Aquaculture research, tập 41, số 1, 2009

Aquaculture Research, 2009, 41, 1^10 doi:10.1111/j.1365-2109.2009.02294.x Promotion of callus propagation by 5-aminolevulinic acid in a Laminaria japonica sporophyte Katsuhiro Tabuchi1, Hiroyuki Mizuta2 & Hajime Yasui3 Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido, Japan Laboratory of Aquaculture Genetics and Genomics, Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido, Japan Laboratory of Marine Industrial Science and Technology, Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido, Japan Correspondence: H Mizuta, Faculty of Fisheries Sciences, Hokkaido University, Minato 3-1-1, Hakodate, Hokkaido 041-8611, Japan E-mail: mizuta@Âsh.hokudai.ac.jp Abstract The eĂects of 5-aminolevulinic acid (ALA) on the induction and growth of callus-like cells in Laminaria japonica were investigated in explants obtained from basal, middle and apical portions along the sporophyte 5-Aminolevulinic acid treatment promoted the induction of callus-like cells in explants obtained from all portions, and the induction rate was higher when a concentration of 50^500 mg L of ALA was used The promotion was especially remarkable in apical explants, and the induction was 10^14 times higher in the 100^500 mg L range than that in the mg L The cell division rate of callus-like cells showed the highest value in the explants cultured with 500 mg L of ALA for 14 days The promotion of the cell division rate by culturing with 500 mg L ALA was also observed under white, blue and red lights The callus-like cells, which were cultured in 500 mg L of ALA for months, had many clear chloroplasts After months, young thalli occurred These results suggest that the ALA treatment is eĂective for stable propagation of callus-like cells in L japonica Keywords: 5-aminolevulinic acid, callus, development, Laminaria, sporophyte Introduction The aquacultural production of Laminaria japonica is the highest of all the seaweeds in the world (Zemke- r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd White & Ohno 1999) Laminariales plants have been widely used in foodstuĂ, medicine, industrial materials, fodder and cosmetics In addition, Laminariales plants have useful chemical components, such as alginate, fucoidan, etc., that allow us to produce individuals with useful characteristics such as high quality, high production, high resistance to disease and environmental stress From this point of view, the development of techniques for plant breeding and the production of useful seed using biotechnology are desired Plant tissue culture is an important technique in both basic and applied studies The technique is also useful and valuable in seaweeds Therefore, many investigations have been carried out in many seaweed species At present, the induction of callus-like cells and regeneration has become possible in L japonica (Saga, Uchida & Sakai 1978; Yan 1984; Wang et al 1998) However, the problem remains that the induction level of callus and its growth rate are lower in seaweed than in higher plants (Aguirre-lipperhaide et al 1995) In addition, a reproducible and well-understood protocol for the production of rapidly growing callus-like cells from sporophyte explants of Laminariales has not yet been achieved (Butler & Evans 1990) 5-Aminolevulinic acid (ALA) is known as a precursor of chlorophyll a, and is naturally distributed in photosynthetic bacteria, algae and plants This compound promotes higher plant growth at low concentrations (Hotta et al 1997), and acts as a herbicide at high concentrations (Rebeiz et al 1984) Additionally, Callus propagation byALA in Laminaria sporophyte K Tabuchi et al this compound has been used to induce colour improvement and tolerance to cold and high salt levels (Sakai et al 2002) The purpose of this study is to evaluate the eĂect of ALA on the induction and growth rate of callus-like cells in the sporophyte of L japonica The eĂect of ALA on the production of callus-like cells in L japonica is also discussed Materials and methods Preparation of young sporophytes The mature sporophytes of L japonica were collected in the coastal area near Hakodate and Hokuto cities in Hokkaido, Japan, from February 2006 and August 2007 These sporophytes were transported to our laboratory The sorus parts were cut from the mature sporophyte using a dissecting knife and stored at 1C in a refrigerator overnight after being wiped with a paper towel and wrapped with a newspaper After 24 h, the sorus part was then placed in 200 mL of seawater to release zoospores The zoospores were then cultured in a polystyrene case (square type, 8.5 17.5 3.5 cm) at 1C, 60 mmol photons m s in a 12-h light:12-h dark (12L:12D) cycle until they developed into sporophytes through the gametophyte stage at the bottom of the culture case Provasolis enriched seawater (Provasoli 1968) without vitamins was used as a medium and renewed once a week during the culture When the sporophytes grew to ca 1mm in length, they were detached from the bottom of the culture case The detached sporophytes were cultured in a polyethylene terephthalate bottle (1L) with aeration and allowed to grow thalli with ca cm blade length at 10 1C, 60 mmol photons m s (12L:12D cycle) The young sporophytes were supplied to the explant collection The seawater used as a medium was Âltered through a glass Âbre Âlter (GA-100, Toyo Roshi Kaisha, Tokyo, Japan) and autoclaved at 121 1C for 20 (ASV-2401, Tiyoda Manufacturing Tokyo, Japan) Bacteriostatic treatment of explants The young sporophytes were cut 0.5 cm from the blade^stipe transition and the apex, and the basal and apical parts were removed The residual central portions of the thallus were subjected to bacteriostatic treatment The segments were placed in 50 mL Aquaculture Research, 2009, 41, 1^10 of seawater with Âne sand, and they were shaken for 15 s This washing procedure was repeated three times The segments were then placed in seawater containing 0.1% of Tween 20, and were shaken for 10 s After the segments were fully washed with seawater, the segments were placed in sterilized seawater including 0.1% of Popiyodon (10% povidone-iodine solution,Yoshida Pharmaceutical,Tokyo, Japan) and agitated After discolored segments were removed, the living segments were placed in an antibiotic mixture (Druehl & Hsiao 1969) After 12 h, the explants (ca mm2) were obtained They were washed with seawater and their sterility was checked using the following procedure The sterility test was carried out using Âve media: STP (Provasoli et al 1957), ST3 (Iwasaki 1965), ESSB1 (Saga 1991), STM (Polne-Fuller & Gibor 1984) and Zobell 2216 (Zobell 1941) Five test tubes containing each medium with 0.4% agar were autoclaved After they were cooled to room temperature, the explants were inserted into each medium and, respectively, cultured at 20 1C under zero (dark) and 60 mmol photons m s for weeks These test tubes were observed under an inverted microscope (Leica DMIL, Wetzlar, Germany) to conÂrm the existence or absence of bacterial colonies Induction conditions on callus-like cells Preliminary experiments were performed to clarify the preferable water temperature, salinity and light intensity in callus induction of L japonica Thirty explants (ca mm2) of the sporophyte were cultured under the basal conditions, which were 10 1C, 27.5 psu and pH 7.8 in a vitamin-free PES medium under 30 mmol photons m s (12L:12D cycle) of illumination under a white Êuorescent lamp (Parck cool, 18 W, National, Osaka, Japan) after the bacteriostatic treatment described above The preferable conditions were determined by varying each factor under the basal conditions The formation of calluslike cells was observed under an inverted microscope during days of culture The callus induction index was estimated according to our previous report (Mizuta et al 2007) The callus occupation rate on a cut face of a square explant was divided into six levels (x) (0%, 0%oxo25%, 25% xo50%, 50% xo75%, 75% xo100% and 100%) We conveniently used the represented values of 0%, 12.5%, 37.5%, 62.5% 87.5% and 100%, which corresponded to six callus occupation levels, to calculate a callus induction in- r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 1^10 Aquaculture Research, 2009, 41, 1^10 Callus propagation byALA in Laminaria sporophyte K Tabuchi et al dex The index (CIn) was calculated using the following equation: CIn%ị ẳ SCi=4N where CIn is the average callus index of the total observed explants, N is the number of observed sporophyte explants and Ci expresses the sum of the callus coverage in each phase of a square explant Accordingly, when a callus occupied four phase of the explants as a whole, the callus induction index was deÂned to be 100% in C1 EĂects of ALA on induction and growth of callus-like cells To study the eĂects of ALA on callus induction in different collection portions of explants, the sporophyte (ca cm in length) was divided into apical, middle and basal portions along the sporophyte axis Forty explants (2 mm2) obtained from each portion were cultured at a density of 20 explants/50 mL at diĂerent ALA concentrations of 0, 1, 5, 10, 50, 100 and 500 mg L under the basal conditions as described above After days, the callus induction index was estimated The culture was continued for about months Twenty other explants (ca mm in length and ca 250 mm in width), which were collected from the basal, middle and apical portions along the thallus, were cultured in vitamin-free PES medium containing 0, 10, 50, 100, 500 and 750 mg L of ALA under the basal conditions to study the eĂect of ALA on the growth of callus After culturing for and 14 days, the Âlamentous callus-like cells were observed under an inverted microscope to estimate the cell division rate Fifteen Âlaments, which were mostly progressive advanced, were selected and the cell number of Âlaments was counted In each condition, the cell division rate was expressed in units/day Moreover,50 other explants were also cultured in a vitamin-free PES medium with and without 500 mg L of ALA under white (peaks at 450, 540 and 610 nm, Parck, National, Osaka, Japan), blue (400^530 nm, peak at 470 nm) and red light (600^ 700 nm, peak at 660 nm) (MIL ^ B18, MIL ^ R18, Sanyo Electric Biochemical, Osaka, Japan) After culturing for 14 days, the callus-like cells were observed under an inverted microscope, and the cell division rate was estimated All data were expressed as the mean1standard deviation The statistical diĂerences in preliminary experiments were determined using a Kruskal^Wallis test, followed by ScheĂes test, because these data did not show homologous variances In the other experiments for evaluation of the eĂects of ALA on the induction and cell division rate of callus-like cells, the statistical analysis was performed using two- or three-way analysis of variance (ANOVA) Results were considered to be signiÂcant at Po0.05 Results There were no bacterial colonies in the sterility test tubes containing ST3, ESS and STM media incubated under both dark and light conditions However, bacterial colonies were observed in one tube of STP medium and three tubes containing Zobell 2216 medium incubated under the light conditions, and in two tubes containing STP medium and three tubes containing Zobell 22 incubated in the dark The callus induction index was signiÂcantly inÊuenced by