OCEANOGRAPHY and MARINE BIOLOGY: AN ANNUAL REVIEW (Volume 44) - Chapter 2 potx

This review aims to give an overview of the role of manganese in aquatic animals, its routes of uptake, and biological effects, mainly focusing on marine crustaceans living in and on the

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Oceanography and Marine Biology: An Annual Review, 2006, 44, 61-83

Taylor & Francis

ROLE, ROUTES AND EFFECTS OF MANGANESE

IN CRUSTACEANS

SUSANNE P BADEN* & SUSANNE P ERIKSSON

Göteborg University, Department of Marine Ecology, Kristineberg Marine Research Station, S-450 34 Fiskebäckskil, Sweden

in biota Manganese may occur in toxic concentrations (10–20 mg l–1) in the bottom water of marinecoastal areas after hypoxia, or more locally (e.g., close to industries) as well as in acidic lakes andaquaculture shrimp ponds Though manganese is an essential metal, it is also an unforeseen toxicmetal in the aquatic environment Although the uptake and elimination of manganese is rapid,manganese affects processes that decrease the fitness of organisms As manganese bioavailabilityincreases, its uptake is predominately through the water The midgut gland, nerve tissue, bloodproteins and parts of the reproductive organs have the highest accumulation factors and are themain target tissues The functional effects of manganese in aquatic environments are still sparselyinvestigated Recent results show that the immune system, the perception of food via chemosensoryorgans and a normal muscle extension are affected at manganese concentrations observed in the field

Geochemical role of manganese

Manganese is the 12th most common element, the fourth most abundant metal and is universallydistributed in the earth’s crust and waters (Anonymous 2005) This metal is involved in a largenumber of chemical processes, due mainly to its redox sensitivity The literature on manganese (Mn)geochemistry in the aquatic environment is immense (Elderfield 1976), whereas literature on theoccurrence and biological effects of manganese in aquatic animals is comparatively sparse Man-ganese concentrations in soil vary from 0.001–7 mg g–1 dry weight (dw), averaging 0.75 mg g–1 dw(Saric 1986) Ocean sediment concentrations vary from approximately 1–50 mg g–1 dw (Elderfield1976) Since the 1800s an intensive and ongoing debate has been centred on the origin and amount

of the manganese flux to the oceans Three main sources have been identified: continental ing (lithogenous origin), submarine volcanism and an upward migration in porewaters as a conse-quence of sediment diagenesis (Elderfield 1976) The anthropogenic supplies of manganese toaquatic biotopes derive mainly from mine tailings and from steel manufacturing industries whereapproximately 90% of total manganese is used as a deoxidising and desulphurising additive and

weather-as an alloying constituent (Saric 1986) Manganese (MnO2) is also widely used in dry cell batteries(Saric 1986), as a contrasting agent for nuclear magnetic resonance tomography, and as an agri-cultural fungicide (Gerber et al 2002) A manganese antiknock additive (methylcyclopentadienylmanganese tricarbonyl (MMT)) was introduced to Canada in 1990 to substitute for lead in fuel,

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SUSANNE P BADEN & SUSANNE P ERIKSSON

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and since 1995 MMT has also been used in several states of the USA (Shukla & Singhal 1984,Davis 1998, Normandin et al 2002) In the rapidly expanding shrimp farming industry of tropicalregions, manganese is added to shrimp ponds in the form of potassium permanganate (KMnO4),

as disinfectant, in concentrations causing potential hazards to life in the ponds and in the coastalzone close to the effluent water (Gräslund & Bengtsson 2001, Visuthismajarn et al 2005).Manganese becomes bioavailable as Mn(II) in water when it is reduced by hypoxic/anoxicconditions in sediment The reduction of manganese dioxides occurs during the degradation ofsedimenting organic matter (Dehairs et al 1989) The process is directly or indirectly microbiallymediated but is fastest when sulphide and Fe(II) are reductants (Johnson et al 1991) In general,the solubility (and bioavailability) of manganese increases with decreasing oxygen tension and pH,but not with increasing temperature (Wollast et al 1979, Faust & Aly 1983)

