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Relationship between differential retention of Escherichia coli and Enterococcus faecalis and variations in enzyme activity in the scallop Patinopecten yessoensis
Marine Pollution Bulletin 60 (2010) 1600–1605
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Relationship between differential retention of Escherichia coli and Enterococcus faecalis and variations in enzyme activity in the scallop Patinopecten yessoensis Bin Li a,b,c, Bijuan Chen c, Zhanhui Qi d, Zengjie Jiang c, Jihong Zhang c, Jianguang Fang c,* a
Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, China Graduate University, Chinese Academy of Sciences, Beijing 100049, China c Key Laboratory for Sustainable Utilization of Marine Fisheries Resources, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China d South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China b
a r t i c l e
i n f o
Keywords: Patinopecten yessoensis Escherichia coli Enterococcus faecalis Bacterial retention Enzyme activity
a b s t r a c t Uptake of Escherichia coli and Enterococcus faecalis and variations of trypsin amylase activity acid phosphatase and superoxide dismutase in tissue of the scallop Patinopecten yessoensis were detected. The results showed that P. yessoensis accumulated E. faecalis in larger numbers and more rapidly than E. coli, both with the highest concentration in the digestive tract and lowest in hemolymph. Compared to E. coli, all scallops exposed to E. faecalis showed significantly higher trypsin and AMS activity. SOD activity in hemocytes and ACP activity in hemolymph was significantly higher in the treatments with 5 log10CFU/ml E. coli than with E. faecalis. But no significant differences in ACP activity of P. yessoensis exposed to a 3 log10CFU/ml inoculum of both bacteria were recorded. In conclusion, the mass retention of gut microflora in P. yessoensis is positively correlated with digestive enzymes activity and negatively correlated with ACP activity in the hemocyte. Ó 2010 Published by Elsevier Ltd.
1. Introduction Scallops are filter-feeders living in a bacteria-rich environment, which, in some cases, may cause marine epizootics and mass mortalities in young bivalves over a short period of time (Laura et al., 2006). Escherichia coli and Enterococcus faecalis are two common bacteria found in the aquatic environment and in the intestines of warm-blooded animals and humans. In summer, they can reach high levels in sewage-polluted seawater and accumulated in high numbers in scallops. Hence, they are suitable indicators to predict fecal contamination in seawater and mollusks (Tallon et al., 2005). About 90% of E. coli is found in the digestive tract and less than 1% in hemolymph (Power and Collins, 1990). Marino et al. (2005) found that Enterococcus durans was quickly taken up by the mussel (Mytilus galloprovincialis) and released more slowly than E. coli or Vibrio cholerae at both 14 and 21 °C. Williams et al. (2009) reported that neither the distribution nor accumulation of live Vibrio campbellii varied with time, except in the hemolymph, and speculated that the gonad may have a major antibacterial defense role in bivalves. The majority of microflora isolates in bivalve shellfish are gram-negative (68%) and aerobic (76%) bacteria, such as Vibrio spp., Pseudomonas spp. and Aeromonas spp., which indicates a * Corresponding author. Tel.: +86 0 532 85822957; fax: +86 0 532 85811514. E-mail address: [email protected] (J. Fang). 0025-326X/$ - see front matter Ó 2010 Published by Elsevier Ltd. doi:10.1016/j.marpolbul.2010.03.017
selective process to sequester and maintain certain species (Kueh and Chan, 1985; Hariharan et al., 1995). Bacteria utilized in probiotic applications, such as Aeromonas media A199 and Roseobacter sp. strain BS107, help to maintain or restore a host’s natural microbial floral and enhance the nutrition of host species through the production of supplemental digestive enzymes (Gillor et al., 2008). However, some xenobiotics may have a negative impact on assimilation efficiency and adaptation possibilities in aquatic animals (Sorokin, 1999). Without specific antibodies, scallop hemocytes play a key role in innate immunity, including recognition, phagocytosis and elimination of invading microorganisms by releasing immune factors, such as lectins, lysozymes, reactive oxygen species and the (anti)oxidases. Studies on modulation of the immune system of bivalves by other organisms have concentrated on the effects of experimental infections on changes in components of plasma or hemolymph. After exposure of the clam Ruditapes philippinarum to Vibrio Pl, hemocyte concentration and peptidase activity increased to a maximum but then decreased to a stable value (Oubella et al., 1994). After an injection with acute virus necrobiotic virus acid phosphatase (ACP), superoxide dismutase (SOD) and catalase (CAT) in the hemocytes of scallop Chlamys farreri were significantly higher than the control but then decreased to normal levels (Xing et al., 2008). SOD activity increased significantly in hemocytes, but not in serum, after an experimental challenge of scallop C. farreri with E. coli (Sun and Li, 1999).
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The Japanese scallop Patinopecten yessoensis is now widely cultivated in northern parts of the Yellow Sea (China). In 2007, production of P. yessoensis in the Dalian area of Liaoning Province reached 1 105 tons (Li et al., 2007). But in summer in recent years high mortality rates have resulted in an abrupt decrease in aquacultural outcome and profit. Heavy occurrence of bacteria may constitute a significant health risk to cultured animals at high temperature or in other specific conditions (Bonadonna et al., 2002). However, the roles of E. coli and E. faecalis in digestion of scallops remain unclear, and information relating to a host’s growth performance, innate immunity and disease outbreaks after a long-term exposure to non-pathogenic fecal bacteria is limited. Bacterial accumulation and variation in digestive and immune enzymes in scallop tissues must be better understood to clarify the interaction between marine microbes and bivalves, and to improve aquaculture environment and seafood safety. Integration of analyzing mollusc digestion and immune response also makes the thorough knowledge of two dominant approaches of bivalves to dealing with the microbial invasion emphasized. The objectives of this study were to investigate the difference of retention of two fecal indicators in scallops, and determine effects of long-term exposure on digestive and immune enzyme activities in P. yessoensis, and evaluate digestive response as a supplement to innate immunity in regulation of bivalve adoptions to a changing intestinal microflora.
