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1. Lobster

1. Lobster

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Effect of environmental parameters on immune response of theIndian spiny lobster,
Panulirus homarus
(Linnaeus, 1758)
Bindhu Verghese
a,
*
,1
, E.V. Radhakrishnan
a
, Abinash Padhi
b
a
Central Marine Fisheries Research Institute, P.B. No. 1603, Cochin 682018, Kerala, India
b
 Department of Biological Science, 600 S. College Avenue, University of Tulsa, Tulsa, OK 74104, USA
Received 17 October 2006; revised 16 January 2007; accepted 26 January 2007Available online 9 February 2007
Abstract
The high export value of the Indian spiny lobster
Panulirus homarus
increasingly attracts the aquaculturists for farming andfattening. However, lack of knowledge on the effect of environmental parameters on the immune system of this animal could resultin high mortality, which ultimately may cause major loss to the industry. Here, we report the effect of salinity (20, 25, 35, 40, and45
&
),pH(5.0,8.0,and9.5),dissolvedoxygen(DO)(1and5 mg L
À
1
),andammonia-Nconcentration (0,0.5,1.5and3 mg L
À
1
)onthe immune response of 
P. homarus
measured in the haemolymph in terms of Total Haemocyte Count (THC), phenoloxidase (PO)activity, and NBT-reduction. Our data showed significant reduction (
 P
<
0.05) in THC, and NBT-reduction at lower (20
&
) andhigher (45
&
) salinities. However, PO activity showed significant disparity, showing an increasing trend from 20 to 45
&
. Signif-icantreduction(
 P
<
0.05)inTHCandPOactivityunderacidicandalkalineconditions,underhypoxiccondition(1 mg L
À
1
),andatthe higher ammonia-N concentrations than their respective optimal conditions were observed. Thus, suggesting that extremeenvironmental parameters can induce modifications in the immune system of the spiny lobster
P. homarus
, which may enhancetheir susceptibility to opportunistic pathogens. The humoral parameters such as THC, PO activity, and NBT-reduction can beused as potential stress indicators for healthy management of spiny lobsters.
Ó
2007 Elsevier Ltd. All rights reserved.
 Keywords: Panulirus homarus
; Total haemocyte count; Phenoloxidase; Superoxide anion production; Environmental parameters
1. Introduction
The Indian spiny lobster
Panulirus homarus
is a truly marine species inhabiting the inshore sea along the southerncoast of India. Live lobsters of this species are exported to Southeast Asian countries. The high demand for live lob-sters in the export market and the decline of wild catches has attracted aquaculturists to venture into lobster farming.Due to lack of hatchery technology, the success of lobster farming largely depends on the fattening of wild caught
* Correspondence to: Department of Biological Science, 600 S. College Avenue, University of Tulsa, Tulsa, OK 74104, USA. Tel.:
þ
1 918 6312765; fax:
þ
1 918 631 2762.
 E-mail address:
1
Present address: Department of Biological Science, 600. S. College Ave. University of Tulsa, OK-74104, USA1050-4648/$ - see front matter
Ó
2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.fsi.2007.01.021Fish & Shellfish Immunology 23 (2007) 928
e
936
 