During oxic conditions in bottom water, sediment porewater may contain Mn concentrations

of 0.16–24.0 mg l–1 (Canfield et al 1993, Aller 1994, Magnusson et al 1996), whereas bottomwater concentrations are between 0.18–16.5 µg l–1 (Laslett & Balls 1995, Hall et al 1996) Duringhypoxia (O2 < 3 mg l–1)), the Mn(II) of the bottom water can increase by several orders of magnitude

to 1.5 mg l–1, as in the Kiel Bight (Balzer 1982), and up to 22 mg l–1 in the anoxic bottom water

of the Orca Basin in the Mexican Gulf (Trefry et al 1984)

This review aims to give an overview of the role of manganese in aquatic animals, its routes

of uptake, and biological effects, mainly focusing on marine crustaceans living in and on thesediment As the biological chemistry of manganese is poorly explored in invertebrates, the relevantmedical and biological literature on basic processes involving Mn in vertebrates is cited

Biological role of manganese

Essentiality

Manganese is an essential trace metal for metabolism belonging to the borderline elements (Mn, Fe,

Co, Ni, Cu, Zn, Cd, Hg, Pb) of the periodic table It lies between the oxygen-seeking elements ofclass A (Na, Mg, K and Ca being the most abundant) and the sulphur- and nitrogen-seeking elements(including heavy metals like Ag, Au and Hg) of class B, and thus exhibits aspects of both classes(Nieboer & Richardson 1980) In its divalent form, Mn(II), manganese has a relatively high affinityfor sulphur or nitrogen in functional groups of proteins and other molecules, which enables Mn tointerfere in a wide spectrum of biological processes (Simkiss 1979, Williams 1981) The divalentMn(II) exchanges water and ligands rapidly and the binding constant of the metal in proteins is weak.Manganese is important as a cofactor or activator of different enzymatic reactions (e.g., electron-transfer reactions, antioxidant defences, and phosphorylation) (Simkiss & Taylor 1989) In the case

of enzymes containing metal ions (mainly Mg(II), Mn(II) and Zn(II)) the metal ion itself can bindwith groupings in the substrate and act as a strain-producing agent by forming a chelated interme-diary compound At the same time the metal ion, because of its positive charge, is an efficientelectrophilic agent that can act as an effective participant in the reaction (White et al 1973).Examples of enzymatic reactions having Mn as an activator are acetyl-CoA carboxylase (the firstreaction in the fatty acid formation in the endoplasmatic reticulum), pyruvate carboxylase (in themitochondrial formation of oxaloacetate), glycylglycine dipeptidase (in the degradation of dena-tured intracellular proteins) and the well-known Mn-super oxide dismutase (Mn-SOD) (a redoxenzyme in the mitochondria facilitating the production of dioxygen) (Cotzias 1958, White et al

1973, da Silva & Williams 1991)

Manganese is mainly accumulated in organelles like the mitochondria, Golgi apparatus andvesicles, whereas concentrations in the cytoplasm are relatively low These concentration gradientsare sustained by metal transporters over the membrane (e.g., Luk & Culotta 2001) The elimination

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ROLE, ROUTES AND EFFECTS OF MANGANESE IN CRUSTACEANS

of Mn(II) from the mitochondria is a slow, energy-requiring, Na-dependent efflux mechanism(Gavin et al 1999)

In general, those metals having an essential biochemical role, such as the metals mentionedabove, are regulated at the individual level, while for non-essential metals such as mercury (Hg),cadmium (Cd) and silver (Ag) there is only weak evidence of controls on accumulation Underconstant ambient conditions, the net balance between inward and outward fluxes of metals providesthe underlying control on tissue burdens and, in general, metals that exchange rapidly tend to beaccumulated less efficiently than metals that exchange slowly Accumulation may give rise to bodyconcentrations in excess of four orders of magnitude above background in non-regulating organisms(Rainbow 1992, 1997)

a chelating treatment of CaNa2EDTA (Hernandez et al 2003) Another much debated theoryconnects excess Mn exposure with the initiation of transmissible spongiform encephalopathy (TSE),also called scrapie in sheep and Creutzfeldts Jacobs disease (CJD) in humans Imbalance of Mnand Cu is established when Mn- and Cu-chelating insecticides (organo-phosphates) are taken up

at the same time, giving a substitution of Cu with Mn as Mn(III) in the CNS prion protein Thissubstitution conforms the prions, preventing their degradation, and TSE may develop (Purdey 2000)