2. Materials and methods 2.1. Preparation of bacteria and experimental animals Scallops (Patinopecten yesoensis) of initial shell length 10.35 ± 0.26 cm and wet weight 107.81 ± 4.72 g were collected from Zhangzi Islands near Dalian (Liaoning Province, China) in December 2008. Samples were stored at 4 °C and transported to the laboratory within 10 h, and then were kept in an concrete pool with the sand-filtered effluent natural seawater at 8 °C for 3 days. The scallops were not fed during the acclimatization. E. coli (O157C83729) and E. faecalis (ATCC29212), purchased from the Microbial Culture Collection Center of Guangdong Institute of Microbiology, were maintained on nutrient agar (Land Bridge, Beijing, China). Bacteria were cultured for 24 h in nutrient broth (Land Bridge) at 36 °C. Then, the culture was centrifuged for 15 min at 3000g, the pellet was washed with 0.01 mol/l phosphate-buffered saline (PBS, 0.5 mM Na2HPO4, 1.5 mM KH2PO4 and 0.13 M NaCl, pH 7.4) and adjusted to an optical density of 1.0 at 550 nm (ca. 9 log10 CFU/ml).
2.2. Uptake of E. coli and E. faecalis by scallops After 3 days, the scallops were placed in plastic uptake tanks (30 40 40 cm) with the static seawater used in the acclimatization period (scallop-to-water ratio of 1:5, wet weight/volume). For exposure treatments, the two bacterial species were added to the tanks at final concentrations of 3 log10 CFU/ml (T1, E. faecalis; T2, E. coli) and 5 log10 CFU/ml (T3, E. faecalis; T4, E. coli). Throughout the experiment the averages of initial bacterial amounts of T1–T4 were 3.36 ± 0.32, 3.72 ± 0.42, 5.25 ± 0.16 and 5.48 ± 0.64 log10 CFU/ml, respectively. The seawater inoculum was changed completely once each day, followed immediately by an inoculation of fresh cultures. As a control group (designated Ctr), an aliquot of PBS was added. Scallops were fed with dry powdered Spirulina at a dose of 0.01% of scallop wet weight once every 2 days. To avoid possible effects of added food on endoenzyme activity, the scallops were starved for 2 days before sampling. Each treatment had three replicates.
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For the experimental infection test, 100 ll of 8 log10 CFU/ml E. faecalis (T5) and E. coli (T6) were injected into adductor muscle. An aliquot of PBS was injected as control group (designated PBS). Seawater was completely exchanged with filtered seawater once each day, and continuously aerated by an air pump at 10 l/min). The experimental conditions were: water temperature 8.39 ± 1.04 °C, salinity 31.67 ± 0.19 psu, pH 7.74 ± 0.16 and dissolved oxygen 8.03 ± 0.46 mg/l. 2.3. Enumeration of bacteria in scallop tissues Enumeration methods followed Chinese industry standards SN0168-92 and SN/T0475-95 with some modifications. For each sample, three scallops were collected for uptake of bacteria in total soft tissue; six scallops in the 5 log10CFU/ml E. coli and E. faecalis treatment groups were sampled for bacteria analysis in tissues. The scallop shells were washed twice with distilled water and then opened with a sterile shucking knife. The total inner content (flesh and liquor) of three scallops was homogenated with a sterile grinder, and a weight of 25 g was removed as the total scallop tissue. Using a sterile scissors six individuals were aseptically dissected to digestive tract, gonads, mantle lobes and gills, and adductor muscles, and were homogenated, respectively, as individual tissue samples. The samples were diluted to 1:10 (w/v) with 0.01 M phosphate buffer. Hemolymph was withdrawn from the posterior adductor muscle with a 1-ml plastic syringe fitted with a 25-gauge needle and stored temporarily at 4 °C as a working solution. Then, 1 ml of accurate decimal dilutions were added to one-off plastic plates of the appropriate selective count media: chromID Coli agar (COLI ID-F, bioMérieux) for E. coli and Pfizer Selective Enterococcus agar (PSE, Land Bridge) for E. faecalis, and incubated at 36 °C for 24 h. For each sample, three groups of six scallops were analyzed. For enumeration of bacteria, the results were expressed as log CFU/ g of sample, and the bioconcentration factor (BCF) and rate of bacterial accumulation (RA) were calculated according to Taylor (1983): BCF = (Ce Ci)/Cs, where Ce = bacterial concentration in scallops at the end of exposure (log10 CFU/g), Ci = initial bacterial concentration in tissue before exposure (log10 CFU/g), Cs = experimental bacterial concentration in seawater inoculum (log10 CFU/ ml). The rate of accumulation was calculated as follows: RA = (bacteria leveltreatment bacteria levelcontrol)/day(s) of bacteria exposure (Taylor, 1983). 2.4. Extract preparation and enzyme activity assay To study effects of long-term exposure to E. coli and E. faecalis on digestion and immune conditions, two digestive enzymes and two immunity-related enzymes were selected. Six scallops were collected from each of the T1–T4 and Ctr groups 21 days after exposure, and from the T5, T6 and PBS groups 18 h after injection. Trypsin and amylase (AMS) activities were recorded in the digestive gland, and ACP and SOD activities were accessed in both hemocyte lysate and cell-free hemolymph. All operations of dissecting tissues and extracting enzyme solutions were done at 4 °C. Spectrophotometry was used to analyze the enzyme activity, and the light path was 1 cm during the test. The digestive glands were dissected from the scallop tissues, and were rinsed with cold distilled water and manually homogenated in the grinders for 2 min. A weight of 1 g of homogenate was diluted in 9 g of physiological saline water (0.86% w/v), and was centrifuged at 5000g for 10 min. The supernatant was recovered and tested within 12 h. Protein content was measured according to Bradford (1976), using the Coomassie staining method. Trypsin activity was tested according to Jin (1995), and amylase according to Worthington (1993), using the iodine staining method. Unit definitions are as follows: one trypsin unit will produce a DA253 of
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0.003 per min at pH 8.0 at 37 °C using N-benzoyl-L-arginine ethyl ester (BAEE) as substrate; one amylase unit will produce the hydrolysis of 10 mg of starch in 30 min at 37 °C. Results of trypsin and AMS activities were quantified as specific activity (U/mg protein, U/mg pro). Hemolymph was withdrawn from the anterior adductor muscle with a 1-ml plastic syringe fitted with a 25-gauge needle and stored temporarily in microcentrifuge tubes maintained on ice. Initial subsamples of pooled hemolymph were centrifuged at 780g for 10 min. The supernatant, corresponding to cell-free hemolymph, was collected, whereas the hemocyte pellet was treated according to Sun and Li (2000). Briefly, pelleted hemocytes were resuspended in the same volume of distilled water for lysis, and then centrifuged at 12,000g for 15 min to obtain hemocyte lysate. Cell-free hemolymph and hemocyte lysate were frozen and stored at 80 °C until analysis. ACP and SOD activities were measured according to the methods of Deng et al. (1991) by the disodium phenyl orthophosphate method and pyrogallol self-oxidation, respectively. Results of ACP and SOD measurement in cell-free hemolymph and hemocyte lysate were expressed as specific activity (U/100 ml and U/ml, respectively).