water temperature, light intensity and salinity (Fig 1) The callus induction index was the highest at 10 1C The high light intensities, which were more than 60 mmol photons m s 1, resulted in a decrease in the callus induction index Calluslike cells were not induced in salinities o25 psu The eĂects of ALA concentration on the callus induction of the explants obtained from diĂerent portions along the sporophyte are shown in Table The collection portion signiÂcantly inÊuenced the callus induction index (Table 2), and the explants obtained from the basal portion had the highest indexes (499.2%) and the lowest indexes were observed in the explants obtained from the apical portion.5-Aminolevulinic acid concentrations considerably inÊuenced the induction index in the explants obtained from the apical and middle portions, and the index increased with elevated levels of ALA The promotion of the induction of callus-like cells by the addition of ALA was remarkable in the explants obtained from the apical portion, and higher indexes were observed in the 50, 100 and 500 mg L ALA concentrations During the culture, the survival rate of basal segments was100% regardless of the ALA concentration for the Ârst days of culture However, there were some bleached explants from the middle and apical portions In particular, this tendency was strong in explants obtained from the apical portion, and the lowest survival rate (25^32.5%) was observed in low ALA concentrations o0 mg L The eĂects of ALA concentration on the cell division rate of callus-like cells are shown in Fig r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 1^10 Callus propagation byALA in Laminaria sporophyte K Tabuchi et al Callus induction index (%) The explant collection portions signiÂcantly inÊuenced the cell division rate (Table 3) similar to the case of the callus induction The cell division rate of callus-like cells was lower (0.14^0.51day 1) in the 100 90 80 70 60 50 40 30 20 10 (a) b ab a c 5C 10C 15C 20C Callus induction index (%) Water temperature (C) 100 90 80 70 60 50 40 30 20 10 (b) a a a b 15 30 b 60 120 Callus induction index (%) Light intensity (àmol photons m 100 90 80 70 60 50 40 30 20 10 s ) (c) b b b ab a n.d a a 15 17.5 20 22.5 Aquaculture Research, 2009, 41, 1^10 explants obtained from apical portions than those (0.21^1.04 day 1) from the middle and basal portions through the culture period The cell division rate in the Ârst days of culturing was increased with increasing ALA, and was the highest in 500 or 750 mg L regardless of the collection portion of the explants The higher cell division rate in 500 mg L was maintained for 14 days of culturing, particularly in the explants obtained from the middle portion of the thallus In contrast, the cell division rate of callus-like cells cultured in 750 mg L decreased to the same level as in the mg L after 14 days, and the colour changed to greenish and some dead cells were observed The features of callus-like cells cultured for weeks in diĂerent ALA concentrations are shown in Fig The callus-like cells grew elongated and branched with increasing ALA concentrations, except under the condition of 750 mg L The calluslike cells continued to grow and the Âlament reached 4300 mm in length from the section after weeks of Table Two-way ANOVA results for the eĂect of ALA concentration and the collection portion of explants on the callus induction in a Laminaria japonica sporophyte n.d 25 27.5 30 32.5 35 Salinity (psu) Figure EĂects of water temperature (a), light intensity (b) and salinity (c) on the callus induction of explants of a Laminaria japonica sporophyte Data show mean1standard deviation (n 30) DiĂerent letters represent statistically signiÂcant diĂerences at P 0.05 by ScheĂes test (P 0.05) nd represents no data Source of variation df ALA concentration Collection portion ALA concentration portion 12 Error 819 Total 839 MS F P-value 29 928.192 30.77 o0.001 415 432.7 427.16 o0.001 8211.127 9.47 o0.001 866.481 Statistically signiÂcant diĂerences at the 1% level ALA, 5-aminolevulinic acid Table EĂect of external ALA concentration on callus-like cells induction in the explants obtained from apical, middle and basal portions of a Laminaria japonica sporophyte ALA concentration (mg L 1) Portion of thallus 10 50 100 500 4.14 8.36 7.27 7.11 35.7 58.2 43.8 Apical ặ ặ ặ ặ ặ ặ ặ Middle 9.87 18.8 16.6 14.7 38.9 40.7 41.4 (25.0) (27.5) (25.0) (32.5) (77.5) (75.0) (77.5) 26.5 36.6 34.8 44.4 50.0 94.1 89.3 ặ ặ ặ ặ ặ ặ ặ Basal 41.4 46.3 45.5 46.5 50.6 22.4 30.4 (35.0) (37.5) (40.0) (50.0) (50.0) (95.0) (85.0) 99.2 100 100 100 100 100 100 ặ ặ ặ ặ ặ ặ ặ 4.94 0.00 0.00 0.00 0.00 0.00 0.00 (100) (100) (100) (100) (100) (100) (100) Data are mean induction index (%) ặ standard deviations (n 40) The numbers in parentheses indicate the survival rate (%) of explants cultured for a week ALA, 5-aminolevulinic acid r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 1^10 Cell division rate (day1) Cell devision rate (day1) Cell devision rate (day1) Aquaculture Research, 2009, 41, 1^10 1.2 Callus propagation byALA in Laminaria sporophyte K Tabuchi et al Table Two-way ANOVA results for the eĂect of ALA concentration and the explant collection portion on the cell division rate of callus-like cells in Laminaria japonica sporophyte cultured for and 14 days (a) 1.0 0.8 0.6 0.4 Source of variation df MS F P-value 10 72 89 1.504 0.245 0.027 0.005 290.69 47.28 5.13 o0.001 o0.001 o0.001 10 72 89 0.297 0.249 0.030 0.015 19.65 16.45 1.97 o0.001 o0.001 0.0490 0.2 0.0 1.2 10 50 100 500 ALA concentration (mg L1) 750 (b) 1.0 0.8 0.6 0.4 0.2 0.0 1.2 10 50 100 500 ALA concentration (mg L1) 750 Statistically signiÂcant diĂerences at Po0.05 Statistically signiÂcant diĂerences at Po0.01 ALA, 5-aminolevulinic acid (c) 1.0 0.8 0.6 0.4 0.2 0.0 (a) Culture for days ALA concentration Collection portion ALA concentration portion Error Total b) Culture for 14 days ALA concentration Collection portion ALA concentration portion Error Total 10 50 100 500 ALA concentration (mg L1) 750 Figure EĂect of external 5-aminolevulinic acid concentration on the cell division rate of callus-like cells in the explants obtained from apical (a), middle (b) and basal (c) portions of a Laminaria japonica sporophyte.White and black columns show the cell division rate of callus-like cells after and 14 days of culturing respectively Data show mean1standard deviation (n 515) being cultured in 500 mg L When the explants were cultured in 500 mg L for month, the callus-like cells were unpigmented and they were often bleached (Fig 4a) In months of culture, the number and size of chloroplasts increased (Fig 4b) The chloroplasts became clear after months (Fig 4c) After months, regenerated thalli were often observed (Fig 4d^f) In addition, there are often two types of thalli The types were characterized by the site of rhizoidal cells One was the thallus that had rhizoidal cells attached to the apical cells of a calluslike Âlament (Fig 4d, e) Another site of rhizoidal cells did not attach to the callus-like Âlament, but the apex of the thallus attached directly to the Âlament (Fig 4f) The eĂects of light quality and ALA treatment on the cell division rate of callus-like cells in the explants obtained from diĂerent portions of sporophyte are shown in Fig The three-way ANOVA result shows statistically signiÂcant eĂects of light quality and ALA treatment on the cell division rate of callus-like cells (Table 4) The cell division rate was high under red light and lower under blue light in all explants regardless of the collection portions Moreover, ALA treatment promoted the cell division rate under all illumination conditions In particular, the rate increased 32^104% in explants from basal and apical portions The explants obtained from the basal portion reached a cell division rate of 1.0 day after the addition of ALA under white and red light conditions Discussion The results of a sterility test showed that bacterial colonies were detected in a part of the media This means that the pre-treatment of explants was not under completely bacteria-free conditions but rather under disinfected conditions The preferable water temperature for callus induction was consistent with that for growth in the whole sporophyte (Okada et al 1985) Exposure to salinity o20 psu resulted in discoloration and erosion of the cells This means that the callus-like cells show the same response as the whole plant (H Mizuta, pers commun.) However, the callus induction was inhib- r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 1^10 Callus propagation byALA in Laminaria sporophyte K Tabuchi et al (a) (b) (c) (d) (e) (f) Aquaculture Research, 2009, 41, 1^10 Figure Callus-like cells in explants obtained from the basal parts of a Laminaria japonica sporophyte The explants were cultured in diĂerent 5-aminolevulinic acid (ALA) concentrations for 14 days (a) ALA mg L 1, (b) ALA 10 mg L 1, (c) ALA 50 mg L 1, (d) ALA 100 mg L 1, (e) ALA 500 mg L and (f) ALA 750 mg L Scale bar:100 mm for all ited in 60 and 120 mmol m s 1, which the growth of whole plants is not inhibited The results showed a negative eĂect of high light intensity on the induction of callus-like cells, although the reason for this was not clear In addition, callus induction occurred even in the dark, but the subsequent growth was inhibited This result indicates that callus induction does not require light irradiation, and that the subsequent growth requires light It is also suggested that the callus-like cells are able to grow autotrophically In this study, ALA promoted the induction and propagation of callus-like cells at concentrations of 450 mg L Particularly, the cell division rate was maintained at the high concentration of 500 mg L 1, which was the same concentration to stimulate the growth of Spirulina platensis (Sasaki et al 1995) In higher plants, ALA promotes plant growth at low concentrations (Hotta et al 1997) and acts as a herbi- cide at high concentrations (Rebeiz et al.1984) In this study, culturing in 750 mg L for 14 days inhibited the propagation of callus-like cells regardless of the explant collection portion along the thallus The growth inhibition by a high ALA level is considered to be due to the herbicidal eĂect of ALA The herbicidal eĂect also seems to become remarkable with culture time In addition, Bindu & Vivekanandan (1998) reported, in the callus of Vigna unguiculata, that low concentrations promoted growth and high concentrations promoted root formation, showing that ALA may have hormone-like activity Therefore, the optimal concentration of ALA treatment might be diĂerent among species The induction and propagation of callus-like cells are often inÊuenced by the explant collection portion from the plant (Notoya 1988) and the collection time (Fries 1980; Kawashima & Tokuda 1990) In previous r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 1^10 Aquaculture Research, 2009, 41, 1^10 Callus propagation byALA in Laminaria sporophyte K Tabuchi et al (a) (b) (c) (d) (e) (f) Figure Morphological changes in callus-like cells in basal Laminaria japonica explants cultured in ALA 500 mg L (a) After month, Âlamentous callus-like cells with undeveloped chloroplasts are shown (b) After months, Âlamentous callus-like cells with developing chloroplasts are shown (c) After months, Âlamentous and swelling