6 antibacterial activity in four marine crustacean decapods copia
Fish & Shellﬁsh Immunology (2002) 12, 371–385doi:10.1006/fsim.2001.0378Available online at http://www.idealibrary.com onAntibacterial activity in four marine crustacean decapods TOR HAUG1*, ANITA K. KJUUL1, KLARA STENSVAG1, ERLING SANDSDALEN2 AND r OLAF B. STYRVOLD1 1 Institute of Marine Biotechnology, The Norwegian College of Fishery Science, University of Tromsø, Breivika, N-9037 Tromsø, Norway and 2 Norwegian Institute of Fisheries and Aquaculture, N-9291 Tromsø, Norway (Received 28 June 2001, accepted 31 August 2001) A search for antibacterial activity in di#erent body-parts of Pandalus borealis (northern shrimp), Pagurus bernhardus (hermit crab), Hyas araneus (spider crab) and Paralithodes camtschatica (king crab) was conducted. Dried samples were extracted with 60% (v/v) acetonitrile, containing 0·1% (v/v) triﬂuoro- acetic acid, and further extracted and concentrated on C18 cartridges. Eluates from the solid phase extraction were tested for antibacterial, lysozyme and haemolytic activity. Antibacterial activity against Escherichia coli, Vibrio anguillarum, Corynebacterium glutamicum and Staphylococcus aureus was detected in extracts from several tissues in all species tested, but mainly in the haemolymph and haemocyte extracts. V. anguillarum and C. glutamicum were generally the most sensitive micro-organisms. In P. borealis and P. bernhardus most of the active fractions were not a#ected by proteinase K treatment, while in H. araneus and P. camtschatica most fractions were sensitive to proteinase K treatment, indicating antibacterial factors of proteinaceous nature. In P. bernhardus the active fractions were generally heat labile, whereas in H. araneus the activities were resistant to heat. Di#erences between active extracts regarding hydrophobicity and sensitivity for heat and proteinase K treatment indicate that several compounds are responsible for the anti- bacterial activities detected. Lysozyme-like activity could be detected in some fractions and haemolytic activity against human red blood cells could be detected in haemolymph/haemocyte and exoskeleton extracts from all species tested. 2002 Elsevier Science Ltd. All rights reserved. Key words: marine bioprospecting, natural products, antibacterial activity, invertebrate immunology, Pandalus borealis, Pagarus bernhardus, Hyas araneus, Paralithodes camtschatica. I. IntroductionMarine invertebrates are constantly exposed to high concentrations of micro-organisms. In crustaceans, the defence system against microbes rests largelyon cellular activities performed by haemocytes such as adhesion, phago-cytosis, encapsulation, nodule formation, and melanisation. The multimericcoagulation and phenoloxidase systems are also considered to be importantdefences in these organisms. Other factors described as part of the immune *Corresponding author: E-mail: firstname.lastname@example.org 3711050–4648/02/$-see front matter 2002 Elsevier Science Ltd. All rights reserved.
