RESEARCH ARTICLE

C. Rosas Æ E. L. Cooper Æ C. Pascual Æ R. BritoR. Gelabert Æ T. Moreno Æ G. Miranda Æ A. Sanchez

Indicators of physiological and immunological statusof Litopenaeus setiferus wild populations (Crustacea, Penaeidae)

Received: 10 April 2003 / Accepted: 26 January 2004 / Published online: 20 March 2004� Springer-Verlag 2004

Abstract Constant environmental changes in oceanic andestuarine systems due to human activity in coastal zonesrequire different tools and strategies for their study to beable to assess the health status of aquatic ecosystems. TheGulf of Mexico has a tremendous ecological importancebecause of its biological diversity. Baseline levels ofblood metabolites and immune components were definedfor wild tropical populations of Litopenaeus setiferus, animportant shrimp species of the Gulf of Mexico. Osmoticpressure, oxy hemocyanin (OxyHc), glucose, proteins,cholesterol, lactate, and triacylglycerols (TAG) wereused as indicators of physiological status; pro phenol-oxydase (proPO) and phenoloxydase (PO) quantificationof hemocytes, as well as respiratory burst, were used toassess the immunological status. Significant differenceswere observed in live weight of juveniles, males, andfemales of L. setiferus with mean values of 10.54, 35.05,and 40.33 g, respectively. A value of 460 mOsm/kg was

recorded in the water where juveniles were sampledand 1,000 mOsm/kg in adult sampling zones. Conse-quently, the osmotic pressure of L. setiferus juveniles(718.02 mOsm/kg) was 22% lower than in adults(925 mOsm/kg); there were no differences between sexes.A significantly lower value in juvenile OxyHc wasobserved. This value was 24 and 16% lower than thatobserved in males and females, respectively. Hemolymphglucose, cholesterol, and triacylglycerol levels were notstatistically different between shrimp stages, showingmedian values of 0.19, 0.32, and 0.39 mg/ml of glucose,cholesterol, and triacylglycerols, respectively. Hemocya-nin was distributed normally in the juvenile–male pop-ulation but not in females. A proportion between 52 to81% of OxyHc of the total protein (OxyHc/Prot) wasobtained for all stages. Digestive gland glycogen showedno normal distribution in juveniles and adults. Digestivegland glycogen was significantly higher in juveniles(median value of 2.64 mg/g) than in adults (median valueof 1.58 mg/g). A higher value of lactate (0.11 mg/ml) wasobserved in juveniles than in males (0.09 mg/ml) andfemales (0.04 mg/ml). Granular cells (GC) correspondedto 36, 44, and 39% of the total hemocytes (TH) observedin juveniles, males, and females. ProPO showed no nor-mal distribution in all stages. A high proPO activity wasrecorded in L. setiferus females that was 95% higher thanin the juvenile–male group. Respiratory burst was di-vided into two groups, juveniles–males and females, andwas distributed normally in both. Female respiratoryburst values were 40 and 45% higher than those observedin the juvenile and male groups, respectively. There wasan increment in OxyHc, proteins, PO, hemocytes, andrespiratory burst with an increase in body weight. Incontrast, the relationship of live weight to glucose, TAG,and proPO revealed no significant regressions. A signif-icant decrease in cholesterol, glycogen, and lactate withan increase in body weight was also observed. We nowhave indicators of nutritional and immune status ofjuveniles and adults that can reveal changes in trophicrelations and health at the population level. This willserve as a basis for using L. setiferus as environmental

Communicated by P.W. Sammarco, Chauvin

C. Rosas (&) Æ C. Pascual Æ A. SanchezLaboratorio de Ecologıa y Biologıa Marina Experimental,Fac. de Ciencias, Universidad Nacional Autonoma de Mexico,Calle 8 A No. 248·5, Vista Alegre Norte, 97130 Merida,Yucatan, MexicoE-mail: crv@hp.fciencias.unam.mx

E. L. CooperLaboratory of Comparative Immunology,Department of Neurobiology, David Geffen School of Medicine,University of California, Los Angeles,Los Angeles, CA 90095-1763, USA

R. Brito Æ R. GelabertFacultad de Ciencias Pesqueras,Universidad Autonoma del Carmen,Cd. del Carmen, Campeche, Mexico

T. MorenoFacultad de Quımica, Universidad Autonoma del Carmen,Cd. del Carmen, Campeche, Mexico

G. MirandaRegion Marina Noreste, PEMEX-Exploracion y Produccion,Cd. del Carmen, Campeche, Mexico

Marine Biology (2004) 145: 401–413DOI 10.1007/s00227-004-1321-y

sentinels, as non-controversial and inexpensive models toevaluate the potential changes that human activitiescould exert on this important marine ecosystem.

Introduction

The effects of constant environmental changes in oceanicand estuarine systems due to human operations incoastal zones require for their study different tools andstrategies to assess the health status of aquatic ecosys-tems. Penaeid shrimp are aquatic organisms that aboundin tropical coastal zones. The white shrimp Litopenaeussetiferus is exposed to different environmental pressures.These include excessive fishing, pollution in estuariesand coastal lagoons due to pesticides used in intensive-agriculture lands, and effects of oil industry and marineoperations developed along the Gulf of Mexico and theAmerican Atlantic coast, among others (Balcom 1994).Unfortunately, scarce information is available on thephysiological status of shrimp and how it could changeif environmental deterioration increases in the areas ofthe shrimp distribution. In their life cycle L. setiferus gothrough several planktonic larval stages, moving towardthe coast where they usually arrive as postlarvae incoastal lagoons and estuaries by means of tidal currents.There they are recruited into the submerged vegetationused as their growth zone (Rosas et al. 1999).