callus-like cells with developed chloroplasts are shown (d^f) After months, regenerated thalli are shown Scale bars:100 mm for all studies, the explants obtained from meristem show higher induction and growth In L japonica, the induction was lower in the explants from the apical portion than those in the middle and basal portions The apical portion is located far from the meristematic region between the stipe and the lamina Therefore, the diĂerence in the induction of calluslike cells among collection portions is considered to be due to age and cell division activity of the explant cells In addition, the nutritional status of explants may inÊuence the induction and growth of callus (Aguirre-Lipperhaide et al 1995) Interestingly, the diĂerence in the cell division rate of callus-like cells among the portions of the thallus seemed to be smaller than that in the induction of callus-like cells These results suggest that the callus-like cells divided independently from the mother explants In other words, the contribution of the mother plant to the propagation of callus-like cells seems to be low compared with its contribution to the induction of callus-like cells It has been reported that the morphological form of callus-masses raised from explants of seaweeds can be divided into two types: pigmented and unpigmented Âlaments (Lawlor et al 1988; Kawashima & Tokuda 1990; Notoya 1997) In this study in L japonica, the callus-like cells cultured with ALA showed typical unpigmented Âlaments early in the culturing period These Âlaments often branched However, the unpigmented cells changed to pigmented Âlaments after months of culturing Chloroplast changes in both size and number in the callus-like cells were also observed in Eckloniopsis radicosa (Notoya 1990) The pigmented cells re- r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 1^10 Callus propagation byALA in Laminaria sporophyte K Tabuchi et al (a) 1.2 Cell division rate (day1) 1.0 (b) (c) 1.2 1.2 White Blue White Red 1.0 1.0 Red 0.8 Aquaculture Research, 2009, 41, 1^10 Red Blue Blue 0.8 0.8 0.6 0.6 0.6 0.4 0.4 0.4 0.2 0.2 0.2 White 0.0 500 500 0.0 500 ALA concentration (mg L1) 500 500 500 ALA concentration (mg L1) 0.0 500 500 500 ALA concentration (mg L1) Figure EĂects of light quality and ALA treatment on the cell division rate in explants obtained from apical (a), middle (b) and basal (c) portions of a Laminaria japonica sporophyte The explants were cultured with and without 500 mg L ALA under white, blue and red light for 14 days Data show mean1standard deviation (n 50) Table Three-way ANOVA results for the eĂect of explant collection portion and light quality on the cell division rate of callus-like cell of explants in Laminaria japonica sporophyte cultured with and without ALA (500 mg L 1) Source of variation df MS F P-value Collection portion Light quality ALA treatment Portion light Portion ALA tr Light ALA tr Portion light ALA tr Error Total 2 2 894 899 8.052 2.283 7.608 0.564 0.532 0.348 0.079 0.009 1893.4 528.26 1760.7 130.56 123.05 80.53 18.39 o0.0001 o0.0001 o0.0001 o0.0001 o0.0001 o0.0001 o0.0001 Statistically signiÂcant diĂerences at the 1% level ALA, 5-aminolevulinic acid diĂerentiated to the thallus through unicellular embryos after months These observations indicate that the development of callus-like cells progresses in the following order: unpigmented cells, pigmented cells, embryogenesis and thallus formation When an embryo developed to a two-celled bladelet, diĂerent types of thalli were observed These thalli were divided into two types based on the site of the rhizoidal cells (Fig 4) One type of thallus had rhizoidal cells located on the far side of the callus-like cells The rhizoidal cells in this type showed good growth Rhizoidal cells of the other type were located on the near side of the callus-like cells Light quality is an important factor in the induction, propagation and re-diĂerentiation of algal callus-like cells The eĂects of diĂerent wave-lengths of light on the callus have been reported in Ecklonia radiata (Lawlor et al 1989) and L japonica (Mizuta et al 2007), in which it was found that the growth of the callus was promoted by red light Lawlor et al (1989) found that the photosynthetic activity was promoted when the wavelength of red light corresponded to the maximum absorption wavelength of chlorophyll a Interestingly, ALA led to callus-like cell growth that was more rapid than the growth observed under the irradiation of red light The doubling rate has been used as an indicator of the propagation of single cells in higher plants In this study, the doubling time in the early period of calluslike cell formation was estimated to be about 1^1.4 days, although the time was inÊuenced by environmental conditions This doubling time was shorter than that (5 days) of free-living gametophytes of Laminaria digitata (Martino et al 2000) Thus, ALA treatment promotes the vegetative propagation of callus-like cells, but it took about three months to obtain in the re-diĂerentiated sporophytes of L japonica Wang et al (1998) also reported that one callus diĂerentiated into a sporophyte of L japonica after r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 1^10 Aquaculture Research, 2009, 41, 1^10 Callus propagation byALA in Laminaria sporophyte K Tabuchi et al months of culture Probably, there is a diĂerent mechanism between vegetative propagation and re-differentiation of the callus-like cells In conclusion, ALA treatment of explants of L japonica sporophyte is useful to produce and propagate callus-like cells stably The ALA treatment will be a good tool for the investigation of callus induction and formation In addition, the thallus that originated from callus-like cells often showed an abnormal form Therefore, development of techniques for obtaining normal morphogenesis from callus-like cells is a future aim Acknowledgments This work was supported in part by a grant-in aid for ScientiÂc Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (no 16580139) and by a grant from the 21st century COE Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan References Aguirre-Lipperhaide M., Estrada-Rodr|Ô guez F.J & Evans L.V (1995) Facts, problems, and needs in seaweed tissue culture: an appraisal Journal of Phycology 31, 677^688 Bindu R.C & Vivekanandan M (1998) Hormonal activities of 5-aminolevulinic acid in callus induction and micropropagation Plant Growth Regulation 26,15^18 Butler D.M & Evans L.V (1990) Cell and tissue culture of macroalgae In: Introduction toApplied Phycology (ed by I Akatsuka), pp.629^645 SPB Academic Publishing bv,The Hague, the Netherlands Druehl L.D & Hsiao S.I.C (1969) Axenic culture of Laminariales in deÂned media Phycologia 8, 47^49 Fries L (1980) Axenic tissue cultures from the sporophytes of Laminaria digitata and Laminaria hyperborean (Phaeophyta) Journal of Phycology 16, 475^477 Hotta Y., Tanaka T., Takaoka H., Takeuchi Y & Konnai M (1997) Promotive eĂects of 5-aminolevulinic acid on the yield of several crops Plant Growth Regulation 22,109^114 Iwasaki H (1965) Nutritional studies of the edible seaweed Porphyra tenera I The inÊuence of diĂerent B12, analogues, plant hormones, purines and pyrimidines on the growth of conchocelis Plant and Cell Physiology 6, 325^336 KawashimaY & Tokuda H (1990) Callus formation in Ecklonia cava Kjellman (Laminariales, Phaeophyta) Hydrobiologia 204/205, 375^380 Lawlor H.J., McComb J.A & Borowitzka M.A (1988) The development of Âlamentous and callus-like growth in axenic tissue cultures of Ecklonia radiata (Phaeophyta) In: Algal Biotechnology (ed by T Stadler, J Mollion, M.-C.Ver- dus, Y Karamonos, H Morvan & D Christiaen), pp 139^ 150 Elsevier Applied Science, London, UK Lawlor H.J., McComb J.A & Borowitzka M.A (1989) Tissue culture of Ecklonia radiata (Phaeophyceae, Laminariales): eĂects on growth of light, organic carbon source and vitamins Journal of Applied Phycology 1,105^112 Martino A.D., Rousseau B., Duval J.C., Noel H & Caron L (2000) EĂect of a cationic cellulosic polymer on gametophytes of Laminaria digitata (Laminariaceae, Phaeophyta): improvement of dispersed free-livingculture Journal of Applied Phycology 12, 83^93 Mizuta H., Kai T.,Tabuchi K & Yasui H (2007) EĂects of light quality on the reproduction and morphology of sporophytes of Laminaria japonica (Phaeophyceae) Aquaculture Research 38,1323^1329 Notoya M (1988) Tissue culture from the explant of Ecklonia stolonifera Okamura (Phaeophyta, Laminariales) TheJapaneseJournal of Phycology (Sorui) 36, 175^177 Notoya M (1997) Chloroplast changes and diĂerentiation of callus cells in Eckloniopsis radicosa (Kjellman) Okamura (Phaeophyta, Laminariales) Journal of Applied Phycology 9,175^178 Notoya M & ArugaY (1990) Tissue culture from the explant of stipe of Eisenia bicyclis (Kjellman) Setchell (Laminariales, Phaeophyta) The Japanese Journal of Phycology (Sorui) 38, 387^390 Okada Y., Sanbonsuga Y & Machiguchi Y (1985) The eĂects of temperature on the growth and shape of the early sporophytes of Laminaria japonica, L ochotensis, L diabolica, L religiosa and L angustata var longissima in culture Bulletin of the Hokkaido Regional Fisheries Research Laboratory 50, 27^44 (in Japanese with English abstract) Polne-Fuller M & Gibor A (1984) Developmental studies in Porphyra I Blade diĂerentiation in Porphyra perforata as expressed by morphology, enzymatic digestion, and protoplast regeneration Journal of Phycology 20,609^616 Provasoli L (1968) Media and prospects for the cultivation of marine algae In: Culture and Collections of Algae Proceedings of U.S ^Japan Conference (ed byA.Watanabe & A Hattori), pp 291^299 Japanese Society of Plant Physiology, Tokyo, Japan Provasoli L., McLaughlin J.J.A & Droop M.R (1957) The development of artiÂcial media for marine algae Archiv ftir Mikrobiologie 25, 392^428 Rebeiz C.A., Montazer-Zouhoor A., Hopen H.J & Wu S.M (1984) Photodynamic herbicides:1 Concept and phenomenology Enzyme MicrobialTechnology 6, 390^401 Saga N (1991) Tissue culture in marine plant In: Labo-Manual Marine Biotechnology (supervised by S Miyachi), ed by N Saga & T Matsunaga), pp 29^43 Shokado,Tokyo, Japan (in Japanese) Saga N., Uchida T & Sakai Y (1978) Clone Laminaria from single isolated cells Bulletin of the Japanese Society of ScientiÂc Fisheries 50, 87 Sakai K., Watanabe M., Tanaka T & Tanaka T (2002) Biosynthesis, biotechnological production and application of r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 1^10 Hepatopancreatic and muscular OTC in white shrimp D Faroongsarng et al limit of quantitations (Table 1) Five replications were performed for each of Âve concentration levels having low relative standard deviations with a good precision And to ensure that OTC in biological matrices is stable during the storage, the spiked biological matrices were stored at 80 1C for months The samples were thawed and determined for OTC The obtained %R was in the range between 90.01% and 99.78%, indicating that OTC in biological matrices was stable up to months of storage In sum, it was found that the determination of OTC in the haemolymph, muscle and hepatopancreas of P vannamei using this method was valid Aquaculture Research, 2009, 41, 143^152 ium (denoted as QI) through each physiological region (the subscript I denoted as B, M or L), and the relative anity of the drug for the haemolymph