lobsters[1]. The wild caught commercial grade lobsters meant for export are maintained carefully in secondary hold-ing centres whereas the undersized lobsters are stocked at high densities in fattening ponds. Although farming trials inindoor systems have shown culture potential for this species[1], the high stocking density coupled with poor waterquality management can lead to devastating consequences on the successful maintenance of these lobsters. Knowl-edge of the immune response of lobsters with regard to water quality and stress could provide insight into developingproper management strategies.Environmental parameters such as temperature, salinity, oxygen, ammonia content, and pH have shown significanteffects on the immune system of crustaceans[2
e
7]. Salinity is one of the major factors that may significantly affectthe physiology of aquatic organism. For example, a reduction in salinity resulted in increased haemolymph total pro-tein and haemocyaninplasma levelin
 Homarusamericanus
[8].Similarly,lower salinity caused significantincrease inoxygen consumption in
Penaeus japonicus
[9]. Wide fluctuation in pH, dissolved oxygen, and ammonia also affectculture conditions[6]. Elevated amounts of environmental ammonia have been reported to affect growth and moultingof crustaceans[10]. Though lethal and sublethal effects of ammonia on metabolism and osmoregulation of shrimpshave been well studied[11,12], recently the focus has been towards understanding the effect of ammonia on their im-mune system[3,13,14]. A decrease in dissolved oxygen (DO) concentration can also hinder the metabolic perfor-mance, growth, and moulting[15]. Decrease in DO concentration can also have a negative effect on crustaceanimmune system[5,6,16]and may decrease resistance to diseases. Although influence of environmental parameterson the immune system of fishes, molluscs, and shrimps have been reported (e.g. refs.[2,5,17,18]), the influence of such factors on the immune response of spiny lobsters are poorly understood.The impact of these environmental parameters can be directly assessed from the immune response of the organism.In decapods, cellular defenses rely on haemocytes with several functions such as coagulation, phagocytosis, encap-sulation[5], and the production of melanin by the phenoloxidase (PO) activating system[9]. Previous studies reported that the PO activities are sensitive to salinity fluctuation; for example, in
Litopenaeus schmitti
, the PO activity is sig-nificantly reduced at lower salinity[4], and in case of 
Penaeus californiensis
its activity increases with increase insalinity[7]. Similarly, Total HaemocyteCount (THC) and NBT-reduction in crustaceans are all affected by fluctuationin salinity, temperature, pH, ammonia content, and dissolved oxygen[6]. The impact of environmental parameters onthe immune response of lobsters can be directly assessed from haemolymph by measuring PO activity, THC, andNBT-reduction. Here, we report the influence of salinity, pH, dissolved oxygen, and ammonia-N on total circulatinghaemocytes, PO activity and NBT-reduction in the spiny lobster
P. homarus
.
2. Materials and methods
 2.1. Animal and experimental setup
The lobsters were collected from Vizhinjam (8.41 N 77.0 E) located on the southwest coast of India and were trans-ported to the Research Centre of Central Marine Fisheries Research Institute (CMFRI) at Calicut, 300 km north of Vizhinjam, in thermocol boxes containing cool saw dust and were acclimatised for two weeks at a temperature of 25
Æ
0.3
C, salinity 35
&
, pH 7.78
Æ
0.2, and dissolved oxygen 4.97
Æ
1.43 ml L
À
1
. The lobsters were maintainedin individual fibreglass reinforced plastic tanks containing 100 L filtered seawater. All experimental lobsters were inthe intermoult stage and weighed 104
Æ
43.88 g. The animals were fed with green mussel (
 Perna viridis
) meat oncedailyat10%ofthebodyweight.Lobstersweregraduallyacclimatisedtoarangeofsalinitiesof20,25,30,40and45
&
either by reducing the salinity with freshwater or by adding salinewater to increase the salinity both at a rate of 2% perhour and maintained for a week in respective salinities. The control animals were maintained at 35
&
. Lobsters for pHstudies were acclimatised to a salinity of 35
&
and maintained for a week at pH 5.0, 8.0, and 9.5, where pH 8.0 wasused as control. Seawater with lower and higher pH was made by adjusting the pH with 1 N HCl or 1 N NaOH, re-spectively. The animals for ammonia stress studies were maintained for a week in different concentrations of ammo-nia-N, 0.5 mg L
À
1
, 1.5 mg L
À
1
, and 3 mg L
À
1
and a control at 0
Æ
0.05 mg L
À
1
. Different concentrations of ammonia-N were prepared by dissolving 38.2 g of NH
4
Cl in 1 L distilled water to make 10,000 mg L
À
1
as stock so-lution. Water was replaced daily to maintain the concentration. For oxygen stress experiments, only two conditionswere noted the hypoxic (1 mg L
À
1
) and the optimal level (5 mg L
À
1
) of oxygen. The oxygen levels were maintainedby controlled aeration and continuous monitoring of the oxygen level. The control groups were maintained at the
929
 B. Verghese et al. / Fish & Shellfish Immunology 23 (2007) 928
e
936 
 