3. Results 3.1. Uptake of E. coli and E. faecalis in scallop Before experimental contamination, the levels of E. coli and E. faecalis in the scallops were undetectable. E. coli and E. faecalis uptake by scallops is presented in Fig. 1 and Table 1. After a 1-day exposure, bacterial numbers in the T1 and T4 group rapidly increased, and then fluctuated slightly. The T2 and T3 groups increased significantly at day 5 and 3, respectively. T3 maintained this level to the end, while T2 recorded significant growth again at day 21 (P < 0.05). The BCF and RA values for treatments with E. faecalis were higher than those with E. coli (Table 1), while both showed higher RA and lower BCF values at a higher bacterial load. Furthermore, the RAs of all treatments decreased significantly with exposure.
3.2. Tissue distribution of E. coli and E. faecalis in scallop The distribution of bacteria in scallop tissue is shown in Fig. 2. The highest concentrations of E. coli and E. faecalis were detected in the digestive tract, were 5.7 and 12.9 times higher than those in total soft tissue, respectively. The lowest numbers of E. coli and E. faecalis were detected in hemolymph, were 6.0% and 13.9% of those in total soft tissue, respectively. Furthermore, the numbers of E. faecalis in hemolymph were 85 times higher than those of E. coli.
2.5. Statistic analysis Significant differences in mean values were determined using one-way analysis of variance (ANOVA) by SPSS 10.0. When overall differences were significant at the 0.05 level, Tukey’s multiple comparisons were conducted to analyze the mean values among all groups.
Fig. 2. Distribution of E. coli and E. faecalis in scallop tissue exposed to high seawater inoculum levels. (a) Total soft tissues; (b) digestive tract; (c) mantle lobes and gills; (d) adductor muscles; (e) gonads and (f) hemolymph. Vertical lines represent standard deviation.
Fig. 1. Retention of E. coli and E. faecalis by scallops in a seawater inoculum of 3 and 5 log10CFU/ml, respectively. Vertical lines represent standard deviation.
Table 1 Bioconcentration factor (BCF) and rate of bacterial accumulation (RA) in scallops versus exposure time (means ± SD). Treatment
Parameter
Time of exposure of scallops to E. coli and E. faecalis (days) 1
3
5
7
14
21
T1
RA BCF
2.469 ± 0.031A* 1.205 ± 0.023a
0.948 ± 0.006B*
0.604 ± 0.020B*
0.335 ± 0.018B*
0.241 ± 0.005B*
0.193 ± 0.004B*
T2
RA BCF
1.151 ± 0.213A* 0.908 ± 0.017b
0.429 ± 0.052B*
0.521 ± 0.021C*
0.415 ± 0.033D*
0.208 ± 0.005E
0.161 ± 0.003E*
T3
RA BCF
3.462 ± 0.021A 0.949 ± 0.002b
1.590 ± 0.018B*
0.969 ± 0.002C*
0.744 ± 0.001D*
0.343 ± 0.001E
0.237 ± 0.001F
T4
RA BCF
3.544 ± 0.018A 0.651 ± 0.003c
1.162 ± 0.033B*
0.722 ± 0.004C*
0.548 ± 0.005D*
0.260 ± 0.006E*
0.170 ± 0.001F*
Superscript letters indicate significant differences with respect to the initial value within one treatment. Asterisks indicate significant differences among four treatments on the same day (P < 0.05).
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3.3. Effect of uptake of E. coli and E. faecalis on trypsin and AMS activity in the digestive gland All treatments with E. faecalis showed significantly higher activities of the trypsin and AMS than with E. coli, except for trypsin activities between injection groups (Fig. 3). Moreover, scallops exposed to 3 log10CFU/ml of both bacteria displayed significantly higher trypsin and lower AMS activities than those exposed to 5 log10CFU/ml (P < 0.001). Except for significantly higher trypsin activity in group T1, all other exposure treatments showed distinctly lower trypsin and AMS activity compared to control (Ctr). For injection groups, there was a significant increase in trypsin activity and a significant decrease in AMS activity compared to control (PBS). The trypsin activity of scallops with healthy digestive glands was more than 83 times higher than AMS activity. These results indicated that uptake of E. coli and E. faecalis at an inoculum greater than 3 log10CFU/ml significantly inhibited digestive enzyme activities. 3.4. Effect of uptake of E. coli and E. faecalis on SOD and ACP activity in hemolymph SOD and ACP activity in cell-free hemolymph was higher than in hemocyte, but no significant difference was observed for ACP activities in the Ctr, PBS, T1 or T2 groups (Fig. 4). After a 21-day exposure to 3 log10CFU/ml inoculum, SOD activities in cell-free hemolymph clearly increased, but decreased significantly in hemocytes (P < 0.05). For treatments with 5 log10CFU/ml E. faecalis, SOD activity in hemocytes was clearly lower, while with 5 log10CFU/ml E. coli was clearly higher compared to control. Only in group T4 ACP activity in hemolymph was distinctly higher compared to control. After 18 h of injection with both bacteria SOD and ACP activ-
Fig. 4. Variations of SOD (A) and ACP (B) activities in scallop hemolymph after uptake of E. coli and E. faecalis. Letters indicate significant differences with respect to control (Ctr). Asterisks indicate significant differences between hemocyte lysate and cell-free hemolymph within one treatment. Superscripts of different letters are significantly different from the corresponding values of group Ctl at P < 0.05. Vertical lines represent standard deviation.
ities in cell-free hemolymph significantly increased. However, in hemocytes SOD activity clearly decreased, and no significance was observed for ACP activity compared to group PBS.