and those tissues (RI) It was assumed that Êow was rate limiting and the drug concentration in the haemolymph rapidly reached equilibrium with the drug concentration in the tissue space of each physiological compartment, i.e., the diĂusion time for the drug to cross the tissue cell membrane and enter the tissue space is relatively fast compared with the transport of the drug via haemolymph Êow As the haemolymph is conserved within the shrimp body, the Êow rates were conditioned as: QB equals QM1QL Using the law of mass balance, the model is then described by a set of three diĂerential equations: Pharmacokinetic analysis VB Intra-sinus administration The intra-sinus administration of OTC uses the circulatory system as a medium to transport the drug to sites of action In the model, the shrimp body was divided into three compartments based on the physiological tissues of interest: the haemolymph, muscle (peripheral tissue) and hepatopancreas, denoted as B, M and L respectively A scheme of the OTC pharmacokinetic model is illustrated in Fig The model also includes a foregut, but as the OTC is directly introduced into the haemolymph, the foregut compartment was not involved in the parameters or variables of the study The drug-time proÂles in the diĂerent compartments were assessed on the basis of the drug Êow rates, where the shrimp haemolymph was the med- Figure Schematic diagram representing the oxytetracycline (OTC) physiological model There are two routes of administraion: intra-sinus (is dose) and oral (oral dose) 146 dCB CM CL ẳ QB CB ỵ QM ỵ QL dt RM RL dCM CM ẳ QM CB QM dt RM   dCL QL ỵ ClL VL CL ẳ QL CB dt RL VM 1ị 2ị 3ị where CI is the drug concentration (mg mL in the haemolymph, or mg g in other tissues) and VI the volume of distribution for each compartment (mL) ClL is the OTC hepatopancreatic clearance (mL h 1) Oral administration As the drug was administered via the gastrointestinal tract, the foregut compartment where OTC is introduced is involved in the model (Fig 2) It is further assumed that OTC is systemically absorbed via the hepatopancreas, and the drug transport from the haemolymph to the foregut is negligible The diĂerential equations describing the rate of OTC levels in haemolymph (Eq (1)) and muscle (Eq (2)) between intra-sinus and oral administrations are identical, while the OTC level in the hepatopancreas is shown in the following equation:   dCL QL ỵ ClL VL ẳ QL CB CL ỵ KG CG 4ị dt RL in which the subscript G represents the foregut compartment while the other variables remain as described for the intra-sinus administration The foregut connects to the hepatopancreas via the hepatopancreatic ducts, i.e., the drug Êows directly from the foregut to the hepatopancreas with an input rate of KG It was impossible to perform the diĂerential equations regression without data manipulation as the r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 143^152 Aquaculture Research, 2009, 41, 143^152 Hepatopancreatic and muscular OTC in white shrimp D Faroongsarng et al sampling time intervals selected were too large It was necessary to perform numerical interpolation of I the data among the time intervals so that DC Dt , which is CI;nỵ1 CI;n ị tnỵ1 tn ị could approach dCI dt , where Dt approaches zero The individual pharmacokinetic proÂle in each of the biological matrices was divided into 2^4 portions to explore the possible best-Ât functions for each of the portions Each was non-linearly Âtted with an empirical function having R2 of not o0.995 (SIGMAs PLOT for WINDOWS v 10.0, Systat Software, Chicago, IL, USA) The best-Ât functions then generated OTC level-time coordinates at pre-assigned 40-s intervals These generated data were gathered to yield the whole proÂle with the best possible superimposition on the original pharmacokinetic proÂle Further data smoothing was performed as needed The data were then imported to a spreadsheet (MS Excel 2007, Microsoft, USA) to calculate the diĂerential function as Table Estimated value of parameters after OTC (oxytetracycline) intra-sinus administration at 10 mg g body weight dose into white shrimps using Eqs (1 0)^(3 0) Pharmacokinetic profile Parameter estimate (SE) CBw CMz CL 0.552 (0.023) 0.396 (0.049) 0.036 (0.007) 0.9373 0.164 (0.013) 0.198 (0.019) 0.8266 0.412 (0.053) 0.105 (0.017) 0.6571 bi for intra-sinus administration or dCL ẳ a3 CB g2 CL ỵ KG CG dt Results Physiological-based model pharmacokinetics of oxytetracycline in PaciÂc white shrimp An attempt to simultaneously Ât the OTC levels in the haemolymph (CB), muscle (CM) and hepatopancreas (CL) with time after intra-sinus and oral administrations was made Instead of the several parameters shown in Eqs (1)^(4), the equations were simpliÂed as dCB ẳ a1 CB ỵ b1 CM ỵ g1 CL dt 1prime; ị dCM ẳ a2 CB b2 CM dt 2prime; ị dCL ẳ a3 CB g2 CL dt 3prime; ị and Coecient of multiple determination square: R2 51 (SSE/ SSTO), where SSE and SSTO are the sum square error and the sum square total of multiple regression according to Eqs (1 0)^ (3 0) wOTC in haemolymph proÂle Âtted into Eq (1 0) zOTC in muscle proÂle Âtted into Eq (2 0) OTC in hepatopancreas proÂle Âtted into Eq (3 0) dCI DCI CI;nỵ1 CI;n ị ẳ lim % t!0 tnỵ1 tn ị dt Dt The numerical data were Âtted with diĂerential equations of the proposed physiological model using commercial software (Systat for WINDOWS V 12, Systat Software) In addition, as OTC entered the hepatopancreas via the foregut in oral administration, the proÂle of the drug in the foregut was simulated based on Ârst-order kinetics using the initial drug concentration of the medicated feed and the absorption constant taken from the literature (0.2986 h 1: Faroongsarng et al 2007) in order to complete the nonlinear regression R2 cI 4prime; ị for oral administration a1 ẳ QB QM QL ;b ẳ ;g ẳ VB RM VB RL VB where a2 ẳ a3 ẳ QM QM ;b ẳ VM RM VM QL QL ỵ ClL ;g ẳ VL RL VL The interpolated data for pharmacokinetic proÂles were numerically Âtted into Eqs (1 0), (2 0) and (3 0) and (1 0), (2 0) and (4 0), yielding ai, bi, gi as well as KG and their standard errors of estimation (SE), as tabulated in Tables and Figures and show the intra-sinus and orally administered OTC pharmacokinetic proÂles according to the simultaneous regressions of Tables and 3, respectively, compared with actual data coordinates The squares of coecients of multiple determinations R2 were in the range of 0.6568^0.9904 (Tables and 3) The equations were the best Âts into the intra-sinus administered haemolymph and orally administered hepatopancreas proÂles with R2 values of 0.9373 and 0.9904, respectively, while the R2 values of the muscle proÂles were the lowest (intrasinus administered R2 of 0.6571 and orally administered R2 of 0.6568) r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 143^152 147 Hepatopancreatic and muscular OTC in white shrimp D Faroongsarng et al Aquaculture Research, 2009, 41, 143^152 Table Estimated values of parameters after OTC (oxytetracycline) oral administration at 50 mg g body weight dose into white shrimps using Eqs (1 0), (2 0), and (3 0) Parameter estimate (SE) Pharmacokinetic profile bi ci KG R2 CBw CMz CL 0.161 (0.020) 0.047 (0.006) 6.781 (0.410) 0.035 (0.015) 0.052 (0.006) 0.009 (0.001) 1.021 (0.028) 194.29 (3.2) 0.9007 0.6568 0.9904 Coecient of multiple determination square: R2 51 (SSE/SSTO), where SSE and SSTO are the sum square error and sum square total of multiple regression according to Eqs (1 0), (2 0), and (3 0) wOTC in hemolymph proÂle Âtted into Eq (1 0) zOTC in muscle proÂle Âtted into Eq (2 0) OTC in hepatopancreas proÂle Âtted into Eq (3 0) Figure Pharmacokinetic proÂles of intra-sinus administered oxytetracycline (OTC) in PaciÂc white shrimps (a) OTC level in haemolymph (b) OTC levels in muscle and in hepatopancreas The lines represent the proÂles according to the physiological model predictions [Eqs (1 0)^(3 0)] Discussion It is observed that the proposed model may be applicable to the simultaneous pharmacokinetic proÂles of OTC administered to P vannamei of the inter-moult C-D0 stage (Tables and and Figs and 4) As can be seen in Tables and 3, the R2 values of the muscle proÂles were relatively low On the one hand, from a statistical point of view, it is common for systemic errors of entrance compartments to be lower than those of peripheral ones when it comes to simultaneous 148 regression, which in turn, might also cause the R2 values of associated muscle proÂles to be lower On the other hand, unlike the typical vertebrate in which the blood circulation is well deÂned as a closed system, the circulatory system of Decapod crustaceans is classed as either open (Dall et al 1991) or partially closed (McGaw 2005) The shrimp cardiovascular system can be subdivided into three parts: (1) the heart, (2) blood or haemolymph and (3) the distribution pathway or arterial system (Guadagnoli, Tobita & Reiber 2007) The haemolymph leaves the r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 143^152 Aquaculture Research, 2009, 41, 143^152 Hepatopancreatic and muscular OTC in white shrimp D Faroongsarng et al Figure Pharmacokinetic proÂles of orally administered oxytetracycline (OTC) in PaciÂc white shrimp compared between actual data coordinates and predicted lines according to the model of Eqs (1 0), (2 0), and (4 0) heart via the arterial system The arteries then subdivide into arterioles, which perfuse all areas of the body The arterioles divide into Âne capillary-like vessels, most blind ending (open), but some Ânishing in complete capillary beds (closed), e.g in the areas of the antennal gland and supraesophageal ganglion After passing through these capillaries, the haemolymph drains into a series of sinuses that guide it to the gills to become reoxygenated, and Ânally to complete the system the oxygenated haemolymph enters the branchio-cardiac veins to the pericardial sinus and then to the heart to begin the circulation again Thus, OTC kinetics in shrimp body could be modelled only to some extent because the model assumption is Êow limited whereas shrimps circulatory system included not only haemolymph Êow but also perfusion Relative anity of drug for haemolymph and tissue By comparison, for the coecients of Eqs (1) with (1 0), (2) with (2 0), (3) with (3 0) and (4) with (4 0), the calculation of the relative anities of drug for haemolymph and tissue (RI) at equilibrium was possible Unfortunately, other parameters such as Êow rates (QI), volumes of distribution (VI) and hepatopancreatic clearances (ClL) could not be explicitly solved because of an insucient number of equations compared with the number of parameters being calculated A prior study performed on vertebrates used the measured blood Êow to the organs to estimate QI in order to cope with the insuciency (Tsuji,Yoshikawa, Nishide, Minami, Kimura, Nakashima, Terasaki, Miyamoto, Nightingale & Yamana 1983) However, the method used in that study could be used with an animal having a closed circulatory system because the distribution of the drug is assumed to be governed by the rate of blood Êow only Nevertheless, the blood Êow measurement for shrimp might not be as appropriate as for vertebrates due to the fact that the crustacean circulatory system is totally diĂerent It was found that the RM and RL values for intrasinus and oral administrations were 0.828 and 6.277, and 0.904 and 14.374 respectively