As Mn(III), manganese is able to accumulate in the brain, likely carried through the brain barrier via transferrin and receptor-mediated endocytosis (Simkiss & Taylor 1989, Aschner &Aschner 1991) Transferrin is a protein containing a Fe-cluster crucial for absorption, transport,storage and excretion of Fe in mammals and is able to cross the otherwise relatively impermeableblood-brain barrier Manganese may mirror Fe and bind to transferrin, not necessarily replacing

blood-Fe, and in this way passes the blood-brain barrier (Aschner & Aschner 1991) Within the brain themain part of Mn(III) appears to release from transferrin and concentrate in certain parts via axonaltransport (Henriksson et al 1999) In freshwater crayfish a structural analogue to the vertebrateblood-brain barrier called the glial perineurium, has been identified The glial perineurium ensuresprotection of the CNS by having a high degree of ion selectivity and regulation (Butt et al 1990)

A direct uptake from the media through the nasal chamber in rats and olfactory chamber of pike

(Esox lucius) followed by axonal transport along primary and secondary neurones into the olfactorybulb has been documented (Tjälve et al 1995, 1996) A similar uptake and transport into nervetissue of invertebrates has not been described to date

Hydrated Mn has an ionic ratio close to that of Ca(II), and its ability to affect various aspects

of neuronal transmission has been ascribed primarily to its mimicry of Ca (Aschner & Aschner1991) Manganese ions are known to affect various steps in the chemical synapses of nerve-muscletransmission in a wide range of animal groups At low concentrations, Mn ions have been found

to pass through Ca channels in a number of different preparations, e.g., giant squid axons (Yamagishi1973), mammalian cardiac muscle (Ochi 1970, 1975; Delahayes 1975), mouse oocytes (Okamoto et al

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1977), starfish eggs (Hagiwara & Miyazaki 1977), larval beetle skeletal muscle fibres (Fukunda &Kawa 1977) and frog skeletal muscle fibres (Palade & Almers 1978) However, at higher concen-trations Mn ions are potent inhibitors of synaptic transmission (Katz & Miledi 1969, Ross & Stuart

1978, Xiao & Bevan 1994) and also act as competitive inhibitors of Ca ion flow through calciumchannels in muscle membranes (Fatt & Ginsborg 1958, Hagiwara & Takahashi 1967, Takeda 1967,Mounier & Vassort 1975) Manganese affects not only the presynaptic site of action but also thepostsynaptic site (Katz & Miledi 1969) This is consistent with earlier studies on the excitation-contraction coupling mechanisms in crustacean muscles, which indicated that Mn ions competewith Ca ions to pass through sarcolemmal calcium channels and thus affect muscle membranedepolarisation (Fatt & Ginsborg 1958, Hagiwara & Nakajima 1966, Chiarandini et al 1970,Mounier & Vassort 1975) More recently, Hirata (2002) presented evidence that Mn(II) can induceDNA fragmentation, a biochemical hallmark for apoptosis, in neuronal cells

Deficiency

The theoretical requirement of manganese for crustaceans has been calculated to be 3.9 µg Mn g1 dw(White & Rainbow 1987) The calculation was based on the animals’ total content, thus includingthe exoskeleton where the majority of the manganese is incorporated into the calcareous matrix

In the literature pelagic crustaceans are reported to have an average muscle and midgut glandconcentration of less than 2 µg Mn g–1 dw and a total manganese body concentration of 1.2–1.4 µg

Mn g–1 dw (Table 1) Even the benthic lobster Nephrops norvegicus from the pristine Faeroe Islandscontains very low Mn concentrations (Table 1) When excluding the exoskeleton and the stomach(which may contain sediment rich in Mn) in these animals, the rest of the body (the soft tissue)contains an Mn concentration of 2.5 µg Mn g–1 dw (n = 32) (S.P Eriksson & S.P Baden, unpublishedobservations) The theoretical required concentration of manganese in the soft tissue of crustaceans

is thus likely to be somewhat overestimated Since no data exist on crustacean manganese deficiency,the precise Mn requirements of Crustacea remain unresolved It is hoped that further investigationswill provide an answer Most field-caught animals contain manganese concentrations well abovethe assumed basic requirements needed and manganese deficiency does not appear to pose a generalthreat to aquatic crustaceans (Table 1)