4. Discussion
Fig. 3. Differential changes in (A) trypsin and (B) AMS activity in the digestive gland of scallops to uptake of E. coli and E. faecalis. Superscripts of different letters are significantly different from group Ctl at P < 0.05. Vertical lines represent standard deviation.
In bivalves, the accumulation of bacteria rapidly reaches a high level but the rates of uptake and depuration vary between genera of invading microorganisms, due to environmental conditions (Le Moullac and Haffner, 2000; Murphree and Tamplin, 1991; Blodgett and Chirtel, 1998) and interaction between microbes and the host immune system (Hong et al., 2006). Our results showed that the RA and the BCF of E. faecalis were both significantly higher than those of E. coli in scallop tissues, and the rate of accumulation gradually decreased with exposure time (Fig. 1 and Table 1). This indicated that it was easier for the scallop to accumulate E. faecalis cells than E. coli, and that the uptake of bacteria in scallop rapidly slowed and retention may approach saturation point. In hemolymph E. faecalis levels were lower than E. coli (Fig. 2), mainly due to antimicrobial activity. Distribution and accumulation of intact bacteria changed with time in the hemolymph but not in other tissues (Williams et al., 2009). The available evidence supports the hypothesis that persistence of bacteria in bivalve tissue largely depends on their sensitivity to the bactericidal activity of the hemolymph (Pruzzo et al., 2005), cell surface properties of the bacteria and environmental conditions (Hernroth, 2003; Hauton et al., 2001; Blodgett and Chirtel, 1998). It was initially reported that long-term exposure to high levels of E. coli and E. faecalis clearly decreased trypsin and AMS activity (Fig. 3). Furthermore, scallops exposed to E. faecalis showed significantly higher trypsin and AMS activity than those exposed to E. coli. These results indicated that uptake of E. coli and E. faecalis at a level of >3 log10CFU/ml significantly inhibited the activity of
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digestive enzymes. The change in digestive enzyme activity varied with the different enzymes and was affected by the number and species of bacteria in scallop tissue. Prieur (1981) also demonstrated both selective ingestion and digestion of microbes by the mussel. Bivalves have an open circulatory and innate immune system, hemocytes and soluble factors operate in a coordinated way to provide protection from invading microorganisms (Rinkevich and Muller, 1996). La Peyre et al. (1995) observed that the concentration of lysozyme varied between different oyster species after exposure to the protozoan Perkinsus marinus. In addition, immune responses to invasion may vary between host species and with growth status (Fei et al., 2008). The characteristics and in vitro function of hemocytes and activity of peroxidase (POD), phenoloxidase (PO) and alkaline phosphatase (ALP) in shellfish immune responses to invasions have been the subject of a number of recent studies (Fisher, 1986; Carballal et al., 1997a,b; Mortensen and Glette, 1996; Xing et al., 2002). Further study on variations in their activity in different types of hemocytes in the scallop Patinopecten yesoensis is required. Physical injury and food supply can alter immune parameters (Laura et al., 2006), and the cells involved in both wound healing and immunity may be limited in their ability to fight disease during active regeneration of wounds (Malham et al., 2003). Nevertheless, this experiment indicated that injection with PBS had little effect on the scallop immune system (Fig. 4), this may be due to differences in experimental animals, bacterial species or sampling point. In addition, the data showed that cell-free hemolymph may have a greater effect on elimination of invasive bacteria than hemocyte, Effects of exposure and injection on variations in enzyme activity were clearly different, which may be due to difference in treatment times and/or injuries caused during administration of injections. Furthermore, scallops exposed to E. faecalis showed higher digestive enzyme activity and lower ACP activity than those treated with E. coli. Therefore, differential retention of E. faecalis and E. coli in scallop could be related to the degree of inhibition of digestive enzyme activities and immune responses in hemolymph. And on the other hand, it can also be deduced that long-term exposure to non-pathogenic bacteria may have a negative effect on digestion, immunity and even growth of the scallop, by upsetting the balance of natural microflora and/or enzyme expression in the digestive tract. However, further research on the interactions between the diversity of natural microflora and development of mollusks and the relationship between exposure to common fecal bacteria and disease outbreaks in bivalves should be paid more attentions. Understanding the immune response in bivalves to invasive infections at cellular and molecular levels offers a theoretical base for exploring molluscan probiotics. Also, due to enhancing the nonspecific defense mechanisms of the host and improving the quality of the aquatic environment, studies on probiotics have received increased attention (Raa, 1996; Li et al., 2007; Bachère et al., 1995; Balcázar et al., 2006). As stated above, the interaction between microbiota, including probiotics, and the host is not limited to the intestinal tract. Probiotic bacteria are not only active on the gills or skin of the host, but also in its ambient environment (Verschuere et al., 2000).
Acknowledgements This work was funded by grants from Special Scientific Research Fund of Agricultural Public Welfare Profession of China (nyhyzx07047) and National High Technology Research and Development Program of China (2006AA100304). The authors thank Wang Wei and Wang Shihuan of Zhangzidao Fisheries Company and Xia
Yuanzheng of Dalian University of Technology for the use of experimental materials and instruments.
