An investigation aimed at establishing the presence or obsence of respiratory (crustacea: Cirripedia)

Comp. Biochera. Physiol. Vol. 91A, No. 4, pp. 849-853, 1988 Printed in Great Britain

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AN INVESTIGATION AIMED AT ESTABLISHING THE PRESENCE OR ABSENCE OF RESPIRATORY PIGMENTS IN BARNACLES (CRUSTACEA: CIRRIPEDIA) M. E. WAITE* and G. WALKER Animal Biology Group, Marine Science Laboratories, Menai Bridge, Gwynedd LL59 5EH, U.K. (Received 18 May 1988) Abstract--1. The haemolymph of balanomorph barnacles and the tissue of the parasitic barnacle, Sacculina carcini, possess neither haemoglobin nor haemocyanin. 2. Very low levels of copper, 0.02-0.03 #g/ml, were found in the haemolymph of Balanus hameri. 3. An iron concentration of 5 #g/ml was recorded in B. hameri haemolymph, which is similar to that reported for other crustaceans.

INTRODUCTION

Crustaceans are an unusual group of animals in that some species possess haemocyanin or haemoglobin, while others do not possess a respiratory pigment (see Wolvekamp and Waterman, 1960; Mangum, 1983; Ghidalia, 1985). Herberts and De Frescheville (1981) have reported the presence of haemocyanin in Sacculina carcini, a parasitic barnacle, while haemoglobin has been recorded in other parasitic barnacles, Septosaccus cuenoti (Prrez and Block-R/iphael, 1946), Peltogaster curvatus, Galathescus squamifera (Fox, 1953) and Briarosaccus callosus (Shirley et al., 1986). A respiratory pigment is absent in the lepa d o m o r p h barnacles, Pollicipes polymerus (Petersen et al., 1974) and Calantica spinosa (Innes, 1985). The only study on the respiratory pigments of balanomorph barnacles is that made by Southward (1963), who found haemoglobin in the muscles of Balanus perforatus and suggested that haemoglobin is also present in the muscles of Elminius modestus and Balanus crenatus. In addition, Southward (1963) believed that there may be a very low level of haemoglobin in the haemolymph of Balanus perforatus. The present study was undertaken to determine whether haemoglobin and/or haemocyanin are present in a variety of balanomorphs, and investigates further the reported presence of haemocyanin in Sacculina carcini.

attached to the horse mussel, Modiolus modiolus, were dredged up off the Isle of Man and then maintained in the laboratory at controlled sea-water temperatures, which were adjusted to follow the seasonal temperature change of the sea off the Isle of Man (see Waite, 1986). Haemolymph was obtained from the balanomorph barnacles either by prizing them off the rocks to which they were attached and absorbing the haemolymph, from the exposed mantle sinus, directly into Whatman paper (see later), or by drilling a hole near the base of a shell plate, using a dental drill, and withdrawing the haemolymph into a syringe. Haemolymph from C. maenas was taken from a sinus of a leg using a hypodermic needle and syringe. Human blood was freshly taken from a finger tip. Sacculina carcini externae were very carefully excized from the abdomen of several C. maenas and then macerated in an eppendorf tube, whilst bodies of B. perforatus were removed from their shells and treated in the same way. Bovine haemoglobin and Limulus polyphemus haemocyanin (Sigma) were dissolved in water and used separately as the haemoglobin and haemocyanin "standards". In early experiments, when the haemocyanin "standard" was not available, freshly collected snail (Helix sp.) hepatopancreas was used as a "standard" along with C. maenas haemolymph, as they are both known to contain haemocyanin. Electrophoresis using starch gels

Balanus balanoides (L.) and Elminius modestus Darwin were both collected from the shores of the Menai Strait and from Dulas Bay, Anglesey, Balanus crenatus Brugui6re from the lower shore at Port Dinorwic, Gwynedd, Balanus balanus (L.) from Oban and Balanus perforatus Bruguirre from near Plymouth. Carcinus maenas (L.) parasitized with Sacculina carcini (Thompson) were collected from Plymouth and supplied by the Marine Biological Association, UK. The crabs and barnacles were maintained in running seawater at ambient temperature. Balanus hameri (Ascanius),

Haemolymph and macerated tissue samples were absorbed into rectangles (I0 x 5mm) of Whatman No. 3 chromatography paper and inserted into the origin of 12% starch gels (Connaught Laboratories). The electrode buffer consisted of 0.1 M Tris, 0.1 M maleic acid, 0.01 M EDTA (Na2), 0.01 M MgCI2.6H20 adjusted to pH 7.4 with 40% NaOH solution. The gel buffer was a 1:9 dilution of the electrode buffer. Gels were run overnight at 90 V, 4°C. Horse spleen ferritin (Sigma) was included on each gel as a marker protein. Naphthalene black was used to stain general proteins on the gel (Smith, 1968) and haemoglobin was visualized using o-toluidine (Smith, 1968). Copper-containing proteins were stained using one of the following: rubeanic acid (=dithiooxamide) (Decleir, 1961), dianisidine (Manwell and Baker, 1963) and cyanide-tetrazolium (Gould and Karolus, 1975).