In recent years, several investigations have quantifiedblood metabolites and hemocyanin as a tool for moni-toring physiological conditions in wild or cultured shrimpexposed to different environmental conditions. Palacios(2000) used triacylglycerols, glucose, cholesterol, andprotein to monitor physiological conditions of wild andcultured populations after repeated spawning of adultfemale L. vannamei. Wild shrimp had a higher fecundityand higher levels of triacylglycerols, cholesterol, protein,and glucose in hemolymph than cultured shrimp. San-chez et al. (2001) reported variations in protein, lactate,glucose, triacylglycerols, cholesterol, and immune re-sponses by assessing hemocytes and pro phenoloxidase(proPO) activities in L. setiferus adult males from wildpopulations and during acclimation at 27 and 30�C. Theirstudy revealed that laboratory conditions produced lowervalues of proteins, triacylglycerols, and cholesterol thanthose of freshly captured shrimp.

Blood metabolites have also been used to monitortemperature effects on adult L. setiferus males. Recentstudies showed a relation between hemolymph metabolicvariables, immune responses, and sperm quality ofshrimp exposed to an extreme temperature (33�C; Pasc-ual et al. 2003b). This high temperature provoked loss ofosmotic capacity and a reduction in (1) blood metabo-lites, (2) hemocytes, (3) proPO activity, and (4) spermquality at day 5 as compared with similar parameters infreshly captured wild shrimp populations. In anotherstudy Rosas et al. (2002) showed that L. vannamei ex-posed at 15& salinity had lower values of blood glucose,

lactate, and oxyhemocyanin (OxyHc) than those ob-served in shrimp maintained at 40& salinity.

The OxyHc, blood protein, glucose, triacylglycerols(TAG), and lactate levels have been studied in L. van-namei juveniles to construct a base line for this speciesmaintained in outdoor (20-m2 ponds) and indoor (90-ltanks) conditions (Pascual et al. 2003a). Type of food hasbeen identified as a dominant factor that affects bloodmetabolites. This suggests that these parameters can beused as reference guidelines for evaluating the physio-logical and immune status of L. vannamei. According toPascual et al.’s (2003a) study a wider spectrum of tropicalcrustacean species should be analyzed to define whetherthe observed differences among species are related toenvironmental characteristics (temperature, salinity,dissolved oxygen, or combinations) or whether they arerelated to morphological and physiological adaptationsindependent of the environmental conditions.

When the OxyHc, blood protein, glucose, and lactatelevels of L. vannamei juveniles were compared with thoseof other crustacean species, it was observed that crabs,lobsters (low-activity species), and closed-thelycumshrimp species (mean-activity species) had lower valuesin comparison to those observed in open-thelycumshrimp species, including L. vannameijuveniles (Pascualet al. 2003a). Blood metabolites and OxyHc could pos-sibly reflect morphological and physiological adaptationof crustaceans as related to shrimp’s specific energeticdemands, that is, show low values for low-activityshrimp and high values for high-activity shrimp.

The shrimp immune system has a strong proteinicbase. Proteins are involved in recognizing foreign glu-cans through the lipopolysaccharide binding protein(LPSBP) and the b-glucan binding protein (BGBP;Vargas-Albores and Yepiz-Plascencia 2000). The clot-ting protein is involved in engulfing foreign invadingorganisms and prevents blood loss upon wounding (Hallet al. 1999; Montano-Perez et al. 1999). Defense reac-tions in shrimp are often accompanied by melanization.The proPO-activating system, mediated by hemocytes, isa zymogen of PO enzyme that catalyzes both o-hydroxylation of monophenols and oxidation of phenolsto quinones leading to the synthesis of melanin (Sritu-nyalucksana and Soderhall 2000). Conversion of proPOto PO is through a serine protease called a pro phenol-oxydase activating enzyme (ppA) that is regulated byanother protein, the a-2 macroglobulin, a trypsininhibitor (Perazzolo and Barracco 1997). The innateimmune response of shrimp also relies upon the pro-duction, in hemocytes, of antimicrobial peptides calledpenaeidins that are active against a large range ofpathogens essentially directed against gram-positivebacteria via a strain-specific inhibition mechanism(Destoumieux et al. 2000). Nutritional status is consid-ered one of the important factors that determines theability of animals to withstand infections; hence, nutri-tional status together with the immune condition isconsidered a good health indicator of cultivated shrimp(Bachere 2000; Merchie et al. 1998; Lopez et al. 2003).

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Recently Rosas et al. (2002) showed that shrimp are welladapted to use protein as a source of energy and mole-cules, indicating that protein metabolism could be thecentral axis of L. vannamei metabolism. In anotherstudy, Pascual et al. (2003b) observed that the immunecondition and nutritional status of wild shrimp L. se-tiferus adult males exposed to an extreme temperaturewere good indicators of the capacity of shrimp to tol-erate environmental stressors; in addition, a reduction inthe nutritional status affected the immune condition andsperm quality, indicating that nutritionally or environ-mentally stressed shrimp can be affected in their repro-ductive capacity, via an imbalance of their generalphysiological status.

This project proposed to use blood metabolites andimmune responses as tools to monitor the physiologicaland immunological status of L. setiferuswild populationsto identify the actual physiological and immunologicalstatus of juveniles and adults ofL. setiferus collected fromthe southeast Gulf of Mexico. Specifically, we obtaineddata to define baseline levels of blood metabolitesand immune responses for wild tropical populations ofL. setiferus. Osmotic pressure, OxyHc, glucose, proteins,cholesterol, lactate, and TAG were used as indicators ofphysiological status, and proPO, quantification of he-mocytes, and respiratory burst served as immunologicalstatus indicators for this shrimp species.