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B. Li et al. / Marine Pollution Bulletin 60 (2010) 1600–1605 Power, U.F., Collins, J.K., 1990. Tissue distribution of a coliphage and Escherichia coli in mussels after contamination and depuration. Applied and Environmental Microbiology 56 (3), 803–807. Prieur, D., 1981. Experimental studies of trophic relationships between marine bacteria and bivalve molluscs. Kieler Meeresforschung, Sonderh 5, 376–383. Pruzzo, C., Gallo, G., Canesi, L., 2005. Persistence of vibrios in marine bivalves: the role of interactions with hemolymph components. Environmental Microbiology 7 (6), 761–772. Raa, J., 1996. The use of immunostimulatory substances in fish and shellfish farming. Review of Fisheries Science 4, 229–288. Rinkevich, B., Muller, W.E.G., 1996. Invertebrate immunology. Progress in Molecular and Subcellular Biology, vol. 15. Springer, Berlin. p. 247. Sorokin, Y.I., 1999. Radioisotopic Methods in Hydrobiology. Springer, New York. pp. 199–217. Sun, H.S., Li, G.Y., 1999. Activity change of seven enzymes in hemolymph in scallop Chlamys farreri after challenge with Escherichia coli. Marine Sciences 5, 40–44. Sun, H.S., Li, G.Y., 2000. Activities and properties of superoxide dismutase and catalase in the hemolymph of Chlamys farreri. Oceanologia et Limnologia Sinica 3, 259–265.
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Tallon, P., Magajna, B., Lofranco, C., Leung, K.T., 2005. Microbial indicators of faecal contamination in water: a current perspective. Water, Air, and Soil Pollution 166, 139–166. Taylor, D., 1983. The significance of the accumulation of cadmium by aquatic organisms. Ecotoxicology and Environmental Safety 7, 33–42. Verschuere, L., Rombaut, G., Sorgeloos, P., Verstraete, W., 2000. Probiotic bacteria as biological control agents in aquaculture. Microbiology and Molecular Biology Reviews 64 (4), 655–671. Williams, H.R., Macey, B.M., Burnett, L.E., Burnett, K.G., 2009. Differential localization and bacteriostasis of Vibrio campbellii among tissues of the eastern oyster, Crassostrea virginica. Developmental and Comparative Immunology 33 (4), 592–600. Worthington, V., 1993. Worthington Enzyme Manual. Enzymes and Related Biochemicals. Worthington Chemical, New Jersey. Xing, J., Zhan, W.B., Li, Z., 2002. Endoenzymes associated with hemocyte types in the scallop (Chlamys farreri). Fish and Shellfish Immunology 13 (4), 271–278. Xing, J., Lin, T.T., Zhan, W.B., 2008. Variations of enzyme activities in the hemocytes of scallop Chlamys farreri after infection with the acute virus necrobiotic virus (AVNV). Fish and Shellfish Immunology 25 (6), 847–852.
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Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Relationship between differential retention of Escherichia coli and Enterococcus faecalis and variations in enzyme activity in the scallop Patinopecten yessoensis Bin Li a,b,c, Bijuan Chen c, Zhanhui Qi d, Zengjie Jiang c, Jihong Zhang c, Jianguang Fang c,* a
Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, China Graduate University, Chinese Academy of Sciences, Beijing 100049, China c Key Laboratory for Sustainable Utilization of Marine Fisheries Resources, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China d South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China b
a r t i c l e
i n f o
Keywords: Patinopecten yessoensis Escherichia coli Enterococcus faecalis Bacterial retention Enzyme activity
a b s t r a c t Uptake of Escherichia coli and Enterococcus faecalis and variations of trypsin amylase activity acid phosphatase and superoxide dismutase in tissue of the scallop Patinopecten yessoensis were detected. The results showed that P. yessoensis accumulated E. faecalis in larger numbers and more rapidly than E. coli, both with the highest concentration in the digestive tract and lowest in hemolymph. Compared to E. coli, all scallops exposed to E. faecalis showed significantly higher trypsin and AMS activity. SOD activity in hemocytes and ACP activity in hemolymph was significantly higher in the treatments with 5 log10CFU/ml E. coli than with E. faecalis. But no significant differences in ACP activity of P. yessoensis exposed to a 3 log10CFU/ml inoculum of both bacteria were recorded. In conclusion, the mass retention of gut microflora in P. yessoensis is positively correlated with digestive enzymes activity and negatively correlated with ACP activity in the hemocyte. Ó 2010 Published by Elsevier Ltd.
1. Introduction Scallops are filter-feeders living in a bacteria-rich environment, which, in some cases, may cause marine epizootics and mass mortalities in young bivalves over a short period of time (Laura et al., 2006). Escherichia coli and Enterococcus faecalis are two common bacteria found in the aquatic environment and in the intestines of warm-blooded animals and humans. In summer, they can reach high levels in sewage-polluted seawater and accumulated in high numbers in scallops. Hence, they are suitable indicators to predict fecal contamination in seawater and mollusks (Tallon et al., 2005). About 90% of E. coli is found in the digestive tract and less than 1% in hemolymph (Power and Collins, 1990). Marino et al. (2005) found that Enterococcus durans was quickly taken up by the mussel (Mytilus galloprovincialis) and released more slowly than E. coli or Vibrio cholerae at both 14 and 21 °C. Williams et al. (2009) reported that neither the distribution nor accumulation of live Vibrio campbellii varied with time, except in the hemolymph, and speculated that the gonad may have a major antibacterial defense role in bivalves. The majority of microflora isolates in bivalve shellfish are gram-negative (68%) and aerobic (76%) bacteria, such as Vibrio spp., Pseudomonas spp. and Aeromonas spp., which indicates a * Corresponding author. Tel.: +86 0 532 85822957; fax: +86 0 532 85811514. E-mail address: [email protected] (J. Fang). 0025-326X/$ - see front matter Ó 2010 Published by Elsevier Ltd. doi:10.1016/j.marpolbul.2010.03.017
selective process to sequester and maintain certain species (Kueh and Chan, 1985; Hariharan et al., 1995). Bacteria utilized in probiotic applications, such as Aeromonas media A199 and Roseobacter sp. strain BS107, help to maintain or restore a host’s natural microbial floral and enhance the nutrition of host species through the production of supplemental digestive enzymes (Gillor et al., 2008). However, some xenobiotics may have a negative impact on assimilation efficiency and adaptation possibilities in aquatic animals (Sorokin, 1999). Without specific antibodies, scallop hemocytes play a key role in innate immunity, including recognition, phagocytosis and elimination of invading microorganisms by releasing immune factors, such as lectins, lysozymes, reactive oxygen species and the (anti)oxidases. Studies on modulation of the immune system of bivalves by other organisms have concentrated on the effects of experimental infections on changes in components of plasma or hemolymph. After exposure of the clam Ruditapes philippinarum to Vibrio Pl, hemocyte concentration and peptidase activity increased to a maximum but then decreased to a stable value (Oubella et al., 1994). After an injection with acute virus necrobiotic virus acid phosphatase (ACP), superoxide dismutase (SOD) and catalase (CAT) in the hemocytes of scallop Chlamys farreri were significantly higher than the control but then decreased to normal levels (Xing et al., 2008). SOD activity increased significantly in hemocytes, but not in serum, after an experimental challenge of scallop C. farreri with E. coli (Sun and Li, 1999).