372 T. HAUG ET AL.system include agglutinins, haemolysins, lysozyme and antimicrobialfactors [1–3]. Antimicrobial activity has been detected in several decapodcrustaceans, including lobster, crabs, shrimps, and freshwater crayﬁsh [4–7].However, little is known about the nature of the antimicrobial factorsin crustaceans, and only a few compounds have been fully characterised.Chattopadhyay et al.  isolated an antimicrobial lectin, named scyllin, fromthe mud crab (Scylla serrata). Antimicrobial peptides have been isolated andcharacterised from the crabs Carcinus maenas  and Callinectes sapidus ,and from the shrimp Penaeus vannamei . Such peptide antibiotics seem tobe important defence molecules in all living organisms, including bacteria,plants, invertebrates and vertebrates . In most of the crustacean speciesstudied, the antimicrobial activity has been located in the haemolymph and/orin the haemocytes. However, potent antimicrobial activity has also beendetected in other organs/tissues [13–15]. The evolution of antibiotic-resistant pathogenic bacteria has stimulated thesearch for alternative antimicrobial agents from natural sources. Manyantimicrobial peptides show a high speciﬁcity for prokaryotes and a lowtoxicity for eukaryotic cells, and their mode of action (destroying mem-branes) is considered unlikely to lead to development of resistance. Theseproperties have favoured their investigation and exploitation as potential newantibiotics [16, 17]. The aim of the present work was to detect and compare antibacterialactivity in di#erent parts of the body of Pandalus borealis Krøyer (northernshrimp), Pagurus bernhardus L. (hermit crab), Hyas araneus L. (spider crab)and Paralithodes camtschatica Tilesius (king crab). They are all members ofthe order Decapoda, with king crab and hermit crab belonging to theinfra-order Anomura, and the shrimp and the spider crab belonging to theinfra-orders Caridea and Brachyura, respectively. II. Materials and MethodsEXPERIMENTAL ANIMALS AND SAMPLE COLLECTION Live specimens of the shrimp P. borealis (220 specimens, average weight 8 g),the crabs P. bernhardus (62 specimens, average weight 14 g) and H. araneus(44 specimens, average weight 200 g) were obtained o# the coast of Tromsø,Norway. P. camtschatica (10 specimens, average weight 1500 g, all male) werecollected from Varangerfjord, Finnmark, Norway. All animals were caught inthe period from March–May 2000, and kept in separate tanks with circulatingseawater until haemolymph and tissue collection. In H. aureus and P.camtschatica haemolymph was collected by entering the unsclerotised mem-brane at the base of the chelipeds and pereiopods with a 21 gauge needleattached to a syringe. In P. borealis and P. bernhardus haemolymph waswithdrawn from the heart with a 25 gauge needle. In the large crabs (H.araneus and P. camtschatica) the haemolymph was immediately centrifuged at800 g at 4 C for 20 min to separate the haemocytes from the plasma (cell-freehaemolymph). After the animals were bled to death, di#erent tissues and partsof the body were sampled, pooled and kept on ice. P. borealis and P.
ANTIBACTERIAL ACTIVITY IN FOUR MARINE CRUSTACEAN DECAPODS 373bernhardus were divided into eggs, cephalothorax (including internal organs),exoskeleton and muscle (abdominal part). From H. araneus; gills, exoskeleton,internal organs, and eggs were collected. P. camtschatica were separated intogills, exoskeleton, muscle, and internal organs. All samples were lyophilisedand frozen separately at 20 C until extraction.EXTRACTION AND SEPARATION OF ANTIBACTERIAL FACTORS Freeze-dried samples (3–50 g) were extracted with 10 volumes (v/w) of 60%(v/v) acetonitrile (ACN; HPLC-grade, SDS, Peypin, France) containing 0·1%triﬂuoroacetic acid (TFA; Fluka Chemie AG, Buchs, Switzerland) for 24 h at4 C. The supernatant was collected, stored at 4 C, and the residue wasextracted once again under the same conditions. The combined supernatantswere incubated at 20 C for 1–2 h to allow two liquid phases, an acetonitrile-rich phase and an aqueous-rich phase, to be formed. The two phases wereseparated, lyophilised and kept frozen at 20 C until further extraction. Theremaining pellet was discarded. Because of small quantities and low watersolubility of several of the acetonitrile-rich fractions, these samples wereexcluded from the study. The aqueous phase fractions were solubilised in acidiﬁed water (pH 4·7) to aconcentration of 100 mg ml 1. Salt was removed from the aqueous