penaeid prawns are important subjects of mariculture and coastal fisheries activities throughout the tropics and subtropics [ 13 ] .  
traditionally , prawn farms located near the coast used directly coastal seawater to rear the prawns without additional processes .
shrimp culture is therefore potentially affected by anthropogenic activities including metal contamination . specifically in coastal mexican waters ,
a great number of basic metal industries represent the mainly heavy metal ( cadmium , lead , nickel , chromium , etc . ) source [ 49 ] .
penaeid prawns have revealed great capacity to accumulate and take up metals from solution , either essential or not essential elements and yet survive in these polluted environments [ 1014 ] .
thus , it is of great concern to investigate detoxificatory processes involved in prawns exposed to metals .
it is well known that the induction of metallothionein which is a cystein - rich protein and has detoxifying properties , occurs in aquatic invertebrates after in situ or laboratory metal exposure ( for review see amiard et al .   and
, physiological markers reflecting the energetics of an organism may contribute to the understanding of the mode of the toxicant [ 1618 ] .
the  metabolic cost  hypothesis suggests that toxic stress induces metabolic changes which may lead to a depletion of its energy reserves resulting in adverse effects on growth and reproduction .
comprehension of the mechanisms related to the sublethal effects caused by toxic metals upon shrimp metabolism would help to develop sensitive and diagnostic tools ( biomarkers ) with a predictable capacity in assessing the toxic effects , thus contributing to better coastal seawater management and sustainable aquaculture .
this study is part of a wider investigation of the regulation and accumulation of the trace metals nickel and lead in a penaeid prawn penaeus vannamei ; metals previously detected in coastal mexican waters [ 48 ] .
nickel has occasionally been interpreted as an essential metal [ 21 , 22 ] , probably as a consequence of its ability to be substituted for other divalent metal ions , particularly zinc , or as a part of an enzymatic structure [ 23 , 24 ] .
this paper specifically investigates both metallothionein - like proteins and energy reserve levels in the hepatopancreas and abdominal muscle of penaeus vannamei , after sublethal exposure of the prawns to dissolved nickel and lead .
juveniles of penaeus vannamei ( between 13.0  cm total length ; mean dry weight 11.0  8.0  mg ; ) were obtained from cultures at aquapacific s.a de c.v . ,
prawns were maintained in artificial sea water ( instant ocean ( io ) , aquarium systems ) at 25 salinity ( ph 7.8 ) , 12  :  12 light / dark periods and 25c as previously described [ 13 , 14 ] .
animals were fed every two days with commercial flakes ( tetramin ) for 15 minutes maximum , before water changes . the use of artificial seawater in all experiments provided physicochemical stability and replicability for trace metal uptake tests .
the dissolved metal concentrations used in laboratory exposure of prawns are environmentally realistic for contaminated mexican coasts [ 27 , 28 ] .
the experimental design involved nine separate experimental groups containing ten prawns each : controls ( not metal exposed ; three groups ) , prawns exposed to 55.5  g  l nickel ( two experimental groups ) , 191  g  l lead ( two experimental groups ) , and same concentration of nickel / lead mixture ( two experimental groups ) in io at 25 salinity and 25c .
the hepatopancreas and abdominal muscle were removed from one control group at the beginning of the experiment , as described by nunez - nogueira and rainbow .
prawns from the remaining two control and metal - treated groups were dissected after 10 days of exposure .
samples destined for biochemical analysis were treated as described by nunez - nogueira et al . .
briefly , samples were transferred into plastic vials to be immediately frozen in liquid nitrogen for a few minutes . after freezing ,
then , samples were homogenized in a buffer solution ( 20  mm tris , 10  mm -mercaptoethanol , 150  mm nacl solution adjusted to ph 8.6 ) .
the soluble and insoluble fractions were separated by centrifugation at 25,074  g for 55 minutes at 4c ( biofuge 28  rs , heraeus sepatech ) .
the cytosolic heat - stable compounds including metallothionein were isolated by centrifugation of the soluble fraction ( 12000  g for 10 minutes at 4c ) after heat - treatment ( 75c for 15 minutes ) . in the heat - denaturated cytosol ,
the amount of mt was determined by differential pulse polarography ( dpp ) according to thompson and cosson   and olafson and olsson .