Based on the model, RI is deÂned as the partition coecient for the distribution of OTC from blood into tissue where the drug in blood leaving the tissue achieves rapid equilibrium with the drug within the tissue (Welling 1986), i.e., RI ẳ CCoI, where CI and Co are the total concentrations in tissues and in blood leaving the tissues respectively As the RM of intra-sinus administration is comparable to that of oral administration (intra-sinus RM of 0.828 vs oral RM of 0.904), it is deduced that OTC distributes in shrimp muscle in an essentially identical manner regardless of the route of administration, i.e., the drug passively distributed from the haemolymph to muscular tissues The accumulation of OTC in muscle is less likely for RM, being slightly lower than unity, i.e., the total concentration is less than or, at best, equal to that in the haemolymph Thus, with an appropriate period of time after stopping dosing, OTC would be disposed out of the shrimp body, resulting in the edible portion being free from drug residue and safe for consumption Previous studies with diĂerent doses and dosing regimens (single dose of 10 mg g body weight with intra-sinus administration: Chiayvareesajja et al 2006) and multiple doses (OTC level of g kg feed with natural feed orally for 14 days: Gomez-jimenez, Espinosaplascencia, Valenzuela-villa & Bermudez-almada 2008) suggested an appropriate withdrawal time of 7^14 days As the tolerance of ppm for OTC in an aquaculture product has been codiÂed previously (FDA/CVM, 2007), it is thus demonstrated in the current study by Fig that at least days could be sucient for withdrawal time, which is comparable to previous Ândings The analysis of oxytetracycline distribution in the hepatopancreas It was found in the case of intra-sinus administration that the total concentration of OTC in the hepatopancreas was approximately sixfold higher than that in the haemolymph (RL,is of 6.277) This Ânding is consistent with that of Chiayvareesajja et al (2006), i.e., r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 143^152 149 Hepatopancreatic and muscular OTC in white shrimp D Faroongsarng et al a signiÂcant amount of OTC in the haemolymph was redistributed into the hepatopancreas Moreover, in oral administration, it was observed that the hepatopancreatic level was the highest compared among the biological matrices tested, which was also consistent with previous reports (Faroongsarng et al 2007; Gomez-Jimenez et al 2008) The hepatopancreatic OTC is not only redistributed from the haemolymph but also directly absorbed from the foregut This could yield a total concentration of OTC in the organ considerably greater than that in the haemolymph, reÊecting an even higher value of RL (RL,oral of 14.374) In addition, previous studies dealing with OTC bioavailability assessment in this and related species showed highly variable results, i.e., the oral bioavailability varied from 43.2% (Penaeus japonicas: Uno, 2004) to 92.2% (Litopenaeus setiferus: Reed et al 2006) Those reports made their assessment based on well-established vertebrate pharmacokinetics As shrimp hepatopancreatic functions include both drug absorption and disposition, the kinetics of the drug may be somewhat diĂerent from vertebrates, where such functions are performed by a separate organ Thus, the previously reported bioavailability variations may be attributed to the variability of not only the extent of absorption but also drug redistribution from the blood to the hepatopancreas USP veterinary pharmaceutical information monographs recommend OTC to be used as an infectious treatment in such aquaculture products as catÂsh and salmonaid (USP, 2003) But infectious disease management might be problematic in shrimp farms as disease progression is rapid and it is dicult to administer the drug due to the fact that infected shrimps feeding behaviour is limited The role of antibiotics as a diseasepreventing agent in shrimp farms was very Ârst experimentally demonstrated in Penaeus aztecus, where it was recommended that the medicated feed containing 0.5^1.0% OTC could prevent the bacterial infection (Corliss, Lightner & Zeian-eldin 1977) Recently, Center for Veterinary Medicine, US FDA approved a few antibiotics including OTC, Êorfenicol, sulfadimethoxine/ trimethoprim and sulfamerazine for use as medicated feeds for disease control in aquaculture (FDA/CVM, 2007) However, the dose level used in the current study was far higher than that in normal practice As a result, the maximum concentration in the hepatopancreas was approximately Âvefold higher than that reported by Gomez-Jimenez et al (2008), which simulated therapeutic use of OTC in an aquaculture-like environment The current study utilized forced feeding, 150 Aquaculture Research, 2009, 41, 143^152 in which the pre-designed dose could be completely introduced into animals, whereas that of Gomez-Jimenez et al (2008) allowed shrimp feed naturally, in which only some portion of the drug entered shrimp body depending on the feeding behaviour, and the medicated feed used in the current study contained OTC at a concentration of 20 g kg feed, which was fourfold higher than that used by Gomez-Jimenez et al (2008) An analysis of OTC redistributing into the hepatopancreas in oral administration was undertaken in the current study It was assumed that the amount of OTC in the hepatopancreas was a linear combination of the amount of drug absorption and the drug redistribution A numerical subtraction of the hepatopancreatic OTC levels simulated according to a reduced model in which the OTC input is solely due to absorption via the foregut, from those of Eq (4 0), could result in a redistribution of OTC in the hepatopancreas, i.e., C0L tị ẳ CL tị CL tị 5ị where C0L tị and CL(t), are the hepatopancreatic levels of OTC redistributed from the haemolymph, and that based on numerical integration of Eq (4 0) respectively CL tị is the hepatopancreatic OTC level simulated according to a reduced model in which the drug input is solely due to oral absorption, whose differential equation is illustrated as follows: dCL ẳ g2 CL ỵ KG CG dt 6ị As OTC was introduced to the same species, C0L should exhibit a kinetic pattern identical to the hepatopancreatic level-time proÂle after intra-sinus administration In order to verify this, C0L was plotted against the OTC levels in the hepatopancreas after intra-sinus administration (CL,is) and is shown in Fig As can be seen, C0L might exhibit a linear relationship with CL,is illustrated by a linear trend line The line passes the origin and exhibits a slope (S; SE) of 0.114 and 0.013 with a coecient of correlation (R2) of 0.922 The inset of Fig illustrates that C0L could be successfully superimposed on the pharmacokinetic proÂle of intra-sinus administered hepatopancreatic OTC It was noted that C0L in the inset of Fig.5 was adjusted by S so as to be comparable with CL,is As intrasinus (Dis) and oral (Doral) doses were 10.0 and 50.2 mg g shrimp body weight, respectively, with essentially the same kinetics, it is presumably suggested that an ideal slope of the plot between C0L and is CL,is would simply be the dose ratio DDoral ị, which is 0.1992 The obtained value of S from the trend line in r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 143^152 Aquaculture Research, 2009, 41, 143^152 Hepatopancreatic and muscular OTC in white shrimp D Faroongsarng et al Figure The plot between hepatopancreatic oxytetracycline (OTC) redistributed after oral administration C0L ị and OTC level after intra-sinus administration (CL,iv) showing a linear relationship (broken trend line) as well as 95% conÂdence prediction intervals (dotted lines) The inset shows the kinetic proÂle of C0L after adjusting by slope of the trend line compared with the actual results from intra-sinus administration Fig deviates from the ideal slope by 42.92%, whereas the relative standard error (RSE: SE/mean) of S is ặ 11.40% It is obvious that the negative deviation is far too high to be solely the result of experimental bias In other words, there might be another cause or causes As OTC enters the circulatory system via the hepatopancreas, which has been recognized as a major site of biotransformation of most xenobiotics in crustacean (James & Boyle1998), the drug may be partially disposed before redistribution Thus, it is hypothesized that OTC might undergo extensive Ârst-pass elimination, resulting in the loss of C0L as shown by a signiÂcant negative deviation In addition, the loss might account for approximately 30^ 40% of the amount of OTC redistributed to the hepatopancreas Oxytetracycline disposition via the hepatopancreas The disposition of OTC in the proposed model is described by hepatopancreatic clearance (ClL) As discussed above, the values of ClL could not be explicitly stated due to the shortage of number of solving equations However, it is possible to compare coecients so as to examine the behaviour of drug disposition via the hepatopancreas Assuming that the volumes of distribution and Êow rates were identical for the drug taken by the animals of the same species, the ratio of coecients between oral and intra-sinus administrations would be g2;oral QL ỵ ClL;oral RL;is % g2;is QL ỵ ClL;is RL;oral 7ị Should there be a linear pharmacokinetic disposiRL;is tion, the ratio of coecients would approach RL;oral for the clearance is identical and the Ârst term of the right-hand side of Eq (7) becomes unity It is observed g RL;is that g2;oral is 9.72 (Tables and 3) whereas RL;oral is 0.44 2;is This signiÂcant discrepancy demonstrates that linear kinetics for OTC disposition is unlikely Furthermore, it was found that the hepatopancreatic OTC levels at each time interval after oral administration were 8.8fold greater than at after intra-sinus counterparts (the reciprocal of slope: S of the trend line in Fig 4) while the oral dose was Âvefold the intra-sinus one It seems that clearance might be dependent on the amount of OTC present It has been reported previously that the hepatic removal of vertebrates exhibited Machelis^Menten-type kinetics, in which the hepatic drug clearance was non-linearly modelled based on substrate concentration dependence (Liu & Pang, 2006) The hepatopancreatic disposition found r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 143^152 151 Hepatopancreatic and muscular OTC in white shrimp D Faroongsarng et al in P vannamei seems to be consistent with the hepatic disposition of vertebrates Thus, it is deduced that shrimp hepatopancreatic drug disposition kinetics may be non-linear and dependent on the amount of drug present in the organ It is necessary that further studies be carried out on hepatopancreatic clearance to better understand the mechanism of OTC disposition In summary, the proposed model was able to address how OTC distributed and was disposed in tissues of interest, for this particular study: muscle and hepatopancreas It was deduced that OTC did not accumulate in muscular tissues, which allows a time for deputation before consumption as food, and the model could also predict OTC disposition behaviour via the hepatopancreas Acknowledgments The authors appreciate the kind assistance of the Department of Biochemistry, Faculty of Science, Prince of Songkla University, for OTC determinations in biological matrices, and the Department of Aquatic