phase bysolid phase extraction. The fractions (maximum 100 ml) were separatelyloaded on to 35 cc Sep-Pak C18 Vac cartridges (Water Associates, MA, U.S.A.)equilibrated in acidiﬁed water (0·05% TFA). After washing with acidiﬁedwater, three stepwise elutions were performed with 10, 40 and 80% (v/v) ACNin acidiﬁed water, respectively. Non-bound material was discarded. Thedi#erent eluates collected were lyophilised and reconstituted with Milli-Qwater (Millipore Corp., MA, U.S.A.) before testing for antibacterial activity.BACTERIAL STRAINS AND GROWTH CONDITIONS The Gram negative bacteria Vibrio anguillarum, serotype O2 (FT 1801, alsocoded as AL 104/LFI 6004), originally isolated from Atlantic salmon by sta# ofthe Norwegian Veterinary Institute (Oslo, Norway), Escherichia coli (ATCC25922), and the Gram positive bacteria Staphylococci aureus (ATCC 9144) andCorynebacterium glutamicum (ATCC 13032) were used as test organisms. V.anguillarum and C. glutamicum were chosen because in preliminary studiesthey were shown to have high sensitivity to various marine extracts. Allisolates were grown at room temperature in Mueller Hinton Broth (MHB;Difco Laboratories, Detroit, U.S.A.).ANTIBACTERIAL ACTIVITY TESTING After solid phase extraction, each fraction was reconstituted in Milli-Qwater and the protein content was determined by the BCA protein assay(Pierce, Rockford, IL, U.S.A.). All samples were diluted to a protein con-centration of 250 g ml 1 and serial two-fold dilutions were made beforeantibacterial activity testing. Aliquots of 50 l test fractions were incubated in
374 T. HAUG ET AL.96-well microtitre plates (Costar, No. 3599; Corning Inc., NY, U.S.A.) with50 l of a suspension of a mid-logarithmic phase culture of bacteria at astarting concentration of 5 103 cells per well in MHB. Incubations wereperformed at room temperature with continuous shaking. Bacterial growthwas assayed by measurement of the optical density at 600 nm using amicrotitre plate reader (THERMOmax; Molecular Devices Corp., CA, U.S.A.)every 6 h for 48 h. Negative controls included media plus sample, and mediaplus milli-Q water. Cecropin P1 (0·5 g ml 1), made synthetically as describedby Kjuul et al. , was used as a positive control. Antibacterial activity wasdetermined when the optical density of the growth control (bacteria plusmedia) reached an absorbance at 600 nm of 0·150–0·200. Fractions wereregarded as active when the optical density was less than 50% of the control,and fractions that were active at protein concentrations of 31·25 g ml 1 orlower were considered as fractions with high activity.PROTEINASE K AND HEAT TREATMENT Fractions showing antibacterial activity were tested for proteinase Ksensitivity. Proteinase K (Promega, Maddison, WI, U.S.A.) was dissolved in50 mM Tris–HCl (pH 7·5) at a concentration of 2·5 mg ml 1. The fractions werediluted in Milli-Q water to a protein concentration of 250 g ml 1. A volumeof 10 l proteinase K solution was added per 50 l sample. The mixture wasincubated at 42 C for 90 min for protein digestion. The temperature was thenelevated to 85 C for 15 min to inactivate the proteinase K. As a control (heattreatment) 10 l 50 mM Tris–HCl (pH 7·5) was added to 50 l of the dilutedsample and subjected to the same treatment as the proteinase K sample.Cecropin P1 (0·5 g ml 1) was used as control to ensure the activity ofproteinase K. The activity in the treated samples were determined in micro-titre plates (Costar) as described above. Fractions that no longer showedantibacterial activity after proteinase K/heat treatment were regarded assensitive.LYSOZYME ASSAY In order to test whether lysozyme-like compounds were present, all fractionswere tested for lysozyme activity according to the method of Nilsen et al. .A standard suspension of Micrococcus luteus (Sigma Chemical Co., No.M-3770, St. Louis, MO, U.S.A.) cell walls (0·2 mg ml 1) was prepared in 50 mMsodium acetate (pH 5·2) and 50 mM NaCl (1:1). The test fractions were dilutedto a protein concentration of 500 g ml 1. Experimental conditions consistedof 1 l sample added to 150 l of M. luteus suspension in microtitre plates(Costar, No. 3599). The activity was determined spectrophotometrically(SPECTRAmax Plus Microplate Spectrophotometer, Molecular DevicesCorp.) by recording the decrease in absorbance at 450 nm (due to cell walllysis) for 6 min at room temperature. Enzyme activity was expressed as units