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The Japanese scallop Patinopecten yessoensis is now widely cultivated in northern parts of the Yellow Sea (China). In 2007, production of P. yessoensis in the Dalian area of Liaoning Province reached 1 105 tons (Li et al., 2007). But in summer in recent years high mortality rates have resulted in an abrupt decrease in aquacultural outcome and profit. Heavy occurrence of bacteria may constitute a significant health risk to cultured animals at high temperature or in other specific conditions (Bonadonna et al., 2002). However, the roles of E. coli and E. faecalis in digestion of scallops remain unclear, and information relating to a host’s growth performance, innate immunity and disease outbreaks after a long-term exposure to non-pathogenic fecal bacteria is limited. Bacterial accumulation and variation in digestive and immune enzymes in scallop tissues must be better understood to clarify the interaction between marine microbes and bivalves, and to improve aquaculture environment and seafood safety. Integration of analyzing mollusc digestion and immune response also makes the thorough knowledge of two dominant approaches of bivalves to dealing with the microbial invasion emphasized. The objectives of this study were to investigate the difference of retention of two fecal indicators in scallops, and determine effects of long-term exposure on digestive and immune enzyme activities in P. yessoensis, and evaluate digestive response as a supplement to innate immunity in regulation of bivalve adoptions to a changing intestinal microflora.
2. Materials and methods 2.1. Preparation of bacteria and experimental animals Scallops (Patinopecten yesoensis) of initial shell length 10.35 ± 0.26 cm and wet weight 107.81 ± 4.72 g were collected from Zhangzi Islands near Dalian (Liaoning Province, China) in December 2008. Samples were stored at 4 °C and transported to the laboratory within 10 h, and then were kept in an concrete pool with the sand-filtered effluent natural seawater at 8 °C for 3 days. The scallops were not fed during the acclimatization. E. coli (O157C83729) and E. faecalis (ATCC29212), purchased from the Microbial Culture Collection Center of Guangdong Institute of Microbiology, were maintained on nutrient agar (Land Bridge, Beijing, China). Bacteria were cultured for 24 h in nutrient broth (Land Bridge) at 36 °C. Then, the culture was centrifuged for 15 min at 3000g, the pellet was washed with 0.01 mol/l phosphate-buffered saline (PBS, 0.5 mM Na2HPO4, 1.5 mM KH2PO4 and 0.13 M NaCl, pH 7.4) and adjusted to an optical density of 1.0 at 550 nm (ca. 9 log10 CFU/ml).
2.2. Uptake of E. coli and E. faecalis by scallops After 3 days, the scallops were placed in plastic uptake tanks (30 40 40 cm) with the static seawater used in the acclimatization period (scallop-to-water ratio of 1:5, wet weight/volume). For exposure treatments, the two bacterial species were added to the tanks at final concentrations of 3 log10 CFU/ml (T1, E. faecalis; T2, E. coli) and 5 log10 CFU/ml (T3, E. faecalis; T4, E. coli). Throughout the experiment the averages of initial bacterial amounts of T1–T4 were 3.36 ± 0.32, 3.72 ± 0.42, 5.25 ± 0.16 and 5.48 ± 0.64 log10 CFU/ml, respectively. The seawater inoculum was changed completely once each day, followed immediately by an inoculation of fresh cultures. As a control group (designated Ctr), an aliquot of PBS was added. Scallops were fed with dry powdered Spirulina at a dose of 0.01% of scallop wet weight once every 2 days. To avoid possible effects of added food on endoenzyme activity, the scallops were starved for 2 days before sampling. Each treatment had three replicates.