*Present address: MAFF, Fisheries Laboratory, Remembrance Avenue, Burnham-on-Crouch, Essex CM0 8HA.

Electrophoresis using cellulose acetate membranes Haemolymph and tissue samples were applied to cellulose acetate membranes (Cellogel, Shandon, Cheshire), using

MATERIALS AND METHODS

849

850

M.E. WA1TE and G. WALKER

either 22mM Tris 135mM glycine pH8.6 or 135mM Tris-50 mM citrat~1.24 mM EDTA pH 7. l as a buffer, and run for 15 and 30 min, respectively at 200 V and 4~C (see Meera Khan, 1971). Horse spleen ferritin was included on some of the membranes as a marker protein. The membranes were stained for proteins with Coomassie brilliant blue (Storey, 1977) or Ponceau S (Cellogel) (0.5 g% in trichloroacetic acid, stained for 5 rain; de-stained in three washes of 5% acetic acid) to visualize general proteins. As the Tris~lycine buffer gave better separation of proteins, this buffer was used for most of the cellulose acetate membranes. Haemoglobin was stained using o-toluidine and coppercontaining proteins were visualized using the cyanidetetrazolium method. Measurement o f copper and iron concentrations in the haemolymph o f B. hameri

Five ml "Aristar" concentrated nitric acid was added to 2 mI B. hameri haemolymph and evaporated to dryness. The dried sample was then taken up in an appropriate volume of deionized water prior to analysis for iron and copper using a Unicam SP90A atomic absorption spectrophotometer. The copper level in the haemolymph of B. hameri was also measured by potentiometric stripping analysis using a radiometer 1SS 820 Ion Scanner (Radiometer, Copenhagen) (see Jagner, 1978; Redpath, 1985). One ml of haemolymph was mixed with I ml of "Aristar" concentrated nitric acid in a beaker and evaporated to dryness on a hot plate. Another 1 ml nitric acid was added to the beaker which was then covered and left on the hot plate overnight. The remaining liquid was then made up to 20 ml with distilled deionized water and analysed. Analysis ofB. hameri haemolymph using a spectrophotometer

Freshly collected B. hameri haemolymph was diluted (1:29) with filtered sea-water and scanned against distilled water, from 200-680nm, in a Unicam SP 800 spec-

trophotometer. Haemoglobin has characteristic absorption maxima at 416422, 540 550 and 576- 586 nm (Goodwin, 1960; Ghidalia, 1985; Shirley et al., 1986), and haemocyanin at 335 345 and 558 570nm (Goodwin, 1960; Arp and Childress, 1981; Herberts and De Frescheville, 1981). RESULTS

Electrophoresis

The n u m b e r of samples which had protein b a n d s visualized with a copper stain on the starch gels was fewer t h a n on the cellulose acetate membranes, indicating that the m e m b r a n e s were more sensitive to the small sample volumes which were used (see Beaum o n t et al., in press). On the starch gels rubeanic acid did not stain any of the separated protein bands; dianisidine visualized copper-containing proteins in only C. m a e n a s h a e m o l y m p h and snail hepatopancreas; the h a e m o c y a n i n " s t a n d a r d " stained clearly with c y a n i d e - t e t r a z o l i u m but none of the barnacle samples stained, o-Toluidine visualized the h a e m o g l o b i n " s t a n d a r d " on the starch gel, but none o f the barnacle h a e m o l y m p h proteins or those from B. p e r f o r a t u s tissue stained. On the cellulose acetate m e m b r a n e s Ponceau S stained the protein b a n d s but with less definition t h a n when using Coomassie brilliant blue. In its favour, however, Ponceau S did not produce a b a c k g r o u n d stain. The p a t t e r n s of protein b a n d s for the haem o l y m p h o f the various species were all different (Fig. 1). O n the cellulose acetate m e m b r a n e s c y a n i d e - t e t r a z o l i u m visualized copper-containing proteins in the h a e m o c y a n i n " s t a n d a r d " , the hae-

O

O

Fig. 1. Electrophoresis of haemolymph and tissue samples on cellulose acetate membrane, stained with Coomassie brilliant blue. (1) Haemoglobin "standard"; (2) haemocyanin "standard"; (3) Carcinus maenas haemolymph; (4) Balanus hameri haemolymph; (5) Sacculina carcini tissues; (6) Elminius modestus (Menai Bridge) haemolymph; (7) Balanus balanoides (Menai Bridge) haemolymph; (8) Balanus crenatus haemolymph; (9) Balanus balanus haemolymph; (I0) haemocyanin "standard".