a mde 150 stand polarographic ( radiometer copenhagen ) tracelab 50 , controlled by the computer software tracemaster 5 through a polarographic analyser pol 150 was used .
the method of standard addition was used for calibration with rabbit liver mt ( sigma chemical co. , st louis , mo , lot .
polarographic determination in heat - denaturated cytosol is an analytical procedure based on several characteristics of mts , but it does not allow , with certainty , the assertion that the target molecule is a true mt unless purification and sequencing are carried out . strictly , therefore , what has been measured is the concentration of proteins with metallothionein properties , that is , metallothionein - like proteins ( mtlp ) .
all glass labware was soaked in 10% hcl , rinsed three times with deionized water and dried in a desiccator protected from atmospheric dust .
the insoluble and soluble fractions were heated ( 75c , 12  h ) with suprapure hno3 acid ( carlo erba ) .
after digestion , metal levels in these acid solutions were determined after dilution with deionized water by flame aas using the zeeman effect ( shimatzu 8600-aa spectrophotometer ) .
standard addition analyses were performed in an isomedium and concentrations of each element were 25  mg metal ml for aas .
the analytical methods were validated by external intercalibrations as previously described nunez - nogueira et al .   and use of reference material tort-2 ( lobster hepatopancreas ) with more than 90% recovery .
total metal concentrations were recalculated from summation of quantities of trace elements in soluble and insoluble fractions determined previously , by combine means procedure .
the results were expressed in gg dry weight of the organ for ni and ngg in case of pb , respectively .
hepatopancreas and abdominal muscle samples were homogenized with a porcelain mortar and pestle at 2c .
the powder obtained was homogenized in a motorized grinder with 1.5  ml citrate buffer ( ph 5.0 ) for glycogen and lipid analyses .
total lipids were determined by the sulfophosphovanillin reaction , according to frings et al . , while glycogen concentration were determined in two aliquots of the homogenate , using enzymatic digestion by amyloglucosidase , according to carr and neff .
olive oil and glycogen from oysters ( sigma type iii ) were used as standards for each method , respectively .
students t - test ( p < .05 ) were performed for protein , lipids , glycogen , and metal concentration comparisons .
tests were developed for small samples according to williams , and carried out in statistica 5.1 for windows ( statsoft inc . ) .
the mtlp concentrations in hepatopancreas and abdominal muscle of juveniles of penaeus vannamei are illustrated in table 1 .
mtlp concentrations were higher in hepatopancreas than in abdominal muscle in all experimental organisms ( control , ni exposed , pb exposed , ni+pb exposed ) . in hepatopancreas , the difference in mtlps concentrations between the control and ni - treated shrimps were not significant , while a significant increase in mtlp concentrations was observed in pb - treated and metal mixture - treated organisms versus controls .
the mtlp concentration ranged from 6.83 to 31.04  mg g in controls , 9.46 to 62.04 in ni - treated , 31.79 to 121.85 in pb - treated , and 36.11 to 94.83 
mg g in metal mixture - treated , respectively . in abdominal muscle , a significant increase in mtlp concentrations was observed in ni - treated and pb - treated prawns in comparison with controls whereas no differences in mtlp concentrations were detected in metal mixture treated organisms versus controls ( apparently due to the presence of two muscle samples ( out of ten ) with higher mtlp concentrations within the pb - treated prawns , suggesting noninfluence of ni and/or pb in mtlp induction in muscle , even when an apparent difference was detected in one metal treated groups ) . the mtlp concentration in muscle tissue ranged from 0.30 to 0.83 in controls , 0.30 to 0.99 in ni - treated , 0.54 to 1.54 in pb - treated , and 0.51 to 0.60  mg g in metal mixture - treated , respectively .
ni and pb concentrations ( total , soluble , insoluble ) in the hepatopancreas and abdominal muscle of penaeus vannamei are indicated in tables 2 and 3 , respectively .
ni concentrations were significantly higher ( p < .05 ) in the hepatopancreas versus the abdominal muscle of prawns , pb was found in major quantities in abdominal muscle . the measured total ni concentration in hepatopancreas ranged from 3.67 to 21.37  g  g ( controls ) , 1.98 to 11.86 ( ni - treated ) , 1.11 to 22.84 ( pb - treated ) , and 3.86 to 56.23  g  g ( mixture - treated ) , respectively . due to a great individual variability ,
no significant differences were observed among groups , including ni - treated . in abdominal muscle , ni concentration ranged from 0.02 to 0.03  g  g in each group and not differences against controls were observed ( table 2 ) .