ANTIBACTERIAL ACTIVITY IN FOUR MARINE CRUSTACEAN DECAPODS 375where one unit represents a decrease in absorbance of 0·001 min 1. Lysozyme-like activity was judged to be present when enzyme activity was more thantwo units per g protein.HAEMOLYTIC ASSAY To test whether the animals contain factors which are toxic to eukaryoticcells, the haemolytic activity in extracts from haemolymph/plasma, haemo-cytes, eggs, muscle, and exoskeleton were determined using fresh human redblood cells (RBC). Four millilitres of blood were collected from a healthyperson into a polycarbonate tube containing heparin to a ﬁnal concentrationof 10 U ml 1. The erythrocytes were isolated by centrifugation at 450 g for10 min and washed three times with phosphate-bu#ered saline (PBS;320 mOsm, pH 7·4) in order to remove plasma and bu#y coat. The cell pelletwas resuspended in 4 ml of PBS. The haematocrit value (Hct) was determinedand the RBC suspension was further diluted to a Hct value of 10%. The test samples were diluted to a protein concentration of 500 g ml 1 andthe test was performed in 96 well U-shaped microtitre plates (NunclonSurface, No. 163320; Nalge Nunc Int., Denmark). To each well was added 40 lPBS, then 50 l test fraction and lastly 10 l of the RBC suspension. Afterincubation in a shaker at 37 C for 1 h the plates were centrifuged at 200 gfor 5 min. The supernatants (60 l) were carefully transferred to new ﬂat-bottomed polycarbonate microtitre plates (Nunc No. 269620, Nalge NuncInt.) and the absorbance of the supernatant was measured at 550 nm. Baselinehaemolysis and 100% haemolysis were deﬁned as the amount of haemoglobinreleased in the presence of PBS and 0·1% Triton X-100 (Sigma), respectively. III. Results The results show that in vitro antibacterial activity is found in all crus-tacean species tested. However, when the antibacterial activity in di#erentSPE-eluates were compared, wide di#erences were found between eluates andorgans as well as between species (Table 1 and 2). Of the four di#erent bacteriatested, C. glutamicum was the most sensitive, while E. coli was the leastsensitive. In P. borealis the highest activity was found in haemolymph (all fractions),but mainly against C. glutamicum. Some activity was also observed in eggs (allfractions), cephalothorax (10 and 80% fraction), muscle (10 and 80% fraction),and exoskeleton (all fractions). Figure 1 shows the time course study of theantibacterial e#ect of the 10% haemolymph extract against C. glutamicum. Adiscernible antibacterial e#ect was evident down to a protein concentration of7·8 g ml 1, and no bacterial growth occurred at a protein concentration of31·3 g ml 1. Activity against E. coli was not detected in any extract, andnone of the antibacterial fractions lost their activity after proteinase Ktreatment. In P. bernhardus high antibacterial activity was found in haemolymph,cephalothorax and exoskeleton. Some activity was also detected in the eggs.The highest antibacterial activity was mainly detected in the 40 and 80% ACN
376 T. HAUG ET AL.Table 1. Antibacterial activity in extracts from Pandalus borealis and Pagurusbernhardus against Vibrio anguillarum (V.a.), Escherichia coli (E.c.), Corynebacterium glutamicum (C.g.) and Staphylococcus aureus (S.a.) Antibacterial activity Organ/tissue Fraction1 Pandalus borealis Pagurus bernhardus (% ACN) V.a. E.c. C.g. S.a. V.a. E.c. C.g. S.a. 10 ++ – +++ + + + + + Haemolymph 40 – – +++ – + – +++ – 80 + – +++ – + – ++ ++ 10 – – + – – – – – Eggs 40 + – + – + – + – 80 – – +++ + + – + – 10 – – – – + – – – Cephalothorax2 40 – – – – + – + – 80 ++ – ++ + + – +++ +++ 10 + – + – – – – – Muscle 40 – – – – – – + – 80 + – + + – – – – 10 + – + – + – + – Exoskeleton 40 – – + – + – + – 80 – – + + – – ++ ++ Abbreviations: – No antibacterial activity at a protein concentration of 125 µg m–1. + Antibacterial activity at a protein concentration of 125 µg m–1. ++ Antibacterial activity at a protein concentration of 62.5 µg m–1. +++ Antibacterial activity at a protein concentration of 31.25 µg m–1. Sensitivity to proteinase K (but not sensitive to heat). Sensitivity to heat (but might be sensitive to proteinase K). 1 Eluates from solid phase extraction. 2 Including internal organs.fractions, and Gram positive bacteria were most sensitive. Only two activefractions (haemolymph—10% and eggs—40%) were sensitive to proteinase Ktreatment, while most of the fractions were sensitive to heat treatment. Onefraction (haemolymph—10%) showed activity against E. coli. No or littleactivity was measured in the muscle fractions.