Material and methods

L. setiferusjuveniles and adults

L. setiferus juveniles were sampled with a trawling net for 5 min infront of Palizada River, in Laguna de Terminos (18�43¢21¢¢N,92�15¢46¢¢W). Juvenile shrimp (n=150) were transported to ourlaboratory in plastic bags with water from the sampling site, sat-urated with oxygen and at a temperature of 25�C. For immediatelaboratory acclimation shrimp were placed in plastic tanks (500 l)for 24 h at 11& salinity (equal to that of the sampling site) beforeany testing.

L. setiferus adults were derived from natural broodstock pop-ulations of L. setiferus caught over the continental shelf of the Gulfof Mexico, near Laguna de Terminos, Campeche, Mexico(18�43¢34¢¢N, 91�55¢14¢¢W) at 4.5 km offshore. To prevent injuryshrimp were caught with a gill net that hooked only on their ros-trum and telson spines. Once caught, shrimp were placed in plasticbags with cold seawater (24�C) with a saturated oxygen atmosphereat a density of 10 shrimp/40 l. Transportation to the laboratoryrequired 2 h. For acclimation shrimp were reared for 24 h in ovaldark ponds (10 m2) at a density of 40 shrimp/pond and providedwith aerated, filtered (20 lm) flow-through seawater at29.7±0.6�C, 32±1& salinity, before any measurement.

Hemolymph sampling

Twenty-four hours after arrival in the laboratory shrimp bloodmetabolites and immune responses were assayed from hemolymphsamples. Before sampling, shrimp were placed in chilled (25�C) andaerated seawater for 5 min to reduce metabolic activity and todecrease the effects of manipulation during sampling. Previousstudies demonstrated that in this way the stress caused by samplingcould be reduced significantly (Rosas et al. 2000). Only shrimp inthe intermolt stage (C stage) were used. Hemolymph (approxi-

mately 500–1500 ll per shrimp depending on the size) was sampledindividually with a chilled syringe needle inserted at the base of thefifth pereiopod, after the shrimp had been dried with a paper towel.To avoid clotting, blood samples were taken without air because airaccelerates the coagulation process. Blood samples of each shrimpwere placed gently on chilled (4�C) plastic foil (Parafilm) over ice.Subsamples were taken from these hemolymph drops. Thirty mi-croliters of the entire hemolymph were used for OxyHc (10 ll) andosmotic pressure (20 ll) measurements. Hemolymph was thencentrifuged at 800 g for 3 min and the supernatant separated fordetermination of plasma contents. Hemocytes were resuspendedgently in cacodylate buffer (100 m M cacodylate, 10 m M CaCl2,pH 7) and centrifuged again at 16200 g for 3 min to be used as asource of the proPO reactions (Hernandez-Lopez et al. 1996).Individual weights (±0.05 g) were measured and molting stageswere observed using the Drach and Tchernigovtzeff (1967) uropodcharacteristics.

Biochemical and physiological indices

For hemocyanin (OxyHc) measurements, 10 ll of hemolymph werediluted immediately with 990 ll of distilled water in a 10-mmcuvette for UV wavelength spectroscopy (1.0 ml; 1-cm path length)and the absorbance measured at 335 nm. Using an extinctioncoefficient of E=17.26 calculated on the basis of functional subunitof 74,000 Da for shrimp hemocyanin, concentrations were deter-mined (Chen and Cheng 1993a, 1993c).

Osmotic pressure (OP) of hemolymph and of seawater weremeasured in a microosmometer with 20 ll of the entire blood pertitration (3 MO-PLUS; Advanced Instruments, Inc., USA). Os-motic capacity (OC) was defined as the difference between osmoticpressure of hemolymph and external medium (Lignot et al. 1999).

Commercial kits were used for glucose (Bayer Sera Pack PlusBO14509-01), lactate (Sigma-cat. 735), cholesterol (Bayer SeraPack Plus BO14507-01), and triacylglycerol (Bayer Sera Pack PlusBO14551-01) determinations, adapted to a micro-plate using 10 llof plasma and 200 ll of enzyme chromogen reagent. Absorbancewas recorded on a micro-plate reader (BIO-RAD model 550) andconcentrations calculated from a standard solution of substrate.Plasma was further diluted 1:100 for protein determination by theBradford (1976) technique adapted to a micro-plate method usingcommercial chromogen reagent (BIO-RAD 500-0006) and bovineserum albumin as a standard solution.

Digestive gland glycogen

Glycogen was measured in the digestive gland of 18-h-fastedshrimp. Glycogen was extracted in the presence of sulfuric acid andphenol (Dubois et al. 1965). The digestive gland was first homog-enized in trichloroacetic acid (5%) for 2 min at 3,300 g. Aftercentrifugation (7000 g) the supernatant was quantified. This pro-cedure was done twice. One microliter of TCA was pipetted into atube and mixed with 5 volumes of 95% ethanol. The tubes wereplaced in an oven at 37–40�C for 3 h. After precipitation, the tubeswere centrifuged at 3,000 rpm for 15 min. The glycogen (pellet) wasdissolved by adding 0.5 ml of boiling water, and then 1 ml ofconcentrated sulfuric acid and phenol (5%) were added and mixed.The contents of the tubes were transferred to a cuvette and read at490 nm in a spectrophotometer.