the distribution of ni among the soluble and insoluble fractions in the hepatopancreas and the abdominal muscle of prawns are shown in figures 1(a ) and 1(b ) , respectively . in hepatopancreas ,
this metal was nearly equally distributed between soluble and insoluble fractions ( figure 1(a ) ) whereas in the abdominal muscle ( figure 1(b ) ) , the soluble fraction was more important than the insoluble fraction .
lead was detected only in the insoluble fraction of both tissues ( table 3 ) .
the mean pb concentrations in all groups were below 0.17  mg  kg in abdominal muscle and 0.003  mg  kg in hepatopancreas . a slight increase ( not significant )
was observed in the hepatopancreas in prawns exposed to this metal , either alone or in a mixture .
the concentration of lipids and glycogen , determined in hepatopancreas and abdominal muscle are shown in figures 2 and 3 , respectively .
lipid concentration in abdominal muscle ranged from 1.08 to 16.45  mg  g dw ( controls ) and from 0.86 to 18.30  mg  g ( metal - treated ) . in hepatopancreas , lipid concentration ranged from 1.85 to 48.95  mg  g dw ( controls ) and from 6.53 to 88.66  mg  g ( metal - treated ) .
no significant differences were observed between controls and treated prawns in the case of lipids .
the range of glycogen was from 0.51 to 23.60  mg  g ( controls ) and 1.60 to 15.05  mg  g ( metal - treated ) in abdominal muscle , while it was from 4.72 to 8.89  mg  g ( controls ) and 1.07 to 11.33  mg  g ( metal - treated ) in hepatopancreas , respectively .
glycogen concentration in abdominal muscle from pb - treated prawns was the only one significantly ( p < .05 ) different from controls . in this case ,
different studies have shown the presence of heavy metals , including ni and pb in mexican coastal waters [ 46 ] .
for example , coastal zones in tabasco and veracruz states ( gulf of mexico ) have shown lead concentration in water between 65 to 210  g  l , while others like cr , hg , and cd between 0.5 and 15  g  l       .
these concentrations can be above the national recommended level ( local legislation ) , as have been observed in the mexican pacific coast .
the pacific white prawn penaeus vannamei spends part of its life cycle in coastal or estuarine areas , making it suitable for metal exposure .
this species showed capacity to induce metallothioneins by metal exposure [ 36 , 37 ] and accumulate metals [ 12 , 25 ] .
total ni concentrations   found here were in good agreement with previous studies that show ni regulation in decapod crustaceans [ 38 , 39 ] , including p. vannamei .
nickel was found almost equally distributed between the soluble and insoluble fraction ( table 2 ) .
these concentrations are within values previously described in penaeid prawns ( including p. vannamei ) [ 40 , 41 ] for these tissues .
the reason of this lack of increase compared to controls must be related to the fact that both metals are regulated or partially regulated ( in case of lead as a nonessential metal ) by this prawn .
previous studies have shown that ni can be regulated by decapods crustacean [ 39 , 42 ] providing a capacity to maintain a minimal trace level concentration in their bodies . in case of pb ,
vogt , and quinitio   suggested that this non - essential metal is eliminated by forming insoluble - lead rich deposits that are excreted after lysosome autolysis , process that can include mt content during protein turnover   ( see below ) . due to
that metal exposures do not exceed the lc-50 for 96 hours in both cases for the experimental conditions here tested , and are far below these values ( 60.54  mg 
l for pb , resp . ) , suggests that such a trace level of exposures is not enough to reach the threshold level of regulation in case of penaeus vannamei without significant accumulation .   according to the results   obtained here ,
mtlp appear to be induced by pb in hepatopancreas under the experimental conditions tested ( table 1 ) .
prawns exposed only to ni in solution did not show a significant increase in tissue concentration , while prawns treated with pb either alone or in a mixture with ni , almost double the amount of mtlps observed in controls and ni - treated groups ( table 1 ) .
most of the lead was detected in the insoluble fraction of the hepatopancreatic samples ( table 1 ) , suggesting a nonsoluble lead - rich deposit .
some authors have suggested that mt 's induction is an intermediate step before insoluble deposits are formed [ 45 , 46 ] .