Manganese in Crustacea — Overview

Manganese is an essential metal and is thus required in at least a minimum concentration for ananimal to be able to fulfil its metabolic functions When discussing the basic body requirements

of manganese, it is, however, also important to differentiate between metabolically active soft tissuesand relatively inert tissues Each tissue is likely to have its own kinetics (reaction rate) of metaluptake and loss, the determination of which can often be valuable when interpreting the biologicalsignificance of metal burdens The interpretation of animal kinetic data and animal metal concen-tration is potentially complicated by a combination of factors including organism condition, growth,food supply, moulting and reproduction cycles, and may also depend directly or indirectly onenvironmental conditions like temperature, oxygen saturation and metal concentration Some tissuemetal concentrations are maintained within a narrow range and for others there may be less tightregulation and even storage Clearly, under such circumstances, increased metal burdens in specifictissues could easily be obscured when analysing whole organisms

The literature on background manganese concentrations in different crustaceans derives fromfield-collected animals from marine and freshwater environments (Table 1) Average total Mn con-centration was 63 µg Mn g–1 dw, with the lowest concentrations found in marine pelagic crustaceansand benthic lobsters from the pristine Faroe Islands The highest total Mn concentration was found

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Table 1 Manganese concentrations found in field-caught crustaceans from pristine areas

Habitat/Order/Species Subhabitat Total Eggs Exo Gills Haem Midg.gl Muscle Ovary Testes References

Marine

Amphipoda

Fialkowski et al 2003 Thoracica

Rainbow & Blackmore 2001

Stomatopoda

Decapoda

et al 2004

unpublished

Martin & Ceccaldi 1976

Carcinus maenas S,B 74–206 92–286 175–282 0.36–0.38 7.5–10 10–24 3.1–19 Martin 1975, Bjerregaard

& Depledge 2002

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SUSANNE P

Table 1 (continued) Manganese concentrations found in field-caught crustaceans from pristine areas

Habitat/Order/Species Subhabitat Total Eggs Exo Gills Haem Midg.gl Muscle Ovary Testes References

Melicertus (as Penaeus)

kerathurus

Sanders et al 1998

Notes: All values are given as µ g Mn g –1 dry weight tissue, except for haemolymph which is in wet weight * Values calculated from wet weight by using ww/dw ratio stated in

the original papers Abbreviations: S-shallow, D-deep, B-benthic, H-hydrothermal vent, P-pelagic, Exo-Exoskeleton, Haem-Haemolymph and Midg.gl.-Midgut gland.

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in a freshwater crayfish (Potamonautes warreni) Highest mean tissue Mn concentration was found inthe animal’s midgut gland (102 µg Mn g–1 dw) and the lowest concentration in the muscle tissue(average 9 µg Mn g–1 dw) All haemolymph values were presented as wet weight (ww) values andwere thus compared as such, giving an average Mn concentration of 0.63 µg Mn g–1 ww The dw/wwratio of haemolymph equivalent to approximately 7–17% (S.P Baden, unpublished observations)

In general, all tissue concentrations showed a high interspecies variability, with the largestdifference (almost 4000-fold) found in the midgut gland of a freshwater, benthic crayfish comparedwith that of a marine, pelagic crab (Table 1) Due to the high interspecies variability, and the factthat often only a few of the tissues are measured in each species, caution should be made whencomparing the mean tissue concentrations at the bottom of Table 1 In two cases, sufficient datawere obtained to statistically compare tissue concentrations in crustaceans of different habitats.The results showed that freshwater decapods had a significantly higher Mn concentration in themidgut gland than marine decapods (one-way ANOVA, df 10, F-value 7.4, p < 0.05), but that nodifference could be observed for the Mn concentration in the exoskeleton of freshwater and marinedecapods (one-way ANOVA, df 8, F-value 0.34, p > 0.05)

The variability within individuals (between tissues) was in comparison lower By ranking thetissue concentrations of Mn in Table 1 for species, where more than two tissues had been measured,the following general relationship between tissues was observed: exoskeleton, gill > egg > testes >ovary, midgut gland > muscle > haemolymph

Even when animals are exposed to elevated Mn concentrations, as in environments that arepolluted (industrial waste), acidic (lakes and rivers) or hypoxic (mainly eutrophic marine areas),the relative relationship between the exoskeleton, gills, midgut gland, muscle and haemolymphholds, though concentrations are higher than in animals from pristine areas (Table 2)