Immune condition

Chemicals and reagents

Because the proPO system can be activated by endotoxins, allglassware was washed with E-Toxa-Clean and all solutions wereprepared using pyrogen-free water. Shrimp salt solution was pre-

403

pared according to Vargas-Albores et al. (1993): 450 m M NaCL,10 m M KCL, 10 m M HEPES, pH 7.3, 850 mosmol kg)1. Theanticoagulant solution for hemolymph extraction was prepared byadding 10 m M of EDTA-Na2 to the shrimp salt solution. A so-dium cacodylate buffer (Cac, 10 m M cacodylate, 10 m MCaCL2,pH 7.0) was used to determine phenoloxydase activity.

ProPO activity

A sample of 50 ll of cacodylate buffer containing degranulatedhemocytes was incubated 3 min at 25�C. Then 50 ll of L-DOPA(3 mg/ml in Cac buffer) was added and the sample incubated10 min, and absorbance was measured at 490 nm (Hernandez-Lopez et al. 1996).

PO activity

A sample of 50 ll of plasma was incubated 3 min at 25�C. Then50 ll of L-DOPA (3 mg/ml in Cac buffer) was added and thesample incubated 10 min, and absorbance was measured at 490 nm(Hernandez-Lopez et al. 1996).

Hemocyte count

A sample of 150 ll of hemolymph was mixed with an Alseversolution (113 m M glucose, 27.2 m M sodium citrate, 2.8 m Mcitric acid, 71.9 m M NaCl), and 10% formalin (v/v). This samplewas chilled (4–8�C) and stored until analysis. Hemocyte werecounted in a Malassez chamber and cellular characterization wasdone taking into account size, form, and granular content of thecells (Le Moullac et al. 1997).

Respiratory burst

Intracellular production of the superoxide anion in hemocytes wasquantified using the NBT (Nitroblue Tetrazolium) reduction toformazan technique (Song and Hsieh 1994). Fifty microliters ofhemolymph diluted in Alsever solution (1:1) were placed in a mi-cro-plate and centrifuged at 300 g for 10 min. Plasma was removedand hemocytes washed with 100 ll of Hank’s solution. Then 100 llof zymosan were added to evaluate active respiratory burst and 100ll of Hank’s solution to evaluate basal respiratory burst. All wereincubated at room temperature for 30 min. After that time, thezymosan and the Hank’s solution were removed and the hemocyteswashed three times with 100 ll of Hank’s solution and stained for30 min with NBT solution (0.3%) at room temperature. After thestained solution was removed, the fixed hemocytes were washedthree times with 100 ll of methanol (70%) and dried for 5 min.Formazan was dissolved with 120 ll of KOH and 140 ll ofDMSO, and the optical density was read at 630 nm using a micro-plate reader (Bio-Rad mod. 550).

Statistical analysis

Descriptive statistical analysis was used to characterize metabolitedata. Normal distribution of blood sample values was analyzedusing a chi-squared test (P<0.05). Thus the base line was con-structed and results expressed as mean±s or as median±quartilerange depending on whether they fit a normal or nonsymmetricaldistribution (Zar 1974). When data did not fit a normal distributionresults were expressed as median±quartile range. When data had anormal distribution, juvenile, male, and female comparisons weredone using an analysis of variance (ANOVA) test. When data didnot have a normal distribution, juvenile, male, and female com-parisons were done using a Kruskal–Wallis nonparametric test

(P<0.05; Zar 1974). When no differences were obtained betweengroups, data were pooled and a new statistical parameter wascalculated and tested for normality and a mean distribution curvecalculated. All the distribution figures were constructed usingclasses that represent an interval of data for each biologicalparameter and were represented as a column in each figure.

Results

Shrimp: sampling, live weight, and maturation stages

A total of 350 shrimp were sampled during 2002, cor-responding to 150 juveniles and 200 adults. Sexualproportion of adults was 1:1 males:females. Of the totalfemales sampled, 69% were in stage II of ovarian mat-uration (immature), 17% in stage III (maturating), and14% in stage IV (mature). Significant differences wereobserved in live weight of juveniles, males, and femalesof L. setiferus with mean values of 10.54, 35.05, and40.33 g, respectively. The frequency distribution of liveweight of juveniles and males showed no normal distri-bution (Fig. 1; Table 1).

Physiological status

Osmotic pressure (OP) was affected by environmentalsalinity. A value of 460 mOsmol/kg was recorded in thewater where juveniles were sampled and 1,000 mOsmol/kg in the adult sampling zone. As a consequence, the OPof L. setiferus juveniles (718.02 mOsmol/kg) was 22%lower than in adults; there were no differences betweensexes. The frequency distribution of OP for juvenilesshowed normal distribution. In contrast adult OP fre-quency distribution did not show a normal distribution(Fig. 1). For this reason a median base-line value of925 mOsmol/kg was proposed for adult L. setiferuscollected during 2002 (Table 1). Osmotic capacity (OC)was significantly different between juveniles and adultswith a value of 224.2 mOsmol/kg for juveniles and)151.89 mOsmol/kg for adults (Table 1). A significantlylower value in juvenile OxyHc was observed. This valuewas 24 and 16% lower than that observed in males andfemales respectively (P<0.05; Table 1). OxyHc wasdistributed normally in all stages (Fig. 1).