lead - insoluble deposits or granules have been observed in terrestrial and aquatic invertebrates [ 4751 ] .
rainbow   highlighted that isopods accumulated metals in detoxified granules ( non - soluble ) with rapid elimination of lead , compared to other metals ( e.g. , cadmium ) .
complementary to vogt and quinitio , our results suggest that lead is mainly stored and detoxified within granules , instead to mtlp 's , perhaps at the antennary gland as in penaeus monodon , and excreted as an apocrine secretion in the urine [ 49 , 50 ] , not only at high - lead level of exposure , as was proved in p. monodon .   in this way
, lead appears to be treated as some essential metals , in respect of quelating cytosolic molecules .
this difference in detoxification between essential and non - essential metals was already observed in p. vannamei   and penaeus indicus , where mtlps appear to be the first detoxification strategy involved in non - essential metals exposure , while essential metals are regulated in cytoplasm by another method ( e.g. , insoluble deposits ) .
it is also considered that mtlps increase slightly with time and doses as a result of a constant turned over . in the abdominal muscle , slight increase in mtlp concentration was observed in ni- and pb - treated , but not in a mixture ( table 1 ) .
no significant relationship ( not shown ) between soluble ni and mtlp was observed . comparing the total metal concentration between controls and treated groups in muscle ,
no significant increase was detected , suggesting that these increases in mtlp could be related to other factors , like normal homeostasis of other essential metals ( e.g. , zn or cu ) and inflammation process caused during experiment procedure , rather than metal exposure [ 15 , 54 , 55 ] .
this idea is also supported by the lack of mtlp induction in muscle in metal mixture - treated group ( table 1 ) .   
it has been proved , that longer period of exposure and higher metal concentrations induced mtlps in crustaceans and aquatic or terrestrial invertebrates [ 36 , 5658 ] .
our results obtained here suggest that it is necessary to carry out bioassays with different metal concentrations near ( below ) the lc50 , to identify the threshold level for mtlp induction for these metals in this species .
a recent work in our lab , revealed that using the following metal concentrations : 15.6 , 31.3 , 62.5 , 125 and 250  mg 
l for pb , for 96 hours of exposure , are higher than the environmentally realistic metal concentrations tested ( 55.5  g  l for ni and 191.2  g  l for pb , resp . ) , and far below the lethal doses obtained , in good agreement with our results , where the trace level of exposure only   inducing minimal or no detectable changes in the metal body concentrations .   
it is widely recognized that both ni and pb share chemical properties and features with class b and borderline metals , like copper , zinc , arsenic , cadmium , and mercury [ 59 , 60 ] . within these metals , cadmium , copper , mercury , zinc , and arsenic
have been identified as metallothionein inductors [ 56 , 57 , 6164 ] .   due to these
the first involves zinc / copper substitution by these metals in the active sites of the cystein - rich proteins ( metal - clusters ) or secondly by direct generation of metal element responses ( mers ) inside the cells . in the first case , nickel or
lead compete against zinc for the active clusters sites of the mt or generate an exchange ni / pb versus zn , inducing and increase in the zinc intracellular concentration and its bioavailability , promoting an homeostatic response by increasing the amount of mts .
this phenomenon has been observed in other invertebrates [ 56 , 63 ] and vertebrates [ 65 , 66 ] , and metal substitution in metallothioneins has been registered in different cases [ 56 , 58 , 64 ] , for example , cadmium in crustaceans . in the case of mers production , the mtlp induction might be related to free - radicals generation and/or oxidative damage that activate a metal transcription factor capable to interact with the promotor region of the mt gene by a positive feedback .
these results are in good agreement with previous nickel and lead reports as mt inductors , supporting that this inductive capacity is present not only in in vitro tests [ 63 , 66 ] . one interesting observation relay on the relationship observed between the amount of mtlps concentration in hepatopancreas of controls and the whole body weight , which almost reaches 3% ( table 3 ) .
metallothioneins are considered as homeostatic proteins , widely present in aquatic and terrestrial invertebrates either from contaminated and noncontaminated sites [ 56 , 61 , 6769 ] .
an explanation could be related to a reduction in the tissue weight , due to   lower quality food used during captivity , or stressful conditions during acclimation , producing a minor difference between tissue mass and mtlp content .