The routes and effects of manganese

In the following sections an up-to-date review on the routes and effects of manganese in crustaceans

is presented In Figure 1 the uptake of manganese from water is described as well as the lation and effects in separate target tissues Existing data on elimination kinetics are describedunder the respective tissue section

accumu-Uptake of manganese from water

For many organisms the key determinant that influences metal accumulation from water is thespeciation of the metal Metals are usually considered more bioavailable as free ions than as complexligands with anions In sea water as much as 58% of the total Mn concentration is free hydratedions whereas 37% is complexed with chloride, 4% with sulphate and 1% with carbonate (Simkiss &Taylor 1989) Hydrated ions are clearly larger than the equivalent ions in a crystal These hydrationproperties of ions in aqueous solution are important in determining the permeability and selectivity

of ions crossing membranes (Simkiss & Taylor 1989)

Of the borderline metals, only Mn has a sufficiently low enthalpy to be able to shed its hydrationand pass through membrane channels The uptake of divalent trace metal ions occurs mainly atpermeable respiratory surfaces, for example gills, and is driven by passive diffusion via ligandbinding occurring through calcium channels (Rainbow 1997)

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through the gills (Rankin et al 1982) The diffusion over the gill membrane is dependent on theconcentration gradient of free metal ions Crustaceans may accumulate essential as well as non-essential metals above the concentration of the medium as the metals may bind to e.g., bloodproteins and thus maintain an inward flux (Baden & Neil 1998) The mean Mn concentration inanimals from pristine areas is 100 µg g–1 dw, but varies from 0.6–508 µg g–1 dw (Table 1) Duringhypoxia in the SE Kattegat, Sweden, in 1995, the mean gill concentration of Mn in Norway lobster

(Nephrops norvegicus) increased by 30 times to1560 µg Mn g–1 (Eriksson & Baden 1998; Table 2).The fraction of absorbed and adsorbed Mn is poorly investigated However, in the SE Kattegat, ablack layer of precipitated Mn on the gills was observed indicating that large amounts of adsorbed

Mn may occur in the field (Baden et al 1990) The effects of the precipitated layer of Mn onrespiration is not yet investigated but it may hamper a normal function and internal hypoxia may

Table 2 Manganese concentrations in field-caught crustaceans from pristine, polluted

(industrial waste), acidic (lakes and rivers) and hypoxic (eutrophic) areas

Habitat/Order/Species Tissue Pristine Polluted Acidic Hypoxic References

Nephrops norvegicus Exoskeleton 223 304 Eriksson & Baden 1998

Freshwater

Decapoda

Young & Harvey 1991

Notes: All concentrations are given as mean µ g Mn g –1 dry weight tissue, except haemolymph which is in wet weight 7044_C002.fm Page 68 Monday, April 17, 2006 1:37 PM

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develop, as has been found by Spicer & Weber (1991) for crustaceans when exposed to other

essential metals like Cu and Zn

Since the gills are part of the exoskeleton, changes in Mn concentration during the moult cycle

follow the same pattern in these two tissues (Eriksson, 2000a) This is further discussed in the

section ‘Exoskeleton’ below

Haemolymph

Having passed the gill epithelium, Mn is transported in the haemolymph to target tissues either

dissolved in the plasma or bound to the haemolymph proteins, predominantly (80–90%) to the

respiratory protein haemocyanin (Baden & Neil 1998) Exposing N norvegicus to realistic

con-centrations of dissolved Mn (5 and 10 mg Mn l–1 for 2 weeks) the haemolymph plasma reaches

the same concentration as the ambient water, whereas the Mn concentrations of the haemocyanin

and whole haemolymph (plasma and haemocyanin) are about twelve and three times higher,

respectively (Baden & Neil 1998) However, when N norvegicus were exposed to Mn

concentra-tions of 60 mg Mn l–1 for 2 weeks the plasma and whole haemolymph reached only 0.5 and 1.5

times the concentration of the ambient water (Selander 1997)

The biological half-life for manganese accumulation in N norvegicus during exposure to 5 and

10 mg Mn l–1 and elimination in undosed sea water is relatively fast in haemolymph (about 24 h

for both processes) (Baden et al 1999)

As the competitive binding of metals by organic ligands (the Irving-Williams series) is stronger

for Cu2+ than Mn2+ (Rainbow 1997), Mn does not replace Cu as apostethic metal in the haemocyanin,

as indicated by a constant Cu concentration with increasing Mn concentration of the haemolymph