Fig. 2. Electrophoresis of haemolymph and tissue samples on cellulose acetate membrane, stained with cyanide-tetrazolium (1) haemoglobin "'standard": (2) Carcinus maenas haemolymph; (3) Balanus hameri haemolymph; (4) Balanus balanoides (Menai Bridge) haemolymph; (5) Carcinus maenas egg mass; (6) haemocyanin "standard"; (7) Elminius modestus (Dulas Bay) haemolymph; (8) Balanus balanoides (Dutas Bay) haemolymph. Some copper-containing protein bands (indicated by arrows) only stained very weekly. The band above number 1 (haemoglobin "standard") could be seen before stainin~

Respiratory pigments in barnacles

Spectrophotometric analysis ofB. hameri haemolymph

Table I. The presence/absence o f copper and iron in various tissue and haemolymph samples run on starch gels and cellulose acetate membranes

Haemoglobin "standard" Haemocyanin "standard" Carcinus maenas h a e m o l y m p h Sacculina carcini whole tissue Balanus balanoides (Menai Bridge) haemolymph Balanus balanoides (Dulas Bay) haemolymph Elminius modestus (Menai Bridge) haemolymph EIminius modestus (Dulas Bay) haemolymph Balanus hameri h a e m o l y m p h Balanus balanus h a e m o l y m p h Balanus crenatus haemolymph Balanus perforatus h a e m o l y m p h Balanus perforatus bodies H u m a n blood Helix sp. hepatopancreas

Copper

Iron

+ + -

+ -

+

-

+

-

-

-

+ + +

N N

851

The scan of B. hameri haemolymph showed none of the characteristic absorption maxima of crustacean haemoglobin or haemocyanin. DISCUSSION

+ , Present; - , absent; N, not tested.

molymph of C. maenas, B. balanoides from Menai Bridge and Dulas Bay, E. modestus from Dulas Bay (Fig. 2) and human blood, o-Toluidine stained only the haemoglobin "standard" on the cellulose acetate membrane. A summary of the tissues and haemolymph samples tested electrophoreticaUy for the presence of copper- and iron-containing proteins is given in Table 1. Measurement of copper and iron concentrations in the haemolymph of B. hameri Atomic absorption analysis gave a mean iron concentration of 5 #g/ml in the haemolymph of B. hameri and a copper level of 0.03 #g/ml or less (the necessary dilution of the haemolymph for analysis put the copper level close to the limit of detection). The mean concentration of copper in the haemolymph of B. hameri was 0.02/~g/ml when measured by potentiometric stripping analysis.

The electrophoresis results show that there are no detectable iron-containing proteins in the haemolymph of any of the balanomorph barnacles tested, and neither are there any iron-containing proteins in the body tissues of B. perforatus or in Sacculina carcini tissues. However, it must be borne in mind that the B. perforatus bodies were only examined by means of starch gel electrophoresis which is a less sensitive technique than that using cellulose acetate membranes. The scan of B. hameri haemolymph with the spectrophotometer showed no characteristic absorption maxima for haemoglobin. However, analysis of B. hameri haemolymph by atomic absorption spectroscopy detected the presence of iron. Various workers have recorded iron in the haemolymph of different crustacean species even though these do not possess haemoglobin (see Table 2). Iron is transported between tissues in vertebrates by transferrin, which is a fl globulin (George, 1982); the crab, Cancer pagurus, is the only invertebrate in which a transferrin-like protein has been found (Guary and Negrel, 1980). Ghidalia et al. (1972) found that the haemolymph of Macropipus puber contains a protein which is able to chelate free iron, while the haemolymph of three species of crayfish, examined by Kazmierczak et al. (1978), contained free iron and not iron bound to proteins. B. hameri haemolymph must contain free iron as electrophoresis does not show the presence of any iron-containing proteins. In barnacles it is thought that iron is transported in the haemolymph and then incorporated in inorganic granules within cells (see Rainbow, 1987). Southward (1963) reported the presence of haemoglobin in B. perforatus whilst P~rez and Bloch-R/iphael (1946) found it in the parasitic barnacle, Septosaccus cuenoti, and Fox

Table 2. Iron concentrations in the h a e m o l y m p h o f some crustaceans Species

Author

Source

/~g/ml

Astacus astacus Astacus leptodactylus Orconectes limosus Cancer irroratus

Kazmierczak et al. (1978) Kazmierczak et al. (1978) Kazmierczak et al. (1978) Martin (1973)

serum serum serum haemolymph

1.38 1.89 3.16 0.9

Balunus hameri

Present study

haemolymph

5.0

Table 3, C o p p e r concentrations in the h a e m o l y m p h o f some selected crustaceans Species