ANTIBACTERIAL ACTIVITY IN FOUR MARINE CRUSTACEAN DECAPODS 377Table 2. Antibacterial activity in extracts from Hyas araneus and Paralithodescamtschatica against Vibrio anguillarum (V.a.), Escherichia coli (E.c.), Corynebacterium glutamicum (C.g.) and Staphylococcus aureus (S.a.) Antibacterial activity Organ/tissue Fraction Hyas araneus Paralithodes camtschatica (% ACN) V.a. E.c. C.g. S.a. V.a. E.c. C.g. S.a. 10 +++ +++ +++ – – – – – Plasma 40 + – + + – – + – 80 ++ – +++ – – – ++ + 10 ++ + ++ + + – – – Haemocytes 40 +++ +++ +++ +++ + – + – 80 +++ +++ +++ – +++ +++ +++ + 10 – – – – – – – – Gills 40 + ++ +++ – – – – – 80 – – ++ – + – – – 10 – – + – – – ++ – Internal organs 40 – – – – – – – – 80 + + + ++ ++ – ++ – 10 + – – – – – + – Exoskeleton 40 ++ ++ +++ – + – +++ – 80 – – + + – – + – Abbreviations: – No antibacterial activity at a protein concentration of 125 µg m–1. + Antibacterial activity at a protein concentration of 125 µg m–1. ++ Antibacterial activity at a protein concentration of 62.5 µg m–1. +++ Antibacterial activity at a protein concentration of 31.25 µg m–1. Sensitivity to proteinase K (but not sensitive to heat). Sensitivity to heat (but might be sensitive to proteinase K). 1 Eluates from solid phase extraction. 2 Including internal organs. High antibacterial activity (against all bacteria) was detected in thehaemocytes and plasma of H. araneus. Activity was also detected in the gills(40 and 80% fraction), the internal organs (mainly 80% fraction), and theexoskeleton (mainly 40 and 80% fraction). No activity was observed in the egg
378 T. HAUG ET AL. 0.3 OD 600 nm 0.2 0.1 0 10 20 30 40 Time (h)Fig. 1. Antibacterial activity of 10% haemolymph extract from P. borealis against C. glutamicum grown in MHB. The optical density at 600 nm was measured in a bacterial suspension of 5 103 cells per well containing bacteria alone (
), or bacteria plus extract adjusted to a protein concentration of 7·8 g ml 1 (), 15·6 g ml 1 (), and 31·3 g ml 1 (), respectively. 0.3 OD 600 nm 0.2 0.1 0 10 20 30 40 Time (h)Fig. 2. Antibacterial activity of 80% haemocyte extract from P. camtschatica against C. glutamicum grown in MHB. The optical density at 600 nm was measured in a bacterial suspension of 5 103 cells per well containing bacteria alone (
), or bacteria plus extract adjusted to a protein concentration of 7·8 g ml 1 (), 15·6 g ml 1 (), and 31·3 g ml 1 (), respectively.extracts (data not shown). In contrast to P. bernhardus and P. borealis, mostof the activities found in H. araneus were sensitive to proteinase K treatment.The 10% fraction from plasma showed higher activity than the 10% fractionfrom haemocytes, and the activity in both fractions were not a#ected byproteinase K treatment. In P. camtschatica, as in the other species, the highest antibacterial activitywas found in the haemocyte extracts (40 and 80% fractions). These fractionswere active against all bacteria tested. Figure 2 shows the time course studyof the antibacterial