Hemolymph glucose, cholesterol, and triacylglycerollevels were not statistically different between shrimpstages and showed no normal distribution (Table 1,Fig. 1). For this reason, median values of 0.19, 0.32, and

Fig. 1 Litopenaeus setiferus. Frequency distribution curves for liveweight, osmotic pressure, oxy hemocyanin (OxyHc), and bloodmetabolites in the entire shrimp population. OxyHc (juveniles,males, and females), juvenile osmotic pressure, juvenile lactatevalues were distributed normally (chi-square; P>0.05). Theremaining hemolymph parameters (adult osmotic pressure; juve-nile, male, and female glucose, cholesterol, and triacylglycerols;juvenile and female protein; male protein; juvenile and adultglycogen; and male and female lactate) were right or left skewed(chi-square; P<0.05)

c

404

405

Table

1Litopenaeussetiferus.Descriptivestatisticsforliveweight(grams),hem

olymphparameters,anddigestiveglandglycogen

ofjuveniles,adultmales,andadultfemales.OxyHc

Oxyhem

ocyanin

Validn

Mean

Confidence

Median

Min.

Max.

Lower

quartile

Upper

quartile

Quartilerange

SD

SE

)95.000%

+95.000%

Liveweight

Juveniles

112.00

10.54

9.48

11.61

9.25

0.80

27.81

6.67

13.68

7.02

5.67

0.54

Males

100.00

35.05

33.40

36.70

34.19

15.31

64.79

29.97

39.21

9.24

8.32

0.83

Fem

ales

60.00

40.33

38.17

42.50

40.04

25.33

64.79

33.64

44.68

39.46

8.38

1.08

Osm

oticpressure,mosm

ol/Kg

Juveniles

102.00

718.02

705.10

730.94

719.00

573.00

914.00

668.00

761.00

341.00

93.00

65.80

Adults

159.00

918.55

909.28

927.81

925.00

715.00

1063.00

895.00

949.00

54.00

59.14

4.69

Osm

oticcapacity,mosm

ol/kg

Juveniles

100.00

224.19

206.18

242.20

242.00

26.00

417.00

147.50

295.00

391.00

147.50

90.75

Adults

159.00

)151.89

)162.52

)141.25

)175.00

)252.00

68.00

)200.00

)118.00

82.00

67.90

5.39

OxyHc,

mmol/l

Juveniles

102.00

1.28

1.21

1.36

1.30

0.31

1.96

1.07

1.53

1.65

0.46

0.39

Males

100.00

1.69

1.61

1.77

1.76

0.70

2.92

1.43

1.92

0.49

0.38

0.04

Fem

ales

60.00

1.53

1.44

1.61

1.52

0.65

2.16

1.30

1.82

1.51

0.33

0.04

Glucose,mg/m

lAllstages

248.00

0.20

0.19

0.22

0.19

0.04

0.70

0.14

0.26

0.13

0.10

0.01

Cholesterol,mg/m

lAllstages

248.00

0.37

0.34

0.40

0.32

0.06

1.20

0.14

0.58

0.44

0.24

0.02

Triacylglycerols,mg/m

lAllstages

247.00

0.44

0.40

0.48

0.39

0.06

2.20

0.21

0.60

0.40

0.32

0.02

Protein,mg/m

lJuveniles

andfemales

348.00

173.07

165.92

180.22

172.07

25.69

417.21

120.85

214.41

93.55

67.79

3.63

Males

148.00

213.07

198.92

227.22

198.04

68.50

429.64

148.53

260.32

111.79

87.09

7.16

Glycogen,mg/m

lJuveniles

82.00

3.10

2.56

3.64

2.64

0.00

20.42

1.69

3.76

20.42

2.07

6.13

Adults

136.00

2.05

1.77

2.32

1.58

0.08

6.49

0.79

3.13

2.33

1.61

0.14

Lactate,mg/m

lJuveniles

44.00

0.11

0.10

0.12

0.12

0.06

0.23

0.08

0.13

0.16

0.04

0.00

Males

57.00

0.19

0.12

0.25

0.09

0.02

1.62

0.05

0.23

1.60

0.17

0.07

Fem

ales

59.00

0.04

0.03

0.04

0.04

0.00

0.10

0.02

0.05

0.10

0.02

0.00

406

0.39 mg/ml were proposed as a base line for glucose,cholesterol, and triacylglycerols, respectively (Table 1).Hemocyanin protein was not normally distributed injuvenile–female and male groups (Fig. 1). Due to thelack of differences between juvenile and female proteinlevels, a median value of 172.07 mg/ml was proposed asa base line. This value was 13% lower than that ob-served in the male group (198.04 mg/ml; P<0.05; Ta-ble 1). Proportions of 52.3 and 81.1% of OxyHc of thetotal protein (OxyHc/Prot) were obtained for male andjuvenile–female groups, respectively.

Digestive gland glycogen showed no normal distri-bution in juveniles and adults (Fig. 1). Digestive glandglycogen was significantly higher in juveniles (medianvalue of 2.64 mg/g) than that observed in adults (medianvalue of 1.58 mg/g; Table 1). Blood lactate showed anormal distribution in juveniles but not in males andfemales (Fig. 1). A higher value of lactate (0.11 mg/ml)was observed in juveniles than in males (0.09 mg/ml)and females (0.04 mg/ml) (Table 1;P<0.01).

Immunological condition

A normal distribution of granular cells was observed injuveniles and females but not in males (Fig. 2). Granularcells (GC) were 36, 44, and 39% of the total hemocytes(TH) observed in juveniles, males, and females, respec-tively (Table 2). Higher GC, hyalin cells (HC), and THconcentrations were observed in males than in juvenilesand females (P<0.05; Table 2). HC were distributednormally in juveniles and females but not in males(Fig. 2).