Author Kazmierczak et Kazmierczak et Kazmierczak et H o r n and K e r r

Panulirus interruptus

Johnston and Barber (1969)

Penaeus esculentus Crangon vulgaris

Smith and Dall (1982) D j a n g m a h and G r o v e (1970)

dialysed haemolymph haemolymph haemolymph

Balunus hameri

Present study

haemolymph

m, Male; L female.

al. (1978) al. (1978) al. (1978) (1963)

Source

Astacus leptodactylus Astacus astacus Oreonectes limosus Callinectes sapidus

serum serum serum serum

/~g/ml 30.87 44.02 55.91 8-173 (m) 16-176 (f) 91.8 25-225 41-200 0.02-0,03

852

M. E. WAITEand G. WALKER

(1953) identified it in two other parasitic barnacle species, Peltogaster curvatus and Galathescus squamifera. Interestingly, however, Fox (1953) did not detect haemoglobin in either Parthenopa subterranea or in Sacculina. Recent work on another parasitic cirripede, Briarosaccus callosus (Shirley et al., 1986), has shown haemoglobin to be present in the haemolymph. The results of the present investigation show that there are no copper-containing proteins in the haemolymph of B. hameri, B. crenatus, B. perforatus or in S. carcini tissues. Copper-containing proteins were, however, found in the haemolymph of B. balanoides collected from Dulas Bay and Menai Bridge, and in the haemolymph of E. modestus from Dulas Bay, but not from Menai Bridge. Dulas Bay receives a high input of copper from the Afon Goch river (dissolved ionic copper = 1600 pg/1, dissolved organically associated copper = 50/~g/1) which flows into it (Foster and Morris, 1971). In contrast, the Menai Strait contains much lower concentrations of copper (dissolved ionic copper= 1.83-3.3/~g/1, dissolved organically associated copper = 0.22~).78 #g/l) (Foster and Morris, 1971). Walker (1977) has found coppercontaining granules in the parenchyma cells of B. balanoides from Dulas Bay, and it seems likely that the haemolymph of these Dulas Bay barnacles contains elevated copper levels as a result of the copper pollution, which is a consequence of past mining activity in this area. The level of copper in the haemolymph of B. hameri is very low compared to that for other crustacean species (see Table 3). The spectrophotometric scan of B. hameri haemolymph did not show any of the characteristic absorption maxima for haemocyanin. It is possible that copper in barnacle haemolymph is due to the presence of a copper-containing enzyme or a protein which transports copper (see George, 1982). The protein mainly responsible for copper transport is ceruloplasmin (Harrison and Hoare, 1982); however, the only marine animal in which ceruloplasmin has been characterized is the fish, Pleuronectes platessa (Syed, 1980). As previously stated, copper in the haemolymph may also be the consequence of environmental pollution. Rainbow et al. (1980) found that copper, in B. balanoides tissues, is bound to high molecular weight proteins and to low molecular weight compounds which may be polypeptides, betaines or amino acids. No traces of copper were found in the S. carcini tissues. Herberts and De Frescheville (1981) have reported the presence of haemocyanin in Sacculina, but they state that it is not known whether the haemocyanin is "totally synthesized by the parasite or if it corresponds to a simple transformation from the crab's respiratory pigment". In view of the results of the present study, in which no haemocyanin could be detected in Sacculina, it seems most probable that the haemocyanin found by Herberts and De Frescheville is really that of the host crab! Indeed, another parasitic barnacle, Briarosaccus callosus, has been shown to absorb small quantities of haemocyanin from its crab host (see Shirley et al., 1986). It also seems most unlikely that S. carcini haemolymph would contain haemocyanin, as other parasitic barn-

acles have been shown to possess haemoglobin (Prrez and BlockoR/iphael, 1946; Fox, 1953; Shirley et al., 1986). The present study has found no evidence for the presence of respiratory pigments in the haemolymph of balanomorph barnacles; this is at variance with the report of haemoglobin in the haemolymph of Balanus perJoratus (Southward, 1963). The lepadomorphs Pollicipes polymerus and Calantica spinosa do not possess a respiratory pigment (Petersen et al., 1974; Innes, 1985) and neither does the parasitic barnacle Sacculina carcini (present study). As barnacles possess a large surface area (balanomorphs = inner mantle lining, branchiae and limbs; lepadomorphs = inner mantle lining, limbs and peduncle) across which oxygen can readily diffuse, then it is plausible that they do not require a respiratory pigment to increase the oxygen carrying capacity of their haemolymph. However, the reason why some parasitic barnacles possess haemoglobin remains enigmatic. Acknowledgements--We are indebted to Dr G. Carvalho and Mrs C. Beveridge, who helped with the electrophoresis, and also wish to thank Drs R. Jones and K. Redpath, who carried out the iron and copper analyses. This work was carried out while one of us (M.E.W.) was in receipt of a SERC studentship.

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