e#ect of the 80% haemocyte extract against C. gluta-micum. The activity was higher with increasing protein concentration and nobacterial growth occurred at protein concentrations higher than 31·3 g ml 1.The activity in plasma was low compared to the haemocyte extracts. Arelatively high activity was also detected in the internal organs (10 and 40%fraction) and the exoskeleton (mainly 40% fraction). No activity wasmeasured in the muscle (data not shown). All active 40% ACN fractions weresensitive to proteinase K treatment, while none of the 10% fractions weresensitive. Of the 80% fractions with antibacterial activity, only the haemocyte
ANTIBACTERIAL ACTIVITY IN FOUR MARINE CRUSTACEAN DECAPODS 379Table 3. Haemolytic activity in extracts from Pandulus borealis, Pagurus bernhardus, Hyas araneus and Paralithodes camtschatica against human erythrocytes (RBC) Haemolytic activity (%)2 Fraction1Organ/tissue Pandulus Pagurus Hyas Paralithodes (% ACN) borealis bernhardus araneus camtschaticaHaemolymph3 10 90 10 10 0 40 30 10 50 0 80 nt4 90 40 100Haemocytes 10 nt nt 40 20 40 nt nt nt4 0 80 nt nt 30 20Eggs 10 100 0 0 nt 40 0 0 20 nt 80 10 0 0 ntMuscle 10 0 0 nt 0 40 20 10 nt 0 80 nt4 20 nt 0Exoskeleton 10 0 0 20 10 40 50 20 50 50 80 100 100 100 100 Abbreviations: nt, not tested; 1, eluates from solid phase extraction; 2, the data are presentedas % haemolysis compared to control (Triton X-100); 3, including haemocytes in P. borealis andP. bernhardus, without haemocytes in H. araneus and P. camtschatica; 4, not tested due to lack ofmaterial.fraction (and only the activity against V. anguillarum) was sensitive toproteinase K. Cecropin P1, an antimicrobial peptide originally isolated from pig intestine, had a bactericidal e#ect on V. anguillarum and E. coli, but showed littleor no activity against C. glutamicum and S. aureus. The activity of cecropinP1 was totally abolished after proteinase K treatment, and heat-treatedproteinase K showed no antibacterial activity. Lysozyme-like activity was detected in some of the crustacean extracts (datanot shown). These were the 80% egg fractions of P. borealis, P. bernhardus andH. araneus, and the 80% gill fraction of P. camtschatica. The activitiesdetected in the egg fractions ranged from 4–8 units per g protein and inthe gill fraction of P. camtschatica a value of 12 units per g protein wasrecorded. Several extracts from the crustaceans were lytic for human erythrocytes.The most abundant activity was detected in the haemolymph/plasma extractsand in the exoskeletal extracts (Table 3). In general, the highest activity wasdetected in the 80% fractions. However, high haemolytic activity (more than50% haemolysis compared to the control) was also detected in 10% fractionsfrom haemolymph and eggs of P. borealis. In addition, high activity could bemeasured in internal organs from H. araneus (10 and 80% fraction) and P.camtschatica (80% fraction), and in the gills (80% fraction) of P. camtschatica(data not shown).