(Baden & Neil 1998)

Removal and displacement of Ca from haemocyanin may change the quaternary structure and

thus the functional properties of the haemocyanin (Van Holde & Brenowitz 1981, Brouwer et al

1983) The binding of Cd and Zn is stronger than Ca and has been shown to replace Ca in the

haemolymph of the blue crab, Callinectes sapidus Even though Mn binds slightly stronger than Ca,

Figure 1 Routes and effects of manganese in a crustacean Dissolved Mn II in water may enter via the gills

or antennules or get precipitated on the exoskeleton Entrance may also occur via the food in a variety of

chemical form Octagonal boxes indicate the route and target tissues of Mn and square boxes indicate the

effects of Mn exposure Observed effects ( √ ) and hypothetical but not yet investigated effects(?).

Mn (ll)

Gills

Midgut gland

Reproductive organs

poetic tissue

Muscle

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no change in Ca concentration of whole haemolymph was found in Nephrops norvegicus with

increasing exposure to Mn of 60 mg l–1 (Selander 1997) This constancy in the whole haemolymph,

however, does not rule out the possibility that Mn has displaced Ca from the haemocyanin to the plasma

An important source of Mn in the ocean is from hydrothermal vents The crustaceans adapted

to live close to these vents may hypothetically contain a higher concentration of Mn than non-vent

crustaceans Professor J.J Childress from the University of California, Santa Barbara, kindly

provided the authors with haemolymph from a vent crab, Bythograea thermydon, which was found

to have Mn concentrations between 0.44 and 1.6 µg g–1 ww These Mn concentrations are within

the range of haemolymph concentration from non-vent crustaceans as seen from Table 1 The

max-imum mean Mn concentrations of 7.35 µg g1 ww in a field-caught crustacean (Nephrops norvegicus)

is reported from the SE Kattegat following a hypoxic period in 1995 (Eriksson & Baden 1998)

The effects of manganese on haemocyanin synthesis and adaptation to hypoxia are described

in a subsequent section discussing the midgut gland, as this is the primary organ for haemocyanin

synthesis (Taylor & Antiss 1999)

The synthesis of haemocytes takes place in the haematopoietic tissue localised as a thin sheet

on the dorsal site of the stomach in crustaceans (Chaga et al 1995) The haemocytes of crustaceans

consist of hyaline, semigranular and granular cells playing an important role in, for example, the

innate immune defence (Ratcliffe & Rowley 1979, Söderhäll 1981, Söderhäll & Cerenius 1992)

Immunotoxicology of invertebrates is an unexplored field and as a result no early investigations

can be cited Recently, Hernroth et al (2004) discovered that when exposed to 20 mg l–1 Mn for

10 days several immunological processes of N norvegicus were affected The number of haemocytes

decreased by 60% Despite the great loss of haemocytes, renewal through increased proliferation

of the haematopoietic stem cells did not appear to occur Additionally, maturation of the stem cells

to immune-active haemocytes was inhibited in Mn-exposed lobsters (N norvegicus) To release the

prophenoloxidase system (ProPO), which is necessary for the immune defence of arthropods, the

granular haemocytes must degranulate This degranulation activity was also significantly suppressed

after Mn treatment Furthermore, the activation of ProPO by the non-self molecule,

lipopolysac-caride, was blocked Probably Mn replaces Ca and thereby inhibits protein required for mobilisation

and activation of the haemocytes

Immune suppression may explain the occurrence of shell disease caused by microbial infection

of the exoskeleton in blue crab, Callinectes sapidus, from North Carolina, U.S (Weinstein et al.

1992) The infection is related to elevated Mn concentrations in the body tissues Similar findings

might explain the high frequency of the parasitic dinoflagellate Hematodinium sp that has been

found in Nephrops norvegicus from the west coast of Scotland (Field et al 1992) In the same area

high concentrations of Mn have been recorded in the tissue of this species (Baden & Neil 1998)

Midgut gland

In contrast to other target tissues, where manganese accumulation reaches an equilibrium

deter-mined by the exposure concentration within 5 days, the midgut gland of N norvegicus continuously

accumulates manganese at a relatively slow rate and does not reach equilibrium after a 3-week

period of exposure This slow accumulation to the hepatopancreas has also been observed for zinc

in Carcinus maenas by Chan & Rainbow (1993) The elimination rate of manganese from the

midgut gland is, however, much faster The biological half-lives for accumulation and elimination

of manganese are about 4 and 1.5 days, respectively (Baden et al 1999) Insoluble granules

containing metals bound with phosphorus or sulphur have been observed in the epithelial cells of

the midgut gland (or comparable organ) in many invertebrates (for review see Ahearn et al 2004)