oxygen saturation of 5 mg L
À
1
. In the hypoxic condition, the dissolved oxygen levels were maintained at 1 mg L
À
1
.All the stress tests were carried out in triplicates.
 2.2. Total Haemocyte Count (THC)
After exposure to each test condition for seven days except for oxygen stress experiments (24 h), haemolymph wasdrawn from each lobster from the ventral sinus of each individual. Haemolymph (0.1 ml) was drawn using a 2 ml sy-ringe containing 0.9 ml of anticoagulant (sodium citrate 0.114 M, sodium chloride 0.10 M, pH 7.45). A drop of hae-molymph in anticoagulant was placed in Neubauer haemocytometer and the THC was made using a phase contrastmicroscope.
 2.3. Phenoloxidase (PO) assay
Although in the spiny lobster (Genus:
Panulirus
) the proPO activating mechanism is similar to other crustaceans,unlike other crustaceans, most of the proPO-activity of spiny lobster has been detected in cell-free plasma[19]. Inaddition, Perazzolo et al.[20]reported a significant level of PO activity in the serum of 
Penaeus paulensis
suggestingthe use of serum for PO activity assay. Considering these evidence, in the present analyses we used serum for PO as-say. PO activity was measured spectrophotometrically by recording the formation of dopachrome from L-dihydrox-yphenylalanine (L-DOPA)[20]. The haemolymph was collected without anticoagulant and kept at 4
C for 5 min andthe clot was broken using a micropestle and repeatedly centrifuged at 2000
Â
g
to remove serum. The serum was im-mediately used for PO analysis. Twenty
m
l serum was pre-incubated with 20
m
l of trypsin (1 mg ml
À
1
) for 30 min at37
Cin96wellmicrotitreplates.Toeach sample20
m
lofL-DOPA(3 mg ml
À
1
)wasaddedandincubatedfor5 minat37
C. Distilled water (260
m
l) was added to slow down the reaction. Activity of PO was detected spectrophotomet-rically using Bio-tek (EL 800) micro plate reader, measuring the formation of red pigment DOPA-chrome after 5, 10,20, and 60 min absorbance at 490 nm. For control, L-DOPA alone was incubated with 0.45 M NaCl to monitor spon-taneous oxidation. PO activity was expressed as IU mg protein
À
1
. The protein concentration of the sample was de-termined by Bradford method[21].
 2.4. Intracellular super oxide anion (O
 2
À
) production assay
Respiratory burst activity of haemocytes was quantified using the reduction of nitroblue tetrazolium (NBT) to for-mazan as a measure of super oxide anion production[22,23]. One hundred
m
l haemolymph was diluted with 400
m
l of anticoagulant and was centrifuged at 300
Â
g
at 4
C for 10 min. The resultant haemocyte pellet was resuspended to10
8
cells per ml in modified complete Hank’s balanced salt solution (MCHBBS) (10 mM CaCl
2
, 3 mM MgCl
2
, 5 mMMgSO
4
and 24 mg ml
À
1
HBSS). One hundred
m
l of haemocyte suspension was added to a flat bottom 96 well micro-litre plates and cyto-centrifuged at 300
Â
g
at 4
C for 10 min. After removing the supernatant, 100
m
l of trypsin(2 mg ml
À
1
) was added and allowed to react for 30 min at 37
C. MCHBBS was added to the remaining haemocytesuspension as a control. NBT (100
m
l, 0.3% in MCHBBS) was added to the haemolymph and incubated for 30 min at37
C. The staining reaction was terminated by removing the NBT solution and adding absolute methanol. After threewashes with 70% methanol, the haemocytes were air dried and coated with a solution of 120
m
l 2M KOH and 140
m
ldimethyl sulfoxide were added to dissolve the cytoplasmic formazan[22,23]. The optical densities of the dissolvedcytoplasmic formazan were measured at 630 nm with a Bio-tek (EL 800) microplate reader. The ratio of OD630 nm of trypsin elicited haemocytes to OD 630 nm of control haemocytes was used as an index for comparingthe effect of different treatments on phagocytic activity.
 2.5. Analyses
Each assay (THC, PO, and NBT-reduction) was repeated at least five times from at least ten individuals for eachdata point. One-way ANOVA followed by Post-Hoc multiple comparisons (except DO analysis) were carried out us-ing the statistical program SPSS ver. 14.0. Significant levels for all analyses were set to
P
<
0.05.
930
B. Verghese et al. / Fish & Shellfish Immunology 23 (2007) 928
e
936 

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