ProPO showed no normal distribution in any of thestages (Fig. 2). A high proPO activity was recorded inL. setiferus females that was 95% higher than in thejuvenile–male group (P<0.05; Table 2). PO in hemol-ymph was 40% higher in adults than in juveniles with amedian value of 0.10 and 0.06 OD490 nm, respectively(P<0.05; Table 2). ProPO per GC (proPO/GC·102)was 35 and 38% higher in the female group than inmales and juveniles, respectively (Table 2).

Respiratory burst, basal (BRB) and activated (ARB),was divided into two groups, juveniles–males and fe-males, and was distributed normally in both groups(Fig. 2). Female respiratory burst values (BRB andARB) were 40 and 45% higher than those observed inthe juvenile and male groups, respectively (P<0.05;Table 2). Similarly BRB per GC (BRB/GC·102) was 40and 52% higher in females than that obtained in juvenileand male groups, respectively (Table 2). ARB/GC·102was 45 and 94% higher in females than that observed injuveniles and males, respectively (Table 2).

Blood parameters and body weight relation

There was an increment in OxyHc, proteins, PO, he-mocytes, and respiratory burst with an increase in body

weight (Figs. 3, 4). Glucose, TAG, and proPO to liveweight relationships revealed no significant regressions.A significant decrease in cholesterol, glycogen, and lac-tate with an increase in body weight was also observed(Fig. 3).

Discussion

Results show that not all hemolymph parameters ofL. setiferus wild shrimp had a normal distribution. In arecent paper Pascual et al. (2003a) observed that onlyhemolymph protein, one of five hemolymph parameters(the others being OxyHc, glucose, triacylglycerols, andlactate), from cultivated L. vannamei shrimp was dis-tributed normally. In another study, Spicer and Baden(2000) reported that only one of five frequency distri-butions of OxyHc generated for Nephrops norvegi-cus,Liocarcinus depurator, and Hyas aranaeus wasnormally distributed and observed that the frequencydistributions are different for different populations.They proposed that extrinsic (dissolved oxygen, salinity,temperature, food availability, etc.) and intrinsic factors(body weight, sex, reproductive condition, moltingstage, etc.) could be determining the shape of the fre-quency curves.

The L. setiferuspopulation was sampled in bothestuarine and oceanic environments with wide salinitydifferences: 11& salinity in juvenile environment and32& salinity in adult environment. Although salinitydifferences were marked and this affected the osmoticpressure and the osmotic capacity, not all hemolymphparameters were grouped according to differences insalinity. Furthermore, we observed that the parametersthat were grouped according to age (juveniles andadults) and as a consequence were affected by salinity(i.e. osmotic pressure, OxyHc, digestive gland glycogen,hemocytes, and plasma phenoloxydase) were skewed tothe left or were symmetrical, suggesting that the shape ofthe curve was not related to this extrinsic factor.

OxyHc was distributed normally in L. setiferus, andprotein, glucose, TAG, and cholesterol were skewedtoward the left. According to these results, the shape ofthe curve could have biological significance. Hemocya-nin (Hc) is a multifunctional protein with a role asoxygen transporter, storage protein, osmolite, ecdysonetransporter, precursor of anti-fungal peptides, and phe-noloxydase-like activity and is an important bloodprotein component (Adachi et al. 2003; Chen and Cheng1993a, 1993b; Destoumieux et al. 2001; Fielder et al.1971; Gellisen et al. 1991; Pascual et al. 2003a). Due toits physiological relevance, Hc is adequately concen-trated in shrimp to produce a symmetrical curve withvalues grouped mainly around the mean value and onlya few shrimp showing extreme low or high concentra-tions. Taking these points into consideration, there areno reasons to assume that the frequency distribution ofthe Hc could have a different from normal distribution,as can hemocyte concentration and respiratory burst.

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408

According to Spicer and Baden (2000) the differencesobserved in the shape of the Hc curve in different crus-tacean populations are related to the range of sampledlive weights rather than to a biologic factor that affectssuch frequency distributions.

In contrast, protein was left skewed and grouped asfemale–juvenile and male groups. Rosas et al. (2002)demonstrated that cultivated shrimp are well adapted touse protein as a source of energy and as molecules forgrowth; to maintain the homeo-osmoticity; and to pro-duce glycogen and glucose through the gluconeogenicpathway. Furthermore, the immune system has a strongprotein base indicating that immune responses dependupon protein metabolism (Hall et al. 1999; Montano-Perez et al. 1999; Sritunyalucksana and Soderhall 2000;

Vargas-Albores and Yepiz-Plascencia 2000). It is inter-esting to note that protein changes with live weight butat the same time there were no statistical differencesbetween females and juveniles, indicating that somemechanisms in which hemolymph protein are enhancedare operating in juveniles. According to manyresearchers, in low salinity, shrimp are obligated tomove amino acids (free amino acids pool: FAAP) fromcells to reduce the osmotic load. Taking into accountthat this mechanism increases the osmotic load in thehemolymph, shrimp should remove FAAP to reduce thisadditional load. Transfer of FAAP to the gastric fluidsinto the digestive gland provides a mean of minimizingthis additional load (Dall 1975). The digestive gland isthought to be an important organ for hemocyanin syn-thesis, and products from this synthesis might cause anincrease in hemolymph protein content in juveniles ofL. setiferus when FAAP is transferred from blood. Achange in osmotic pressure and higher hemocyanin andprotein concentrations were observed in L. vannameijuveniles exposed to 15& salinity as compared withshrimp maintained at 35& salinity (Rosas et al. 2002),putting in evidence that FAAP in excess could be usedfor the synthesis of Hc and blood protein.