380 T. HAUG ET AL. IV. Discussion A screening for antibacterial activity in di#erent organs and tissues of fourcrustacean species was conducted. The results show that all species testedpossess antibacterial activity in vitro. Antibacterial activity has previouslybeen described in a wide range of crustacean species [7, 15, 21–24]. In most ofthe species studied, only the haemolymph and/or the haemocytes have beentested for activity. The present study demonstrated the presence of anti-bacterial factors in several other tissues, such as gills, eggs, exoskeleton andinternal organs. Whether the same antibacterial factors are responsible forthe activity in all organs, is unknown. However, the major activity was mainlylocated in the haemolymph and/or the haemocytes. In fact, in P. borealis, H.araneus and P. camtschatica, some haemolymph/haemocyte fractions showedactivity with protein concentrations as low as 8 g/ml 1 (data not shown).This is comparable to studies in the shore crab, C. maenas, where haemocytelysate supernatants had antibacterial activity at protein concentrationsof approximately 2 g ml 1 . Some of the other tissues collected willnecessarily contain some haemolymph and haemocytes, but any dominante#ect on activity is not expected. High antibacterial activity could be detected in the gills of H. araneus,whereas little or no activity was detected in P. camtschatica. The process ofremoval of bacteria from the crustacean circulation involves the formation ofa large number of haemocyte-bacteria aggregations in the gill lamellae .The antibacterial activity in haemolymph/haemocytes of crustaceans can alsobe inducible [5, 23]. Several authors have described aggregations of haemo-cytes in the gills of crustaceans in which viruses, bacteria or fungal sporeshave been injected [25–28]. Therefore, if the H. araneus in the present studyhad an ongoing microbial infection, it is possible that the antibacterialactivity detected in the gills is of haemocyte origin. In H. araneus and P. camtschatica, some antibacterial activity was found inthe internal organs. The cephalothorax from P. bernhardus and P. borealis,which mainly consists of exoskeleton and internal organs, also showedactivity. Antibacterial activity has previously been found in internaltissues, such as seminal plasma of S. serrata  and hepatopancreas ofHomarus americanus . In the latter study some activity was also detectedin the haemolymph, but negligible activity was found in the haemocytes.These ﬁndings indicate that antibacterial factors are also produced incrustacean tissues other than in haemolymph/haemocytes. In insects, the fatbody (a functional equivalent of the mammalian liver) seems to be animportant organ for synthesis of antibacterial compounds, although anti-bacterial factors are produced in granular haemocytes and other tissues aswell [12, 29]. In other arthropods, like myriapods and chelicerates, thehaemocytes seem to be the main tissue for production of antibacterial agents[30, 31]. All animals in the present study were caught in their spawning period, andthe eggs sampled were ready to hatch. Some of the egg fractions from P.borealis and P. bernhardus showed antibacterial activity, mainly againstmarine bacteria. There was no detectable activity in the eggs from H. araneus.
ANTIBACTERIAL ACTIVITY IN FOUR MARINE CRUSTACEAN DECAPODS 381Antimicrobial activity has previously been found in the eggs of various marineinvertebrates [32, 33]. The antibacterial activity might be due to factors of theinnate immune system. However, the activity might also be of microbialorigin. The antimicrobial activity detected in the eggs of the decapodsPalaemon macrodactylus and H. americanus was demonstrated to be due tobacterial symbionts [34, 35]. Micro-organisms might in some cases also beresponsible for the antibacterial activity detected in the gills, exoskeleton andeven within the internal organs, although this is probably not likely for mostof the samples. The exoskeleton showed antimicrobial activity in all species tested. Theexoskeleton of crustaceans is composed mainly of chitin, a polymer ofN-acetyl-glucosamine covalently bound to protein. Biologically, a deacetylasetransforms chitin to chitosan by hydrolysing the acetamido groups ofN-acetyl-glucosamine . It has been reported that both chitin and chitosanfrom crustaceans possess antimicrobial activity [36, 37]. However, chitin isinsoluble in water and acid while chitosan is relatively insoluble in water, butsoluble in acid . Due to these properties, it is unlikely that chitin and/orchitosan are responsible for the antibacterial activity detected in this study.Several exoskeletal proteins have been characterised from crustaceans. Someof these proteins are cationic, low molecular weight peptides [38, 39], which ischaracteristic for antimicrobial peptides. However, none of these peptideshave been tested for antibacterial activity (Andersen, pers. comm.). The detection of antibacterial activity in the exoskeleton suggests that thisactivity is important in the defence against micro-organisms present in themarine environment. It has been reported that most of the bacteria residing onthe carapace of C. sapidus are susceptible to the hosts antibacterial factors,and crabs having depressed levels of this antibacterial activity were in higherrisk of developing shell disease . The solid phase extraction method separates compounds according to theirhydrophobicity. As antibacterial activity was detected in 10, 40 and 80%fractions, it is reasonable to assume that multiple factors are responsible forthe antibacterial activity. Some of the organic phase fractions (acetonitrile-rich phase), containing fat, carotenoids and other lipophilic compoundspossessed antibacterial activities (data not shown). The stock solutions of theaqueous phase fractions were based on protein concentration, and dilutionswere made from these stock solutions. Some fractions may contain a highproportion of non-active proteins, and it is therefore a possibility that someactive components are missed due to non-inhibitory concentrations. In H. araneus, most of the active fractions were sensitive to proteinase Ktreatment. Since enzymatic digestion destroys the antibacterial activity, theactive molecules are most likely of proteinaceous nature. Antibacterialpeptides and proteins have previously been identiﬁed in C. sapidus ,C. maenas [9, 41], S. serrata , Penaeus setiferus , P. vannamei  andParachaeraps bicarinatus , and thereby seem to represent a common featurein the defence system of crustaceans. It must be emphasised that the heatlabile fractions might also be sensitive to proteinase K treatment. The heattreatment was included in the test to ensure that the antibacterial activitydetected was not caused by proteinase K itself.