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For the experimental infection test, 100 ll of 8 log10 CFU/ml E. faecalis (T5) and E. coli (T6) were injected into adductor muscle. An aliquot of PBS was injected as control group (designated PBS). Seawater was completely exchanged with filtered seawater once each day, and continuously aerated by an air pump at 10 l/min). The experimental conditions were: water temperature 8.39 ± 1.04 °C, salinity 31.67 ± 0.19 psu, pH 7.74 ± 0.16 and dissolved oxygen 8.03 ± 0.46 mg/l. 2.3. Enumeration of bacteria in scallop tissues Enumeration methods followed Chinese industry standards SN0168-92 and SN/T0475-95 with some modifications. For each sample, three scallops were collected for uptake of bacteria in total soft tissue; six scallops in the 5 log10CFU/ml E. coli and E. faecalis treatment groups were sampled for bacteria analysis in tissues. The scallop shells were washed twice with distilled water and then opened with a sterile shucking knife. The total inner content (flesh and liquor) of three scallops was homogenated with a sterile grinder, and a weight of 25 g was removed as the total scallop tissue. Using a sterile scissors six individuals were aseptically dissected to digestive tract, gonads, mantle lobes and gills, and adductor muscles, and were homogenated, respectively, as individual tissue samples. The samples were diluted to 1:10 (w/v) with 0.01 M phosphate buffer. Hemolymph was withdrawn from the posterior adductor muscle with a 1-ml plastic syringe fitted with a 25-gauge needle and stored temporarily at 4 °C as a working solution. Then, 1 ml of accurate decimal dilutions were added to one-off plastic plates of the appropriate selective count media: chromID Coli agar (COLI ID-F, bioMérieux) for E. coli and Pfizer Selective Enterococcus agar (PSE, Land Bridge) for E. faecalis, and incubated at 36 °C for 24 h. For each sample, three groups of six scallops were analyzed. For enumeration of bacteria, the results were expressed as log CFU/ g of sample, and the bioconcentration factor (BCF) and rate of bacterial accumulation (RA) were calculated according to Taylor (1983): BCF = (Ce Ci)/Cs, where Ce = bacterial concentration in scallops at the end of exposure (log10 CFU/g), Ci = initial bacterial concentration in tissue before exposure (log10 CFU/g), Cs = experimental bacterial concentration in seawater inoculum (log10 CFU/ ml). The rate of accumulation was calculated as follows: RA = (bacteria leveltreatment bacteria levelcontrol)/day(s) of bacteria exposure (Taylor, 1983). 2.4. Extract preparation and enzyme activity assay To study effects of long-term exposure to E. coli and E. faecalis on digestion and immune conditions, two digestive enzymes and two immunity-related enzymes were selected. Six scallops were collected from each of the T1–T4 and Ctr groups 21 days after exposure, and from the T5, T6 and PBS groups 18 h after injection. Trypsin and amylase (AMS) activities were recorded in the digestive gland, and ACP and SOD activities were accessed in both hemocyte lysate and cell-free hemolymph. All operations of dissecting tissues and extracting enzyme solutions were done at 4 °C. Spectrophotometry was used to analyze the enzyme activity, and the light path was 1 cm during the test. The digestive glands were dissected from the scallop tissues, and were rinsed with cold distilled water and manually homogenated in the grinders for 2 min. A weight of 1 g of homogenate was diluted in 9 g of physiological saline water (0.86% w/v), and was centrifuged at 5000g for 10 min. The supernatant was recovered and tested within 12 h. Protein content was measured according to Bradford (1976), using the Coomassie staining method. Trypsin activity was tested according to Jin (1995), and amylase according to Worthington (1993), using the iodine staining method. Unit definitions are as follows: one trypsin unit will produce a DA253 of
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0.003 per min at pH 8.0 at 37 °C using N-benzoyl-L-arginine ethyl ester (BAEE) as substrate; one amylase unit will produce the hydrolysis of 10 mg of starch in 30 min at 37 °C. Results of trypsin and AMS activities were quantified as specific activity (U/mg protein, U/mg pro). Hemolymph was withdrawn from the anterior adductor muscle with a 1-ml plastic syringe fitted with a 25-gauge needle and stored temporarily in microcentrifuge tubes maintained on ice. Initial subsamples of pooled hemolymph were centrifuged at 780g for 10 min. The supernatant, corresponding to cell-free hemolymph, was collected, whereas the hemocyte pellet was treated according to Sun and Li (2000). Briefly, pelleted hemocytes were resuspended in the same volume of distilled water for lysis, and then centrifuged at 12,000g for 15 min to obtain hemocyte lysate. Cell-free hemolymph and hemocyte lysate were frozen and stored at 80 °C until analysis. ACP and SOD activities were measured according to the methods of Deng et al. (1991) by the disodium phenyl orthophosphate method and pyrogallol self-oxidation, respectively. Results of ACP and SOD measurement in cell-free hemolymph and hemocyte lysate were expressed as specific activity (U/100 ml and U/ml, respectively).
3. Results 3.1. Uptake of E. coli and E. faecalis in scallop Before experimental contamination, the levels of E. coli and E. faecalis in the scallops were undetectable. E. coli and E. faecalis uptake by scallops is presented in Fig. 1 and Table 1. After a 1-day exposure, bacterial numbers in the T1 and T4 group rapidly increased, and then fluctuated slightly. The T2 and T3 groups increased significantly at day 5 and 3, respectively. T3 maintained this level to the end, while T2 recorded significant growth again at day 21 (P < 0.05). The BCF and RA values for treatments with E. faecalis were higher than those with E. coli (Table 1), while both showed higher RA and lower BCF values at a higher bacterial load. Furthermore, the RAs of all treatments decreased significantly with exposure.
3.2. Tissue distribution of E. coli and E. faecalis in scallop The distribution of bacteria in scallop tissue is shown in Fig. 2. The highest concentrations of E. coli and E. faecalis were detected in the digestive tract, were 5.7 and 12.9 times higher than those in total soft tissue, respectively. The lowest numbers of E. coli and E. faecalis were detected in hemolymph, were 6.0% and 13.9% of those in total soft tissue, respectively. Furthermore, the numbers of E. faecalis in hemolymph were 85 times higher than those of E. coli.
2.5. Statistic analysis Significant differences in mean values were determined using one-way analysis of variance (ANOVA) by SPSS 10.0. When overall differences were significant at the 0.05 level, Tukey’s multiple comparisons were conducted to analyze the mean values among all groups.
Fig. 2. Distribution of E. coli and E. faecalis in scallop tissue exposed to high seawater inoculum levels. (a) Total soft tissues; (b) digestive tract; (c) mantle lobes and gills; (d) adductor muscles; (e) gonads and (f) hemolymph. Vertical lines represent standard deviation.
Fig. 1. Retention of E. coli and E. faecalis by scallops in a seawater inoculum of 3 and 5 log10CFU/ml, respectively. Vertical lines represent standard deviation.