Table 2 L. setiferus. Descriptive statistics for immune response for juveniles, adult males, and adult females. Pro Po Pro-phenoloxidase;OD optical density; PO phenoloxidase; GC granular cells; HC hyalin cells; BRB basal respiratory burst; ARB activated respiratory burst

Valid n Mean Confidence Median Min. Max. Lowerquartile

Upperquartile

Quartilerange

SD SE

)95.000% +95.000%

ProPO, OD 490 nmJuveniles and males 143.00 0.20 0.16 0.25 0.09 0.01 1.62 0.04 0.24 0.20 0.28 0.02Females 45.00 0.39 0.23 0.55 0.11 0.00 1.89 0.06 0.40 1.89 0.53 0.08

PO, 490 nmJuveniles 86.00 0.06 0.05 0.08 0.06 0.00 0.46 0.03 0.07 0.46 0.04 0.00Adults 102.00 0.12 0.10 0.14 0.10 0.03 1.01 0.08 0.12 0.04 0.11 0.01

GC, cells/ml·103Juveniles 74.00 6.80 6.17 7.44 6.80 1.20 13.80 4.80 8.60 12.60 3.80 0.44Males 55.00 13.31 11.68 14.93 12.00 5.80 26.60 7.80 18.20 10.40 6.00 1.48Females 30.00 9.85 7.87 11.84 8.90 2.60 24.20 6.20 12.20 21.60 5.31 0.97

HC, cells/ml·103Juveniles and females 104.00 13.87 12.50 15.25 13.20 0.80 35.40 9.00 17.90 8.90 7.08 0.69Males 40.00 18.50 15.55 21.45 16.20 8.00 48.80 12.20 21.60 9.40 9.21 1.46

Total cells, cells/ml·103Juveniles 74.00 20.07 17.86 22.27 18.60 2.00 46.40 14.00 25.40 44.40 11.40 1.32Males 55.00 31.63 27.89 35.36 27.40 14.60 75.40 21.80 37.60 15.80 13.80 1.86Females 30.00 25.42 21.82 29.02 25.10 5.40 44.00 18.60 29.60 38.60 9.65 1.76

BRB, OD 630nmJuveniles and males 37.00 0.15 0.13 0.17 0.14 0.08 0.28 0.11 0.17 0.06 0.06 0.01Females 15.00 0.21 0.18 0.25 0.20 0.13 0.37 0.17 0.23 0.23 0.06 0.02

ARB, OD 630nmJuveniles and males 37.00 0.22 0.19 0.25 0.20 0.10 0.43 0.16 0.28 0.12 0.09 0.02Females 15.00 0.32 0.27 0.38 0.35 0.15 0.50 0.26 0.38 0.35 0.09 0.02

ProPO/GC·102Juveniles 8.82 8.82Males 9.01 8.33Females 12.18 11.23

BRB/HC·102Juveniles and males 10.81 10.61Females 11.35 12.34

ARB/HC·102Juveniles and males 15.86 15.15Females 17.30 21.61

Fig. 2 L. setiferus. Frequency distribution curves for hemocytesand their products for shrimp populations. Juvenile and femalegranular cells, juvenile–female hyalin cells, female total cells, andrespiratory burst values were distributed normally (chi-squar-e;P>0.05). Juvenile, male, and female pro phenoloxydase (proPO),juvenile and male total cells, male granular cells, male hyalin cellswere left skewed (chi-square; P<0.05). OD optical density

b

409

Glucose, digestive gland glycogen, cholesterol, lac-tate, and TAG were consistently left skewed. In thissense, Pascual et al. (2003a) showed that these blood anddigestive gland molecules are not distributed symmetri-cally in L. vannamei. The biological explanation for thisdistribution is related to shrimp metabolism. Rosas et al.(2001) showed that hemolymph glucose depends upondietary carbohydrates (CHO) and that shrimp havelimitations in processing and absorbing glucose due tothe limited capacity of amylase and the digestive glandfor glycogen storing. In another study Pascual et al.(2004) observed that synthesis of Hc could be related toamino acid synthesis following the transaminationpathway in wild L. vannamei. In these shrimp, bloodlactate elevation together with Oxy Hc suggested thatglycolysis–lactate–pyruvate–amino acids–Hc pathwaycould be activated, accounting for the consistently lowglucose and lactate values observed in L. setiferus(present article) and L. vannamei wild populations(Rosas et al. 2002).

Cholesterol and TAG blood levels are directly relatedto type of food and to reproductive condition (Palacios2000; Palacios et al. 1999; Pascual et al. 2003a). Due toits rapid absorption in tissue, hemolymph cholesterol isconsistently low as has been observed in the presentstudy and in L. vannamei (Pascual et al. 2003a). Lipids

have apparent digestibility coefficients that exceed 90%,indicating that the transport and absorption processesare fast, which might be related to the limited ability ofshrimp for de novo synthesis of polyunsaturated andhighly unsaturated fatty acids (D’Abramo 1997; Gonz-alez-Felix et al. 2003a, 2003b). In this sense, there are noreasons to assume that the frequency distribution ofglucose, digestive gland glycogen, cholesterol, and tria-cylglycerols should be symmetrical if shrimp absorb andaccumulate these nutrients rapidly to compensate fortheir limited capacity for synthesis. In consequence,shrimp will have blood levels of these molecules mainlynear zero and only a few shrimp will have high levels inthe hemolymph in an attempt to preserve all thesenutrients in tissues and not in the hemolymph.