The granules scavenge and detoxify surplus metals, and are later eliminated through exocytosis

Several marine snails have been shown to eliminate manganese this way (Simkiss 1981, Nott &

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Nicolaidou 1994) Although no such granules have yet been described in manganese-rich crustaceans,the surplus of manganese is clearly delivered to the midgut gland for net accumulation as indicated

in Rainbow (1997) and Baden et al (1999) Accumulation is also demonstrated by the relativelyhigh levels of Mn in midgut glands from different species of crustaceans (Table 1) The highestmean tissue concentrations found in the literature are from the midgut glands of a marine hermit

crab, Clibanarius erythropus (1596 µg Mn g–1 dw) and a freshwater crayfish, Procambarus clarkii

(1677 µg Mn g–1 dw), both collected in areas with known anthropogenic input (Gherardi et al.

2002, Nott & Nicolaidou 1994) Unfortunately, no background data are available for either of thesespecies which is why they have not been included in Table 2 However, unpublished data on

background Mn concentrations in another marine hermit crab, Pagurus bernhardus, varied from

15–28 µg Mn g–1 dw in the midgut gland (Andersson 1993), and the highest overall midgut glandbackground concentration published is 374 µg Mn g–1 dw in a freshwater crayfish, Potamonautes

warreni (Steenkamp et al 1994; Table 1).

The synthesis of haemocyanin is primarily recognised to take place in the midgut gland(Taylor & Antiss 1999, for review) In a recent study the combined and separate effects of hypoxia(2.5 mg l–1) and manganese (20 mg l–1) on the haemocyanin concentration were investigated after

an exposure period of 2 weeks Crustaceans adapt to hypoxia by increasing or decreasing (depending

on the initial value) the haemocyanin concentration, presumingly to an optimal concentration(Spicer & Baden 2001) A simultaneous exposure to manganese affects this adaptation by preventingthe synthesis of haemocyanin (Baden et al 2003)

Muscle

The manganese concentration of the muscle tissue remains relatively constant throughout the moultcycle and is less dependent on the exposure concentration of Mn compared with other tissues(Bryan & Ward 1965, Baden et al 1995, Baden & Neil 1998, Eriksson & Baden 1998, Bjerregaard &Depledge 2002) This constancy is especially interesting since the muscle is a metabolically activetissue with high mitochondrial content Calculations indicate that an increase in Mn concentration

of muscle tissue after exposure to elevated Mn concentrations can, in principle, be explained bythe increase in Mn in the extracellular haemolymph of the muscle tissue (Hille 1992, Baden et al.1995) A plausible explanation for the relatively stable concentration in the muscle cells themselves

is, thus, either that turnover rates of manganese in these cells are high enough to disguise increaseduptake (at least for the exposure concentrations that have so far been studied) or that the metalnever enters the muscle cells but remains in the extracellular haemolymph

Normal muscle concentrations of Mn lie in the range of 0.4–8.0 µg Mn g–1 dw with the exception

of the extremely high values of 24 µg Mn g–1 found in small Carcinus maenas by Bjerregaard &

Depledge (2002) and 87 µg Mn g–1 found in the freshwater crayfish, Potamonautes warreni by

Steenkamp et al (1994) Many values in the literature are stated as wet weight concentrations withthe primary objective being risk assessment of heavy metals in human food Taken that the dailyrecommended intake for humans is 2.5–5 mg Mn day–1, a person would have to eat ca 1 kg of

crustacean meat just to fulfil the daily requirement Manganese at natural levels in crustaceans isthus not likely to pose a threat for human consumption

When lobsters (Nephrops norvegicus) are exposed to 10 mg Mn g–1 their muscle extension andthus most probably (consequently) the swimming capacity is affected as will be discussed underthe section ‘Nervous system’

Exoskeleton

Due to its chemical properties, manganese is found in highest concentrations in the calcified parts

of crustaceans, mainly in the exoskeleton, gills and the gastric mill of the stomach (Bryan & Ward