weight is a factor proved that affect the mt concentration in soft tissues of aquatic invertebrates [ 67 , 68 ] , however , this appears to be scarcely possible due to the fact that mean weight of the hepatopancreas was between 8 and 9  mg ( dry weight ) in all experimental groups , including controls , representing nearly 6% of the whole body weight , in good agreement to the proportion established in healthy decapods .
the hepatopancreas of p. vannamei showed traces of pb and relatively amounts of ni in controls ( 0.002 and 6.41  g  g , resp . ) , which might be triggering the defensive and regulatory mechanisms of mts , explaining why the mtlp concentration in this group almost reaches 3% of the tissue weight , highlighting that previous metal exposures increase the original amount of mtlps .
higher concentrations of energy reserves , mainly lipids , and mtlps in the hepatopancreas are in good agreement with its function of main organ for metabolic reserves and metal - binding proteins .
energy reserves are of importance considering ( among others ) its use in detoxification processes [ 7274 ] . in this respect ,
the concentration of lipids ( figure 2 ) and glycogen ( figure 3 ) did not change in the hepatopancreas . in abdominal muscle , a slight increase in the lipid and glycogen fraction occurred , suggesting a possible effect of pb on these reserves , but no correlation was observed with metal concentration in the tissue .
toxic pb exposures have been considered a cause for a decrease in metabolic rates in postlarvaes of p. indicus ( according to satyavathi   and chinni and yallapragada ) , causing perhaps   the higher glycogen concentration observed in the abdominal muscle , as a result of lower degradation rates by glycogen phosphorylase and an increase in the carbohydrate synthesis by the glycogen synthase .
however , the low pb concentration detected in the muscle , suggested that other stressful factors could be involved , and not necessarily related to the metal presence in the tissue .
previous studies in prawns and other crustaceans , have shown that under some particular stressful conditions ( e.g. , salinity and metal conditions ) , the energy budget can be modified in opposite direction . in some cases ,
energy production can be altered by metals like mercury and zinc by increasing the lactate dehydrogenase activity , enzyme involved in energy production , as was observed in the crab carcinus maenas .
the fact that no significant changes were observed in the hepatopancreas , which showed to be the main target organ for pb , suggests that the concentration of metal in solution tested in this study is not sufficient to compromise the amount of energy available in these tissues , and it is enough to cover the detoxification process trigger under the stress simulated in the hepatopancreas , or involves alternative mechanisms to mitigate the adverse effect .
it is possible that higher metal concentrations , near lethal concentrations and longer exposure periods , could induce significant changes in the energy reserve , as has been observed in p. indicus exposed to pb , but further studies are required in p. vannamei .
this study shows the first results of mtlp quantification and energy reserves in the estuarine prawn p. vannamei exposed in the laboratory to ni and pb . it is assumed that the mtlp induction in the hepatopancreas is caused by metal stress ( presence of pb and ni ) and did not influence the energy metabolism .
this protein induction appears to be related either to the detoxification process of this non - essential metal ( pb ) and by essential metal regulation ( ni ) . in case of ni
, the mtlp concentration could be considered as the basal level for cellular homeostasis of the essential metal .
the experimental conditions tested do not modify the energy budget present in both tissues at the sublethal exposure tested .
mtlp concentrations could not be considered as a biomarker , at least in the hepatopancreas of p. vannamei , for the realistic metal concentration tested here , in environmental risk studies .
further studies involving different metal concentrations ( just below the lethal level ) and exposure times will help to define the threshold level for mtlps and energy reserves changes in this species .
 this study analyzed the changes in metallothionein - like proteins ( mtlps ) and energy reserves ( ers ) in hepatopancreas and abdominal muscle of the white prawn penaeus vannamei . 
 realistic metal concentration exposure for 10 days to ni and pb in solution revealed that juvenile prawns partially induce mtlp in hepatopancreas after pb exposure . 
 ni was distributed equally between soluble and insoluble fractions , while pb was present only in the insoluble fraction , suggesting different detoxification strategy . 
 no changes in lipids and glycogen concentration were detected under these experimental conditions in both tissues analyzed . 
 mtlp could not be considered as a suitable indicator for lead exposure in hepatopancreas . 
1. Introduction
2. Material and Methods
3. Metal and Metallothionein-Like Proteins Analyses
4. Energy Reserves Analyses
5. Results
6. Discussion
7. Conclusions