeus penicillatus (Chen and Lin, 1991), Penaeus chinensis(Chen and Lin, 1992), P. semisulcatus (Kir and Kumlu, 2006) and Lito-penaeus vannamei (Lin and Chen, 2001; Li et al., 2007). However,interestingly, Rebelo et al. (2000) observed that ammonia-N toxicity tothe estuarine crab, Neohelice (Chagmagnathus) granulata, increased atboth low and high salinities. Such a result may indicate species-specificdifferences or the absence of an acclimation period in their experimentleading to sudden hypo- and hyper-osmotic shock/stress.

It is common for acute ammonia-N toxicity tests on aquatic animals toutilize high ammonia-N concentrations to inducemortalities for purposesof measuring the relative toxicity or toxicity patterns which can then beapplied toecological risk assessmentmodels (Naitoet al., 2003).However,typically, animals in nature are less likely to encounter these upper endconcentrations used for acute toxicity tests while the lower end ofconcentrations may be more realistic. In polluted estuarine systemsammonia-N levels rarely exceed5 mg L−1 (Rebelo et al. 1999;Weihrauchet al., 1999), although sediments with high organic contents, wherebenthic crustaceans may bury, have been reported to reach as high as39.2 mg L−1 ammonia-N (Weihrauch et al., 1999). Furthermore, salinitieson river/estuarine systems that serve as habitats for early P. pelagicusjuveniles can fluctuate from b10‰ to N40‰ depending on the tides,month and distance from the ocean (de Lestang et al., 2003).Consequently, the current study also utilized lower ammonia-N concen-trations at different salinities to investigate the closely linked processes ofgill Na+/K+-ATPase activity andammonia-Nexcretionof earlyP. pelagicusjuveniles to regulatehaemolymphosmolality/ions andammonia-N levels.

To the best of our knowledge no other investigators have measuredthe haemolymph ammonia-N levels of crustaceans at differentammonia-N and salinity combinations. However, Lin and Chen (2001)speculated that the significantly higher ammonia-N toxicity ofL. vannamei at low salinities was the result of increased ammonia-Nintake. The results of our current experiment supports this suggestionsince at a low salinity of 15‰, the haemolymph ammonia-N levels of the

crabs exposed to 20, 40 and 60 mg L−1 ammonia-N were significantlyhigher compared to the crabs exposed to the sameammonia-N level at ahigh salinity of 45 ‰.

One of the mechanisms to reduce haemolymph ammonia-N incrustaceans is through active ammonia-N excretion fuelled by gill Na+/K+-ATPase activity (Weihrauch et al., 2004). Indeed, this likely explainsincreased gill Na+/K+-ATPase activity in response to in vitro elevatedammonia-N levels for decapod crustaceans including Macrobrachiumolfersii (Furriel et al., 2004), Callinectes danae (Masui et al., 2002, 2005)and C. ornatus (Garçon et al., 2007) as well in vivo increases ofammonia-N to gill Na+/K+-ATPase activity from P. chinensis and M.nipponense juveniles (Chen andNan, 1992;Wanget al., 2003). Similarly,in the current experiment, gill Na+/K+-ATPase activity of P. pelagicusjuveniles generally increased with elevated ammonia-N levels, likelycontributing to the haemolymph ammonia-N levels being lower thanthe experimental test solutions. However, consistent Na+/K+-ATPaseactivity increases at all salinitieswere onlyobservedon the anterior gills,while Na+/K+-ATPase activity in response to ammonia-N significantlyincreased only on the posterior gills of the crabs at a salinity of 30‰. Thisindicates the anterior gills are likely tobemore responsible to ammonia-N excretion, perhaps due to being unburdened by osmoregulatoryrequirements. This finding is in agreement with the experiment byWeihrauch et al. (1999) who demonstrated higher Na+/K+-ATPaseactivity and ammonia-N excretion occurred on perfused anterior gills ofthe crab, Cancer pagurus, in response to ammonia-N (Weihrauch et al.,1999). However, in the same study, the difference between ammonia-Nexcretion on the posterior and anterior gills were negligible for thestronger osmoregulating crabs of Carcinus maenas and Eriocheir sinensis(Weihrauch et al., 1999). Since both C. pagurus and P. pelagicus are bothweak osmoregulators (Romano and Zeng, 2006;Weihrauch et al., 1999)this characteristicmay also contribute to thesefindings although furtherresearch is likely necessary.