382 T. HAUG ET AL. In P. borealis and P. bernhardus, on the other hand, most of the activefractions were resistant to proteinase K treatment. The antibacterialcomponents in these fractions are therefore probably of non-proteinaceousnature. In P. bernhardus, the active fractions were generally heat labile,whereas in H. araneus the activities were resistant to heat. Heat labileantibacterial factors have been detected in several crustaceans [5, 42, 43].However, Relf et al.  isolated an antibacterial protein of 11·5 kDa whichwas active even after heating for 10 min at 100 C. These results indicatethat crustaceans have developed a variety of defence molecules againstpathogenic micro-organisms and the kind dominating vary among thedi#erent species. Since many fractions (especially from P. bernhardus) were sensitive to heattreatment, the antibacterial activity might be of enzymatic character.Lysozyme-like activity could be detected in all of the species tested in thisstudy, but only in the eggs from P. bernhardus, P. borealis and H. araneus, andin the gills from P. camtschatica. Lysozyme(s) might therefore be responsiblefor the antibacterial activity detected in these extracts. In contrast, noantibacterial activity was detected in the eggs from H. araneus. Attempts todemonstrate lysozyme activity in crustaceans have so far given equivocalresults, and no lysozyme has so far been puriﬁed and characterised. Lysozyme-like activity has been detected in the cysts (dormant eggs) of the marinebranchiopod Artemia franciscana , and in the haemolymph of severalfreshwater crayﬁsh [43, 44]. A common feature of these factors is that thehighest activity is demonstrated at pH values of 6·2–8·0, and the activity isheat labile. In the present study we examined for lysozyme activity at pH 5·2.It is therefore possible that we, under other experimental conditions, wouldhave detected higher activities. On the other hand, no lysozyme-likeactivity was detected in the haemolymph of P. setiferus , P. vannamei and C. sapidus . Based on previous reports and our results, the eggs seemto be a better target for lysozyme search than haemolymph in marinecrustaceans. Some of the fractions, especially of the haemolymph/plasma and exoskel-eton, contained high haemolytic activity. Cantacuzène  reported in 1912the presence of haemolytic factors in serum of Eupagurus prideauxii, which isclosely related to P. bernhardus. Since then, haemolytic activity has beendetected in Maia squinado , Panulirus argus , Penaeus californiensis, and in Nematopalaemon tenuipes . In the latter study, the activecompound was shown to be a steroid. In the present study, several extractsshowed both antibacterial and haemolytic activity. Whether the same com-pound(s) are responsible for both activities remains to be clariﬁed. From apharmaceutical point of view it is advantageous that antibacterial drugs haveno side e#ects, such as haemolytic activity. In conclusion, this study shows that a number of marine decapod crus-taceans contain factors with antibacterial activity, particularly in the haemo-lymph and/or the haemocytes. This property seems to be a common featurethroughout the order. Di#erences between active extracts regarding hydro-phobicity and sensitivity to heat/proteinase K treatment indicate that severalcompounds are responsible for the antibacterial activities. This study has also
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