Science, Faculty of Natural Resources, Prince of Songkla University, for providing the shrimp samples Special thanks are due to Dr D Patterson for English language revision and Mr Wibul Wongphuwarax for statistical regression consultancy The authors are also grateful to theThailand Research Fund for Ânancial support References Chiayvareesajja S., Chandumpai A.,Theapparat Y & Faroogsarng D (2006) The complete analysis of oxytetracycline pharmacokinetics in farmed PaciÂc white shrimp (Litopenaeus vannamei) Journal of Veterinary Pharmacology and Therapeutics 29, 409^414 Corliss J.P., Lightner D.V & Zeian-Eldin Z.P (1977) Some effects of oral doses of oxytetracycline on growth, survival and disease in Penaeus aztecus Aquaculture 11, 355^362 Dall W., Hill B.J., Rothlisberg P.C & Staples D.J (1991) The Biology of the Penaeidae-Advances in Marine Biology 27 Academic Press, London, UK, 489pp Faroongsarng D., Chandumpai A., Chiayvareesajja S & Theapparat Y (2007) Bioavailability and absorption analysis of oxytetracycline orally administered to the standardized moulting farmed PaciÂc white shrimps (Penaeus vannamei) Aquaculture 269, 89^97 Food and Drug Administration, Center for Veterinary Medicine (2007) FDA/CVM Approved Drugs for Use in Aquacul- 152 Aquaculture Research, 2009, 41, 143^152 ture Available at http://www.fda.gov/cvm/drugsuseaqua htm (accessed May 2009) Gomez-Jimenez S., Espinosa-Plascencia A., Valenzuela-Villa F & Bermudez-Almada C (2008) Oxytetracycline (OTC) accumulation and elimination in hemolymph, muscle and hepatopancreas of white shrimp Litopenaeus vannamei following an OTC-feed therapeutic treatment Aquaculture 274, 24^29 Guadagnoli J.A.,Tobita K & Reiber C.L (2007) Assessment of the pressure-volume relationship of the single ventricle of the grass shrimps, Palaemonetes pugio Journal of Experimental Biology 210, 2192^2198 James M.O & Boyle S.M (1998) Cytochromes P450 in crustacea Comparative Biochemistry and Physiology^C 121, 157^172 Liu L & Pang K.S (2006) An integrated approach to model hepatic drug clearance European Journal of Pharmaceutical Sciences 29, 215^230 McGaw I.J (2005) The decapod crustacean circulatory system: a case that is neither open nor closed Microscopy and Microanalysis 11,18^36 Reed L.A., Siewicki T.C & Shah J.C (2004) Pharmacokinetics of oxytetracycline in the white shrimp, Litopenaeus setiferus Aquaculture 232,11^28 Reed L.A., Siewicki T.C & Shah J.C (2006) The biopharmaceutics and oral bioavailability of two forms of oxytetracycline to the white shrimp, Litopenaeus setiferus Aquaculture 258, 42^54 Sangrungruang K., Chotchuang A & Ueno R (2004) Comparative pharmacokinetics and bioavailability of oxytetracycline in giant tiger prawn Fish Science 70, 467^472 Tsuji A., Yoshikawa T., Nishide K., Minami H., Kimura M., Nakashima E.,Terasaki T., Miyamoto E., Nightingale C.H & Yamana T (1983) Physiologically based pharmacokinetic model for -lactam antibiotics I: tissue distribution and elimination in rats Journal of Pharmaceutical Sciences 72, 1239^1252 Uno K (2004) Pharmacokinetics of oxolinic acid and oxytetracycline in kuruma shrimp, Penaeus japonicus Aquaculture 230, 1^11 Uno K., Aoki T., KleechayaW.,Tanasomwang V & Ruangpan L (2006) Pharmacokinetics of oxytetracycline in black tiger shrimp, Penaeus monodon, and the eĂect of cooking on residues Aquaculture 254, 24^31 USP (2003) USP veterinary pharmaceutical information monographs ^ antibiotics Journal of Veterinary Pharmacology andTherapeutics 26(Suppl 2), 1271 Verri T., Mandal A., Zilli L., Bossa D., Mandal P.K., Ingrosso L., Zonno V.,Vilella S., Ahearn G.A & Storelli C (2001) Dglucose transport in decapod crustacean hepatopancreas Comparative Biochemistry and Physiology ^A 130, 585^606 Welling P.G (1986) Pharmacokinetics Processes and Mathematics American Chemical Society, Washinton, DC, US 290pp r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 143^152 Aquaculture Research, 2009, 41, 153^156 doi:10.1111/j.1365-2109.2009.02303.x SHORT COMMUNICATION The effect of Lactococcus lactis on the abundance of aeromonads in the rearing water of the goldfish, Carassius auratus (Linnaeus) Haruo Sugita,Takashi Fujie,Tomoko Sagesaka & Shiro Itoi Department of Marine Science and Resources, Nihon University, Kameino, Fujisawa, Kanagawa, Japan Correspondence: H Sugita, Department of Marine Science and Resources, Nihon University, Kameino 1866, Fujisawa, Kanagawa 2528510, Japan E-mail: sugita@brs.nihon-u.ac.jp The intestinal tract of freshwater Âsh is known to be primarily colonized by the genus Aeromonas (Sugita, Tanaka, Yoshinami & Deguchi 1995) These bacteria have been reported to occur at high densities in the intestinal tracts of freshwater Âsh and are continually supplied to rearing water through defecation (Sugita, Ushioka, Kihara & Deguchi 1985) Under conditions of host stress, these bacteria have been reported to become opportunistic pathogens causing aeromonasis (Walters & Plumb 1980) Rearing water is considered to constitute the primary vehicle for disease transmission in cultured Âsh species, and the gills, skin and intestines are known to constitute the main portals of entry for Aeromonas into the Âsh host (Ventura & Grizzle 1987) Consequently, suppression of these bacteria in the rearing water is likely to result in a decrease in the occurrence of aeromonasis Recently, considerable interest has been generated in the use of probiotic agents in aquaculture (Sugita, Ohta, Kuruma & Sagesaka 2007) In a previous study, we isolated Lactococcus lactis from the intestinal tract of the Amur catÂsh, Silurus asotus, for use as a probiotic candidate that eĂectively inhibited the growth of representatives of the genus Aeromonas (Sugita et al 2007) Subsequently, we found that another strain, C7, isolated from the Amur catÂsh, showed higher antibacterial activity than the above-mentioned bacteria In this study, therefore, the eĂect of L lactis strain C7 on aeromonads in the rearing water of the goldÂsh, Carassius auratus, was examined r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd The 16S rDNA sequence of strain C7 was determined and analysed (Tsuchiya, Sakata & Sugita 2008) The antibacterial activity of strain C7 was determined using the double-layer method according to a manual of Ishida and Sugita (2006) BrieÊy, strain C7 was incubated for 24 h at 25 1C in Trypticase soy (TS) broth (BBL, Sparks, MD, USA) Macrocolonies of this bacterium were formed on TS agar (TS broth solidiÂed with 1.5% agar) plates by inoculating mL droplets of the above culture using a multipoint inoculator After incubation at 25 1C for 48 h under aerobic conditions, the macrocolonies were killed with chloroform vapour for 15 Five Aeromonas species as target strains were individually incubated for 24 h at 25 1C in TS broth and diluted until the optical density at 610 nm reached 0.2 (approximately 108 CFU mL 1) The cultures were further diluted 100^200-fold with TS soft agar (TS broth with 1.0% agar) and poured over the plates (4.5 mL each plate) After incubation at 25 1C for 48 h, the antibacterial activity was deÂned as the diameter of clear inhibitory zones around a macrocolony This procedure was repeated Âve times Two glass tanks (Tanks and 2; 60 35 30 cm) equipped with recirculating water systems were Âlled with 50 L of tap water and maintained at 20 ặ 1C A total of kg of pebbles (about mm in diameter) were used as the Âlter material of each tank, and the Êow rate of the water was maintained at L Five goldÂsh weighing 2.0 ặ 0.2 g were transferred into each tank after allowing sucient 153 Probiotic eĂect of L lactis on Aeromonas H Sugita et al time for the recirculating systems to become established No clinical signs or characteristics of infection were observed in all Âsh used The pelleted diet (Fuji Flour Milling, Shizuoka, Japan) supplemented with strain C7 at a density of 108 CFU g 1, referred to as the probiotic diet, was fed to the Âsh in the experimental tank The control diet had no bacterial supplement The Âsh in the experimental and control tanks were fed 0.5 g of their respective foods daily for 64 days GoldÂsh in Tank (control group) were fed the control diet for 64 days, while the goldÂsh in Tank (probiotic group) were fed the control diet on day 0, followed by the probiotic diet on days through 57 and then the control diet again for an additional days (days 58^64) During the experimental period, there was 20% mortality of the goldÂsh in each tank, although no symptoms of bacterial infection were observed in the dead Âsh The rearing water was sampled at the middle layer of each tank using a sterile pipette once per sampling time, and the sample was placed in a sterile test tube The sample was plated in duplicate in four dilutions on 1/20 PYBG (1/20 peptone-yeast extractbeef extract-glucose) agar (Sugita et al 1985), DHL (deoxycholate-hydrogen sulphide-lactose) agar for Aeromonas (Eiken Chemical, Tokyo, Japan) and MRS (de Man-Rogosa-Sharpe) agar (Difco, Becton and Dickinson, MD, USA) for L lactis and incubated at 25 1C for days under aerobic conditions The presence of Aeromonas on DHL agar was conÂrmed using gram staining, cellular morphology, oxidase production and glucose fermentation, as described by Ishida and Sugita (2006) The presence of L lactis on MRS agar was conÂrmed using PCR according to the method of Pu, Dobos, Limsowtin and Powell (2002) The lower limit of detection for Aeromonas and L lactis was 100 CFU mL Strain C7 is a facultative anaerobe with ovoid, Gram-positive cells that ferment glucose, is nonmotile and produces both catalase and oxidase Phylogenetic analysis based on16S rDNA sequences showed that strain C7 has similarities of100 and 98.4% to Lactococcus lactis subsp lactis (AB100803) and L lactis subsp cremoris (AB100802), respectively (Fig 1), strongly suggesting that strain C7 is closely related to L lactis subsp lactis The partial sequence (641bp) of the 16S rDNA from strain C7 was deposited in the DDBJ/GenBank/EMBL Database libraries underAccession Number AB375867 Strain C7 is referred to as L lactis subsp lactis C7 in the rest of this paper Lactococcus lactis subsp lactis C7 exhibited a high inhibitory activity against Âve Aeromonas species, 154 Aquaculture Research, 2009, 41, 153^156 Figure Neighbor-joining phylogenetic tree based on partial 16S rDNA sequences of strain C7 and related microorganisms Numbers on the branches represent percentage (! 