Interestingly, ammonia-N excretion at all ammonia-N levels wassignificantly higher for early P. pelagicus juveniles subjected to asalinity of 15‰ than those subjected to 45‰, although in contrast, thehaemolymph ammonia-N levels of the crabs subjected to 15‰ weresignificantly higher at 20, 40 and 60 mg L−1 ammonia-N compared tothose within the same ammonia-N treatment at 45‰. This likelyindicates that additional coping mechanisms are being utilized moreheavily at high salinities. Previous investigations have demonstrated achange in metabolic processes from ammontelism to ureotelism atincreasing salinities for Scylla serrata (Chen and Chia, 1996) andMarsupenaeus japonicus (Lee and Chen, 2003), and a similarphenomenon of detoxifying ammonia-N to urea may have occurredin the current study. Certainly further research is required to confirmthis. However, since different osmoregulatory strategies are necessaryat hypo- and hyper-osmotic conditions it would appear beneficial forcrustaceans to prevent excessive Na+ absorption, in exchange forNH4

+ excretion, at high salinity conditions.Despite both gill regions being involved in ammonia-N excretion, gill

specialization for respiration and osmoregulation on the anterior andposterior gills, respectively has been established based on Na+/K+-ATPase activity responses to salinity (Péqueux, 1995; Kirschner, 2004;Freire et al., 2008). The results of the current experiment are inagreement with these previous investigations. Anterior gill Na+/K+-ATPase activity of early P. pelagicus juveniles showed no significantsalinity response and remained consistently low whereas the posteriorgill Na+/K+-ATPase activity of the crabs subjected to a salinity of 15‰after over a week (acclimation period plus 96-h) were 2.6 and 1.8 foldgreater than those at30‰ and45‰, respectively. This result of increasedgill Na+/K+-ATPase activity at low salinities, to increase haemolymphNa+ and Cl− uptake, is typical for estuarine and marine crustaceans(Holliday, 1985; Piller et al., 1995; Castilho et al., 2001; López-Mañaneset al., 2002; Genovese et al., 2004; Torres et al., 2007; Lucu et al., 2008).However, gill Na+/K+-ATPase activity is unlikely to playa dominant rolein hypo-osmoregulation for P. pelagicus juveniles since this activity did

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not significantly change at high salinities, and is a similar finding withother crustacean species including Neohelice (Chasmagnathus) granu-latus (Genovese et al., 2004), Scylla paramamosain (Chung and Lin,2006), Carcinus aestuarii (Lucu et al., 2008). Interestingly, at the highsalinity of 45‰, the haemolymph K+ and Ca2+ of P. pelagicus juvenileswere hyper-regulated, which is in contrast to haemolymph K+ hypo-regulation of S. serrata at hyper-osmotic conditions (Chen and Chia,1997). Furthermore, there was no significant effect of ammonia-Nexposure to thehaemolymphosmolality, Na+, K+orCa2+ levels despitea general trend of increased gill Na+/K+-ATPase activity at elevatedammonia-N levels. It is unclear as to the exactmechanisms contributingto these results, however, other transport channels (e.g. Na+/K+/2Cl−,Ca2+-ATPase, K+-ATPase) and/or the antennal glandmay have played arole.

Evidence from the current experiment indicates that P. pelagicusjuveniles are relatively well adapted to elevated ammonia-N exposuresince, regardless of the salinity level or ammonia-N concentration thatinduced morbidity, ammonia-N excretion and gill Na+/K+-ATPaseactivitywere still functioningwhile thehaemolymph ammonia-N levelsremained substantially lower than the experimental test solutions.These findings may be linked with the burying behavior often adoptedby P. pelagicus in nature, which was suggested to be an importantcharacteristic for the development of an effective mechanism tomaintain the haemolymph ammonia-N levels below that of theenvironment (Weihrauch et al., 2004). However, changes to theexternal salinity was demonstrated to significantly influence thiscapacity which likely explains the increased ammonia-N toxicity ofearly P. pelagicus juveniles at lower salinities. Due to the seeminglycontradictory results of increased haemolymph ammonia-N levels butincreased ammonia-N excretion of the crabs at low salinities, experi-mentation on potential detoxification processes, changes to themetabolism and gill permeability are warranted. Such experimentsmay to help further elucidate the underlying adaptive mechanisms ofcrustaceans to elevated ammonia-N levels when subjected to differentsalinity conditions.

Acknowledgements

This project was funded by Dr. Thomas Romano (2008) and theGraduate Research Scheme, James Cook University (2008). We wouldlike to thank the two anonymous reviewers for their constructivecomments.

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