It is interesting to note that a nonsymmetric fre-quency distribution curve was observed for proPO andPO, indicating that at the time of sampling, manyshrimp had granular cells with low proPO activity andfew shrimp had high proPO activity. The proPO con-centration can vary, increasing or decreasing dramati-cally during an infection or during stress depending onthe immunological status of shrimp.

In the case of the white shrimp L. setiferus the mag-nitude of the immunological condition has reproductiveimplications. Previous research demonstrated that theloss of reproductive capacity of adult L. setiferusmalesassociated with melanization and degeneration of thereproductive tract syndromes, induced in captivity, isrelated to an increment in the proPO per granular cell(ProPO/GC) ratio. A reduction in hemocytes togetherwith an increment in proPO activity and an increase in

Fig. 3 L. setiferus. Osmotic pressure, OxyHc and blood metabo-lites as a function of body weight of shrimp population. The solidlines are the best fit for all the data. Dashed lines indicate the 95%confidence interval of the regression model. OxyHc and protein tolive weight relations were positive; whereas cholesterol, glycogenand lactate to live weight relations were negative

410

proPO/GC proportion coincided with a reduction inspermatic cells when shrimp were exposed to 33�C for5 days (Pascual et al. 2003b). We observed that tem-perature stress provoked an increment in immune re-sponse and loss of the mechanisms that control theimmune system. From that study, we concluded thatproPO-filled hemocytes migrated to the vas deferens,where spermatic cells are attacked in their travel to theampule, before spermatophore formation. These previ-ous results could explain why, in the present article,whereas granular and hyalin cells were significantlyhigher in males, the proPO activity and respiratory burstwere lower than in females and similar to juveniles ofL. setiferus. According to Bachere (2000) and Johanssonet al. (2000), granular cells are filled with differentzymogens that, together with proPO and hyalin cells,work to defend shrimp against foreign material. ProPOis synthesized in hemocytes whereas hemocytes aresynthesized in the hematopoietic tissue; hence, the twoprocesses develop at different times according to shrimpdefense requirements. It is obvious from the present re-sults that adult males of L. setiferus have high hemocyteconcentration but a lower immune condition, reflectingan adaptation associated with the maintenance of arelatively low immune system and a high reproductivecapacity.

OxyHc and protein in L. setiferus increased with liveweight, indicating that protein metabolism changes withthe age of shrimp. In contrast to the relative stability ofthe sea, the waters of estuaries are characterized bysudden and often extensive changes in salinity andtemperature. Furthermore, a depletion of dissolvedoxygen can occur in brackish water following tempera-ture changes or in the presence of heavy organic load,affecting the physiological condition of L. setiferus(Rosas et al. 1997, 1998). In this sense, shrimp such asL. setiferus, whose juveniles live in estuarine environ-ments, must be adapted to cope with continuous orirregular fluctuations of environmental factors. Inshrimp, several physiological and immunological func-tions are activated during a saline change. In lowsalinity, shrimp require protein as a source of aminoacids to maintain the osmotic pressure since duringsalinity acclimation, a very rapid change in free aminoacid content occurs (Claybrook 1983; Gerard and Gilles1972). According to Rosas et al. (2001), protein metab-olism is basic to ensure that amino acids allow shrimp tocompensate for their osmolarity. For this reason shrimpare more dietary-protein-dependent in diluted environ-ments and the protein hemolymph levels are lower injuveniles living in low salinity than in adults living in thesea. Taking into account that Hc is considered a mole-cule that stores amino acids, it is not surprising that Hcfollowed the same behavior of proteins, with low valuesin juveniles and high values in adults.

Estuarine systems offer protection and food to juve-niles. For this reason the estuarine system has beenclassified as a nursery zone for many crustacean species.

Fig. 4 L. setiferus. Hemocytes and their products as a function ofbody weight of shrimp population. The solid lines are the best fit forall the data. Dashed linesindicate the 95% confidence interval of theregression model. All immune responses were positively related tolive shrimp weight, indicating that immune responses increase withage. ARB Activated respiratory burst; BRB basal respiratory burst

411

A negative relation between cholesterol, lactate, anddigestive gland glycogen to body weight has been ob-served in L. setiferus revealing that juveniles living inestuarine systems are accumulating reserves. It is inter-esting that these three nutrients are basic for growththrough their role during molting. Cholesterol is thebase of membrane construction, participating in colla-gen synthesis, and as a precursor of ecdysone hormoneduring molting processes, and glycogen and lactateparticipate actively in glucose metabolism as well as inchitin synthesis. Thus, it is to be expected that thesemolecules are accumulated in juveniles when the prin-cipal activity during this stage is growth (Ceccaldi 1998;Teshima 1998).

The Gulf of Mexico has a tremendous ecologicalimportance because of its biological diversity. At thesame time, the oil industry, fisheries industry, andtransportation are growing, increasing pressures onmarine and coastal ecosystems (Ridgwaya and Shimm-ieldb 2002). Our results in the present article could serveas the basis for using L. setiferus as environmental sen-tinels, as non-controversial and inexpensive models toevaluate the potential changes that human activitiesexert on this important marine ecosystem. Currently, thepresent report provides available indicators of nutri-tional status of juveniles and adults and indicators ofimmune responses that can reveal changes in trophicrelations or health problems at the population level.

Acknowledgements This research was partially financed by a UC-MEXUS grant to C.R. and E.L.C. We thank Tomas Jesus Garcıafor L. setiferus sampling.

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