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1965, Baden et al 1990, 1995, Eriksson & Baden 1998, Eriksson 2000a) Depending on thethickness of an animal’s exoskeleton the vast majority of manganese is found in this tissue, as it

contains more than 98% of the total Mn content of the decapod lobsters Homarus gammarus (Bryan

& Ward 1965) and Nephrops norvegicus (Baden et al 1995) The manganese incorporated in the

matrix of the exoskeleton is believed to have little effect on the animals

The manganese concentration of the exoskeleton changes during the moult cycle, and lobsters

(N norvegicus) collected in the field show a step-wise increase in average Mn concentration from

postmoult, intermoult to premoult (Eriksson & Baden 1998) The crustacean moult cycle is inated temporally by the intermoult phase, with brief periods of postmoult and premoult There is,however, no correlation between the contemporary environmental Mn(II) concentration of ambientsea water and that of the exoskeleton in field-caught intermoult lobsters (Eriksson & Baden 1998)

dom-It was thus proposed that the amount of Mn found in the exoskeleton of intermoult individualsprimarily depends on the Mn concentration to which the animals are exposed during the calcificationprocess at postmoult, rather than the current ambient Mn concentrations (Eriksson & Baden 1998,

Eriksson 2000a) During growth, the shell of the barnacle Balanus amphitrite has been shown to

incorporate Mn in direct proportion to the concentration of the sea water (Hockett et al 1997).Unlike most crustaceans, the calcified shells in barnacles grow more or less continuously (Bourget &Crisp 1975), thus having continuous calcification In most crustaceans, however, calcification occurs

during a short postmoult period To test the theory, newly moulted Nephrops norvegicus were

exposed to flow-through sea water with <0.06 mg Mn l–1 (controls) or 10 mg Mn l–1 for 20 days(S.P Eriksson, unpublished observations) The animals were sacrificed and the Mn concentrationwas measured in the exoskeleton and in the cast exuviae (exuviae were removed immediately aftermoulting, prior to Mn addition) The cast exuviae showed no difference (one factor ANOVA, F1,8 =0.09, P = 0.77, n = 5) between the control group and the (later) Mn-exposed group; Mn concen-trations were 352 ± 70 and 326 ± 55 (mean ± SE) µg Mn g–1 dw, respectively After 20 days thenewly calcified intermoult exoskeletons showed significant differences between the two groups(one factor ANOVA, F1,8 = 151, P < 0.001, n = 5) The Mn-exposed animals had exoskeletal Mnconcentrations of 2524 ± 201 µg Mn g–1 dw (mean ± SE) whereas the control animals containedonly 44 ± 8 µg Mn g–1 dw In comparison, an earlier study on intermoult animals also exposed to

10 mg Mn l–1 dw for 20 days showed a modest increase from 200 µg Mn g–1 to 290 µg Mn g–1

dw (Baden et al 1999) The results, though not extensive, thus appear to support the theory thatintermoult exoskeleton Mn concentrations are mainly the result of prevailing Mn concentrationsduring the calcification process

In contrast, the increase from intermoult to premoult found in N norvegicus is thought to be

the result of exoskeletal breakdown (Eriksson 2000a) During premoult, crustaceans degrade andresorb some of the old cuticle Cuticle components, such as calcium, are stored for later use inhardening of the new ‘shell’ (Aiken & Waddy 1992) The breakdown of the old cuticle results in adecreased dry weight/wet weight ratio which in turn also leads to an apparent increase in Mnconcentration from intermoult to premoult (Eriksson 2000a)

Moulting has been suggested as one possible way for decapods (Homarus gammarus, Palaemon

elegans, Systellaspis debilis) to dispose of excess unwanted metals (Bryan & Ward 1965; Ward

1966; White & Rainbow 1984a,b, 1987; Swift 1992) Although crustaceans on occasion eat part

or all of their cast exuviae, preliminary data on Mn uptake from food suggests that Mn incorporated

in exoskeletal parts is not easily accessible when ingested, as described in the section of Mn uptakefrom food Moulting might thus serve as an important regulator of the Mn content providing thereare low Mn(II) concentrations in the water at the time of moult

Manganese precipitations on the hard-shelled exoskeleton are visible as persistent black dots

mainly in crevices as observed after hypoxia on Nephrops norvegicus in the SE Kattegat (Baden

et al 1990) Being insoluble, the precipitation of Mn on the exoskeleton is a potential biomarker

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