50%) of 1000 bootstrap replications A bar represents the evolutionary distance (Knuc) of 0.1 Bacillus subtilis was used as an outgroup The DDBJ/EMBL/ GenBank accession numbers are shown in parentheses Table Antibacterial activities of Lactococcus lactis subsp lactis C7 against Âve target bacteria Target bacteria Activity (mm) Aeromonas caviae ATCC 15468 A hydrophila ATCC 7966 A jandaei ATCC 49568 A sobria ATCC 43979 A veronii ATCC 35624 8.8 9.3 8.2 10.1 6.7 ặ ặ ặ ặ ặ 1.0 1.0 1.0 0.6 0.5 Mean diameters ặ SE of inhibitory zones (n 5) The diameters of macrocolonies ranged from 4.3 to 4.4 mm compared with the Ândings of a previous report (Sugita et al 2007) (Table 1) During the study period, changes in the bacterial population size of the total bacteria, Aeromonas, and L lactis in the goldÂsh-rearing water of the control and the probiotic diet tanks were determined (Fig 2) The total bacterial densities in the rearing water of the control group ranged from 4.3 103 to 4.6 105 CFU mL Aeromonas densities increased from o1 100 on days and to 5.0 100^3.9 103 CFU mL on days 8^64, and no L lactis was detected in the rearing water Aeromonas densities were observed to increase with rearing time in the carp, Cyprinus carpio, in a previous study (Sugita et al 1985) Meanwhile, in the rearing water of the probiotic group, total bacterial densities ranged from 8.7 103 to 6.6 104 CFU mL While no L lactis was detected on day 0, L lactis was observed r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 153^156 Aquaculture Research, 2009, 41, 153^156 Figure Population density changes for total bacteria (), Aeromonas () and L lactis (&) in the rearing water of (a) Tank (control group) and (b) Tank (probiotic group) An arrow indicates the period when the probiotic diet was orally administered on day at a density of 3.3 103 CFU mL and the density Êuctuated thereafter in the range of 7.1 101 to 3.4 103 CFU mL 1.These results strongly suggest that L lactis detected in the rearing water of the probiotic group (Tank 2) was derived from the probiotic diet Aeromonas densities ranged from o1 100 to 100 CFU mL with the probiotic diet, which suggests that the oral administration of L lactis to the goldÂsh had the eĂect of reducing Aeromonas densities in the rearing water Aeromonas densities in Tank were signiÂcantly lower than that of Tank on days through 57 (Po0.05 using a paired t-test) In addition, L lactis was detected at 1.7 102 CFU mL in the rearing water of Tank (probiotic group) days after the probiotic diet was replaced with the control diet (day 64), while no L lactis was detected inTank1 (control group) This shows that L lactis could remain for a relatively long time in the goldÂsh intestines Probiotic eĂect of L lactis on Aeromonas H Sugita et al One of the pioneering studies on the application of probiotics in aquaculture, by Nogami and Maeda (1992), reported that daily supplements of the bacterial strain, PM-4, successfully inhibited vibriosis in the developing Japanese swimming crab, Portunus trituberculatus However, in order to obtain an inhibitory eĂect, the authors were required to add large quantities of probiotic bacterial culture to the rearing water daily, which is impractical at the scale of aquaculture ponds Consequently, the present study was undertaken to determine whether the opportunistic bacteria in the goldÂsh-rearing water were inhibited by the oral administration of L lactis subsp lactis C7 The Ândings showed that the addition of L lactis subsp lactis C7 as a probiotic strain successfully inhibited the proliferation of Aeromonas in the rearing water during the experimental period The population of Aeromonas may be induced to decrease through the eĂects of either (i) the intestinal tract of the goldÂsh, (ii) the rearing water after defecation or (iii) a combination of these, being induced by the probiotic eĂects of the L lactis subsp lactis C7 Thus, the results show that L lactis subsp lactis C7 is an excellent probiotic candidate against the opportunistic genus Aeromonas Large-scale experiments using this bacterial strain, to determine whether the eĂects can be scaled up for aquacultural use, would be a logical next step in the near future because this was a preliminary study Acknowledgments This research was supported, in part, by a Grantin-Aid for ScientiÂc Research from the Japan Society for the Promotion of Science References Ishida Y & Sugita H (ed.) (2006) Methods in Microbiology for Marine Environmental Assessment (Revised edition) Koseisha Koseikaku,Tokyo (in Japanese) Nogami K & Maeda M (1992) Bacteria as biocontrol agents for rearing larvae of the crab Portunus trituberculatus Canadian Journal of Fisheries and Aquatic Science 49, 2373^7376 Pu Z.Y., Dobos M., Limsowtin G.K.Y & Powell I.B (2002) Integrated polymerase chain reaction-based procedures for the detection and identiÂcation of species and subspecies of the Gram-positive bacterial genus Lactococcus Journal of Applied Microbiology 93, 353^361 r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 153^156 155 Probiotic eĂect of L lactis on Aeromonas H Sugita et al Sugita H., Ushioka S., Kihara D & DeguchiY (1985) Changes in the bacterial composition of water in a carp-rearing tank Aquaculture 44, 243^247 Sugita H., Tanaka K., Yoshinami M & Deguchi Y (1995) Distribution of Aeromonas species in the intestinal tracts of river Âsh Applied and Environmental Microbiology 61, 4128^4130 Sugita H., Ohta K., Kuruma A & Sagesaka T (2007) An antibacterial eĂect of Lactococcus lactis isolated from the intestinal tract of the Amur catÂsh, Silurus asotus Linnaeus Aquaculture Research 38,1002^1004 Tsuchiya C., Sakata T & Sugita H (2008) Novel ecological niche of Cetobacterium somerae, an anaerobic bacterium 156 Aquaculture Research, 2009, 41, 153^156 in the intestinal tracts of freshwater Âsh Letters in Applied Microbiology 46, 43^48 Ventura M.T & Grizzle J.M (1987) Evaluation of portals of entry of Aeromonas hydrophila in channel catÂsh Aquaculture 65, 205^214 Walters G R & Plumb J A (1980) Environmental stress and bacterial infection in channel catÂsh, Ictalurus punctatus RaÂnesque Journal of Fish Biology 17,177^185 Keywords: Lactococcus lactis subsp lactis, probiotics, goldÂsh, Aeromonas, antibacterial eĂect r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 153^156 Aquaculture Research, 2009, 41, 157^160 doi:10.1111/j.1365-2109.2009.02305.x SHORT COMMUNICATION Method of ethanol anaesthesia and individual marking russac, 1831 for oval squid (Sepioteuthis lessoniana Fe in Lesson 18301831) Yuzuru Ikeda, Chikatoshi Sugimoto, Hajime Yonamine & Yota Oshima Department of Chemistry, Biology and Marine Sciences, University of the Ryukyus, Okinawa, Japan Correspondence:Yuzuru Ikeda, Department of Chemistry, Biology and Marine Sciences, University of the Ryukyus, Okinawa, 903-0213, Japan E-mail: ikeda@sci.u-ryukyu.ac.jp Cephalopods are commonly used as experimental animals, however the anaesthesia that is often necessary for surgical treatments such as in neuroscience or tagging experiments for Âsheries science can be problematic for this group, particularly for squid There have been chemical agents applied as anaesthetics for cephalopods, e.g solutions of urethane (Messenger 1968; Young 1971) and ethanol (Froesch & Marthy 1975), but they sometimes result in the animals climbing out the tank and/or inking violently In contrast cold water can be a good anaesthetic without traumatizing octopus (Andrews & Tansey 1981), however it is not sucient anaesthesia for certain operations such as brain lesions because the tissue hardens after anaesthesia Messenger, Nixon and Ryan (1985) tested anaesthetic eĂect of magnesium in some cephalopods and found it suitable However, in Messenger et al (1985) study, duration of survival after anaesthesia was relatively short with the longest survival at days for European cuttleÂsh Sepia ocinalis, 46 h for Loligo forbesi, overnight for European common squid Alloteuthis subulata, days for common octopus Octopus vulgaris, and day for curled octopus Eledone cirrhosa On the other hand, Japanese common squid Todarodes paciÂcus was successfully anaesthetized with cold water (Sakurai, Ikeda, Shimizu & Shimazaki 1993; Bower, Sakurai, Yamamoto & Ishii 1998), enabling marking experiments or long distance transportation of this species Unfortunately, this method is not suitable for cephalopods without a strong thermal tolerance likeT paciÂcus which moves through a wide range of thermal layers in a single day (Hamabe 1964) So far, there is r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd not a universally perfect cephalopod anaesthesia available, hence each anaesthetic needs to be tested on each species before Ârst use Individual identiÂcation is a useful and necessary capability for experimental studies in general, but we have few techniques available for individually marking cephalopods: e.g plastic ribbon tags in Japanese common squidT paciÂcus (Sakurai et al.1993) and formaldehyde-free nail varnish in European cuttleÂsh S ocinalis (Warnke 1994) Recently, Replinger and Wood (2007) applied visible implant Êuorescent elastomer (VIE) tags for tagging Caribbean reef squid Sepioteuthis sepioidea, and they were successful in tagging captive and wild individuals for a signiÂcant duration (56 days in captivity) In this paper, we report the methods of rapid ethanol anaesthesia and marking with Visible Implant Fluorescent Elastomer tags for oval squid Sepioteuthis lessoniana Forty-six young and subadult S lessoniana (identiÂed as Shiro-ika by the number of ovum in a single egg case; Segawa, Hirayama & Okutani 1993) that had been raised from hatching in a circular tank with closed water system (20 or 50 L volume, MULTIs HYDENSE Aqua, Tokyo, Japan) were used for the experiment (Table 1) For the anaesthesia, squid were individually collected from the circular tank with a circular container Âlled with seawater, then transferred into a plastic box (110 165 40 mm) Âlled with 1.0%, 1.5% or 2.0% ethanol in seawater (on 11 and one occasions, respectively, we used 1.5% and 2.0% ethanol solution, we used1.0% ethanol solution for other cases, see Table 1) The ethanol solution in the plastic box was renewed after every three squid 157 Anaesthesia and individual marking for squid Y Ikeda et al Aquaculture Research, 2009, 41, 157^160 Table Summary of oval squid Sepioteuthis lessoniana individuals used in the anaesthesia and marking experiment Marked site/numbers at Marked site/numbers Duration (days) between Survival (days) Mantle Squid No. 1st anaesthesia at 2nd anaesthesia 1st and 2nd anaesthesia after 1st anaesthesia length (mm) Age (days) 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 Head/1 Head/1 Arm (r) base/2 Arm (r) base/2 Arm (r) base/2 Arm (l) base/2 Arm (l) base/2 Arm (l) base/2 Arm (l) base/2 Arm (r) base/2 Arm (r) base/2 Arm (r) base/2 Dorsal mantle/1 Dorsal mantle/1 Dorsal mantle/1 Head/1 Head/1 Dorsal mantle/1 Head/1 Head/2 Head/2 Head/2 Head/2 Head/2 Head/2 Head/2 Head/1 Head/2 Head/2 Head/2 Head/2 Head/2 Head/2 Head/2 Head/1 Head/2 Head/2 Head/2 Head/2 Head/2 Head/2 Head/2 Head/2 Head/2 Head/2 Head/2 Head/1 Arm (r) base/2 Arm (r) base/2 Arm (l) base/2 Arm (l) base/2 Arm (l) base/2 Arm (l) base/2 Head/2 Arm (r) base/2 Arm (r) base/2 Head/2 Head/1 Head/2 Head/2 Head/2 Head/2 Head/2 Head/2 Head/2 Head/2 Head/2 28 28 28 28 28 28 28 28 28 28 15 15 15 6 6 6 6 41 43 59 40 40 41 41 58 59 59 41 41 31 48 75 54 100 13 13 16 27 28 31 32 48 34 54 40 42 56 73 76 77 86 62 15 19 20 22 22 23 23 23 32.8 72.1 30.4 76.6 99.6 65.2 65.0 69.4 57.2 91.5 69.6 92.1 101.8 98.4 82.8 91.2 106.2 119.8 140.4 71.1 67.9 69.4 75 82 100.3 89.3 91.2 92.3 119.8 93.2 97.6 107.3 127.1 122.4 117.8 124.7 72.8 116.6 93.5 111 103.6 97 103.5 96.1 93.2 96.8 66 107 68 109 125 106 106 107 107 124 125 125 135 135 99 116 143 122 168 96 96 99 110 111 114 115 116 117 122 123 125 139 156 159 160 169 105 159 132 136 137 139 139 140 140 140 Anaesthetized ethanol concentration in seawater, No 1, 2.0%; No 2^12, 1.5%; No 13^46, 1.0% were anaesthetized After having been transferred into the ethanol solution, squid exhibited a dark colour on the whole body and moved in the box vigorously, before becoming immobile and the body colour changing to a more transparent pattern with a dark band on the mantle and head (between eyes) in ca 10^20 s, at which time the squid appeared to be 158 under anaesthesia (Fig 1a) The anaesthesia process lasted around 30 s, with squid usually not inking, and thus not appearing to be stressed Anaesthetized squids were scooped up by hand, and then the VIE tags consisting of a liquid elastomer (Northwest Marine Technology, Shaw Island,WA, USA) were injected either in the dorsal mantle, head or right side or left r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 157^160 Aquaculture Research, 2009, 41, 157^160 Anaesthesia and individual marking for squid Y Ikeda et al side of the base of the arm (Fig 1b) The combination of body part tagged and colour of the VIE tags (red, orange, yellow and green) enabled individual identiÂcation The marked squid were transferred into a bowl (200 mm, L volume) Âlled with seawater, where squid began to ventilate actively, and appeared to arouse in 20^30 s After this arousing process, squid were released into the circular tank where it soon began to swim as usual (Fig 1c) The whole process from anaesthesia to releasing lasted 2^3 for each squid In case visibility of the colour of the VIE tags became weak as time lapsed, we carried out a second marking under anaesthesia days, 15 days or 28 days after the Ârst anaesthesia and marking test in the same manner as the Ârst test described (Table 1) After this treatment, the colour of the VIE tags were still clearly visible (Fig 1d) and squid survived up to 100 days after the original anaesthesia and marking (Table 1) We were able to identify, based on the VIE tags, each marked squid swimming in the tank and recorded behavioural data on the social organization (to be reported later in another study) The smallest individual (Squid No 1) that had been anaesthetized with 2% ethanol solution died during anaesthesia (Table 1) Another individual (Squid No 3) died in days post anaesthesia (Table 1) The former case revealed 2% of ethanol seems to be critical concentration for S lessoniana resulting in death Our pilot examination of anaesthesia with 2% ethanol solution also resulted in young S lessoniana dying in 1^2 days (data not shown) Handling is another problem for squid being stressed during individual marking Squid can be damaged in air condition, because of no respiration, during tag injection (the latter case) The remaining 44 squid anaesthetized with 1.0% ethanol solution (n 34) or 1.5% ethanol solution (n 510) survived for 40^100 days after the original anaesthesia and marking, even though some of them received a second anaesthesia/marking test (Table 1) Since most of these marked squid were also interrupted and collected from the tank due to another study, the maximum survival duration after anaesthesia/marking may be longer Body size in dorsal mantle length and age of examined squid at death, respectively, ranged 32.8^140.4 mm and 66^169 days (Table 1) Figure Ethanol anaesthesia and individual marking of oval squid Sepioteuthis lessoniana (a) Squid soaked in ethanol seawater solution, (b) squid injected with the Visible Implant Fluorescent Elastomer tags in dorsal mantle, (c) squid released after marking, (d) squid after marking (marks on centre of the head and near left eye), note, dark pattern on mantle and head of squid in (a) Arrow in (c) indicates marking r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 157^160 159 Anaesthesia and individual marking for squid Y Ikeda et al Aquaculture Research, 2009, 41, 157^160 The present study revealed that ethanol solution in seawater is useful and can be a quick anaesthesia for young and subadult S lessoniana A hundred days is the longest record for survival after anaesthesia/ marking for squid so far reported, beside similar record for S sepioidea (Replinger & Wood 2007, but this lacked anaesthesia) Ethanol seawater solution is easy to make, and thus is applicable for various types of experiments for cephalopods The very clear change in body patterns during ethanol anaesthesia is a useful criterion to check that the cephalopod has undergone the physiological process of anaesthesia (Hanlon 1982) The present study also conÂrmed that VIE tags, widely applied for Âsh and recently used in squid (S sepioidea; Replinger & Wood 2007), can be also useful for cephalopods Additionally we were also successful in marking Pharaoh cuttleÂsh Sepia pharaonis (Y Ikeda & A Segawa pers comm.) withVIE tags Site of tagging did not aĂect survival of S lessoniana, providing various potential marking patterns and combinations for marking many individuals Since many cephalopods, especially squids, are obligatory schoolers and not well in isolation (Hirtle, DeMont & ODor 1981), easily recognized markings presently reported are crucial for meaningful quantitative growth studies of multiple animals in social groups The methods presented here for anaesthesia and individual marking will enable many behavioral experiments and simplify growth studies for cephalopods Bower J.R., Sakurai Y., Yamamoto J & Ishii H (1998) Transport of the ommastrephid squid Todarodes paciÂcus under cold-water anesthesia Aquaculture 170,127^130 Froesch D & Marthy H.J (1975) The structure and function of the oviducal gland in octopods (Cephalopoda) Proceedings of the Royal Society B 188, 95^101 Hamabe M (1964) Study on the migration of squid (Ommastrephes sloani paciÂcus Steenstrup) with reference to the age of the moon Bulletin of theJapanese Society of ScientiÂc Fisheries 30, 209^215 Hanlon R.T (1982) The functional organization of chromatophores and iridescent cells in the body patterning of Loligo plei (Cephalopoda: Myopsida) Malacologia 23,89^119 Hirtle R.W.M., DeMont M.E & ODor R.K (1981) Feeding, growth, and metabolic rates in captive short-Ânned squid, Illex illecebrosus, in relation to the natural population Journal of ShellÂsh Research 1,187^192 Messenger J.B (1968) The visual attack of the cuttleÂsh, Sepia ocinalis Animal Behaviour 16, 342^357 Messenger J.B., Nixon M & Ryan K.P (1985) Magnesium chloride as an anesthetic for cephalopods Comparative Biochemistry & Physiology 82C, 203^205 Replinger S.E & Wood J.B (2007) A preliminary investigation of the use of subcutaneous tagging in Caribbean reef squid Sepioteuthis sepioidea (Cephalopoda: Loliginidae) Fisheries Research 84, 308^313 Sakurai S., IkedaY., Shimizu M & Shimazaki K (1993) Feeding and growth of captive adult Japanese common squid, Todarodes paciÂcus, measuring initial body size by cold anesthesia In: Recent Advances in Cephalopod Fishery Biology (ed by T Okutani, R.K ODor & T Kubodera), pp 467^476 Tokai University Press,Tokyo Segawa S., Hirayama S & Okutani T (1993) Is Sepioteuthis lessoniana in Okinawa a single species? In: Recent Advances in Cephalopod Fishery Biology (ed by T Okutani, R.K ODor & T Kubodera), pp 513^521 Tokai University Press,Tokyo Warnke K (1994) Some aspects of social interaction during feeding in Sepia ocinalis (Mollusca: Cephalopoda) hatched and reared in the laboratory.Vie Milieu 44, 125^ 131 Young J.Z (1971) The Anatomy of the Nervous System of Octopus vulgaris Clarendon Press, Oxford, 690p Acknowledgments We acknowledge G T Pecl for reviewing the manuscript We thank Y Abe, S Kobayashi, M Kobayashi, M Matsumura, K Shimazoe, H Isono and T Higa for their cooperation in squid maintenance This study was supported by KAKENHI (18580188), which we are most grateful References Andrews P.L.R & Tansey E.M (1981) The eĂect of some anesthetic agents in Octopus vulgaris Comparative Biochemistry and Physiology 70C, 241^247 160 Keywords: oval squid, marking, anaesthesia, ethanol, visible implant Êuorescent elastomer r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 157^160 [...]... et Schlegel) Aquaculture Research 35, 494^500 r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 19^26 Aquaculture Research, 2009, 41, 27^34 doi:10.1111/j.1365-2109 .2009. 02298.x A novel multiplex PCR method for detecting virulent strains of Vibrio alginolyticus Shuang-Hu Cai1,2,Yi-Shan Lu1,2, Zao-He Wu1,2, Ji-Chang Jian1,2 & Yuang-Cong Huang1,2 1 Fisheries... Sinica 30, 553^558 (in Chinese with English abstract) r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 27^34 Aquaculture Research, 2009, 41, 35^44 doi:10.1111/j.1365-2109 .2009. 02299.x Genetic linkage map of the pearl oyster, Pinctada martensii (Dunker) Yaohua Shi1,2,3, Hong Kui2, Ximing Guo3, Zhifeng Gu1,Yan Wang1 & Aimin Wang1 1 Key Laboratory of Tropic... embryology of the neon goby, Gobiosoma oceanops Copeia 1072, 477^481 r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 11^18 Aquaculture Research, 2009, 41, 19^26 doi:10.1111/j.1365-2109 .2009. 02296.x Edwardsiella tarda infection in Korean catfish, Silurus asotus, in a Korean fish farm Jin-Ha Yu1, Jung Jo Han2, Kwon Sam Park3, Kwan Ha Park1 & Sung Woo Park1 1... (1941) Studies on marine bacteria I The cultural requirements of heterotrophic aerobes Journal of Marine Research 4, 42^75 r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 1^10 Aquaculture Research, 2009, 41, 11^18 doi:10.1111/j.1365-2109 .2009. 02295.x Reproduction, early development and larviculture of the barber goby, Elacatinus figaro (Sazima, Moura & Rosa... Æ 1.7 1C The eggs measured in average 2.1 in long-axis diameter and 0.7 mm in short-axis diameter h.p.f., hours post fertilization 16 r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 11^18 Aquaculture Research, 2009, 41, 11^18 Barber goby earlier development and culture M E Meirelles et al Figure 3 Barber goby larval development: (a) Newly hatched larvae:... with evidence of fungus and protozoa Only spawnings where the eggs were not partially or totally eaten by their parents were considered r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 11^18 Aquaculture Research, 2009, 41, 11^18 The incubation period, hatching rate and interval between spawnings of the same pair were recorded The egg size and yolk sac diameter... PCR PreMix (Bioneer) with universal primers 27F (5 0 -AGAGTTTGATCMTGGCTCAG-3 0) and 1492R (5 0 -TACGGYTACCTTGTTACGACT T-3 0) (Pratten, r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 19^26 Aquaculture Research, 2009, 41, 19^26 Wilson & Spratt 2003) The PCR condition was as follows: initial denaturation at 95 1C for 5 min,30 cycles of denaturation at 94... showing an enlarged kidney with various size abscesses (arrows) and protrusion of the vent (arrowhead) r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 19^26 21 Edwardsiella tarda infection in Korean cat¢sh J H Yu et al Aquaculture Research, 2009, 41, 19^26 Figure 2 Histopathlogy of diseased cat¢sh, Silurus asotus (a) Liver showing necrotizing hepatic... Gram-nega- 22 Experimental infection The result of experimental infection is shown in Table 3 All dead ¢sh showed typical external and inter- r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 19^26 Aquaculture Research, 2009, 41, 19^26 Edwardsiella tarda infection in Korean cat¢sh J H Yu et al Table 1 Comparison of biochemical characteristics between Edwardsiella... antibiotics It is feasible that the antibiotics tested in this study can be considered in controlling the E tarda infections Edwardsiella r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 19^26 Aquaculture Research, 2009, 41, 19^26 tarda usually has a higher resistance to antibiotics because the bacteria have a long history as a causative agent frequent exposure