Table 1 Bioconcentration factor (BCF) and rate of bacterial accumulation (RA) in scallops versus exposure time (means ± SD). Treatment
Parameter
Time of exposure of scallops to E. coli and E. faecalis (days) 1
3
5
7
14
21
T1
RA BCF
2.469 ± 0.031A* 1.205 ± 0.023a
0.948 ± 0.006B*
0.604 ± 0.020B*
0.335 ± 0.018B*
0.241 ± 0.005B*
0.193 ± 0.004B*
T2
RA BCF
1.151 ± 0.213A* 0.908 ± 0.017b
0.429 ± 0.052B*
0.521 ± 0.021C*
0.415 ± 0.033D*
0.208 ± 0.005E
0.161 ± 0.003E*
T3
RA BCF
3.462 ± 0.021A 0.949 ± 0.002b
1.590 ± 0.018B*
0.969 ± 0.002C*
0.744 ± 0.001D*
0.343 ± 0.001E
0.237 ± 0.001F
T4
RA BCF
3.544 ± 0.018A 0.651 ± 0.003c
1.162 ± 0.033B*
0.722 ± 0.004C*
0.548 ± 0.005D*
0.260 ± 0.006E*
0.170 ± 0.001F*
Superscript letters indicate significant differences with respect to the initial value within one treatment. Asterisks indicate significant differences among four treatments on the same day (P < 0.05).
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3.3. Effect of uptake of E. coli and E. faecalis on trypsin and AMS activity in the digestive gland All treatments with E. faecalis showed significantly higher activities of the trypsin and AMS than with E. coli, except for trypsin activities between injection groups (Fig. 3). Moreover, scallops exposed to 3 log10CFU/ml of both bacteria displayed significantly higher trypsin and lower AMS activities than those exposed to 5 log10CFU/ml (P < 0.001). Except for significantly higher trypsin activity in group T1, all other exposure treatments showed distinctly lower trypsin and AMS activity compared to control (Ctr). For injection groups, there was a significant increase in trypsin activity and a significant decrease in AMS activity compared to control (PBS). The trypsin activity of scallops with healthy digestive glands was more than 83 times higher than AMS activity. These results indicated that uptake of E. coli and E. faecalis at an inoculum greater than 3 log10CFU/ml significantly inhibited digestive enzyme activities. 3.4. Effect of uptake of E. coli and E. faecalis on SOD and ACP activity in hemolymph SOD and ACP activity in cell-free hemolymph was higher than in hemocyte, but no significant difference was observed for ACP activities in the Ctr, PBS, T1 or T2 groups (Fig. 4). After a 21-day exposure to 3 log10CFU/ml inoculum, SOD activities in cell-free hemolymph clearly increased, but decreased significantly in hemocytes (P < 0.05). For treatments with 5 log10CFU/ml E. faecalis, SOD activity in hemocytes was clearly lower, while with 5 log10CFU/ml E. coli was clearly higher compared to control. Only in group T4 ACP activity in hemolymph was distinctly higher compared to control. After 18 h of injection with both bacteria SOD and ACP activ-
Fig. 4. Variations of SOD (A) and ACP (B) activities in scallop hemolymph after uptake of E. coli and E. faecalis. Letters indicate significant differences with respect to control (Ctr). Asterisks indicate significant differences between hemocyte lysate and cell-free hemolymph within one treatment. Superscripts of different letters are significantly different from the corresponding values of group Ctl at P < 0.05. Vertical lines represent standard deviation.
ities in cell-free hemolymph significantly increased. However, in hemocytes SOD activity clearly decreased, and no significance was observed for ACP activity compared to group PBS.
4. Discussion
Fig. 3. Differential changes in (A) trypsin and (B) AMS activity in the digestive gland of scallops to uptake of E. coli and E. faecalis. Superscripts of different letters are significantly different from group Ctl at P < 0.05. Vertical lines represent standard deviation.
In bivalves, the accumulation of bacteria rapidly reaches a high level but the rates of uptake and depuration vary between genera of invading microorganisms, due to environmental conditions (Le Moullac and Haffner, 2000; Murphree and Tamplin, 1991; Blodgett and Chirtel, 1998) and interaction between microbes and the host immune system (Hong et al., 2006). Our results showed that the RA and the BCF of E. faecalis were both significantly higher than those of E. coli in scallop tissues, and the rate of accumulation gradually decreased with exposure time (Fig. 1 and Table 1). This indicated that it was easier for the scallop to accumulate E. faecalis cells than E. coli, and that the uptake of bacteria in scallop rapidly slowed and retention may approach saturation point. In hemolymph E. faecalis levels were lower than E. coli (Fig. 2), mainly due to antimicrobial activity. Distribution and accumulation of intact bacteria changed with time in the hemolymph but not in other tissues (Williams et al., 2009). The available evidence supports the hypothesis that persistence of bacteria in bivalve tissue largely depends on their sensitivity to the bactericidal activity of the hemolymph (Pruzzo et al., 2005), cell surface properties of the bacteria and environmental conditions (Hernroth, 2003; Hauton et al., 2001; Blodgett and Chirtel, 1998). It was initially reported that long-term exposure to high levels of E. coli and E. faecalis clearly decreased trypsin and AMS activity (Fig. 3). Furthermore, scallops exposed to E. faecalis showed significantly higher trypsin and AMS activity than those exposed to E. coli. These results indicated that uptake of E. coli and E. faecalis at a level of >3 log10CFU/ml significantly inhibited the activity of
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digestive enzymes. The change in digestive enzyme activity varied with the different enzymes and was affected by the number and species of bacteria in scallop tissue. Prieur (1981) also demonstrated both selective ingestion and digestion of microbes by the mussel. Bivalves have an open circulatory and innate immune system, hemocytes and soluble factors operate in a coordinated way to provide protection from invading microorganisms (Rinkevich and Muller, 1996). La Peyre et al. (1995) observed that the concentration of lysozyme varied between different oyster species after exposure to the protozoan Perkinsus marinus. In addition, immune responses to invasion may vary between host species and with growth status (Fei et al., 2008). The characteristics and in vitro function of hemocytes and activity of peroxidase (POD), phenoloxidase (PO) and alkaline phosphatase (ALP) in shellfish immune responses to invasions have been the subject of a number of recent studies (Fisher, 1986; Carballal et al., 1997a,b; Mortensen and Glette, 1996; Xing et al., 2002). Further study on variations in their activity in different types of hemocytes in the scallop Patinopecten yesoensis is required. Physical injury and food supply can alter immune parameters (Laura et al., 2006), and the cells involved in both wound healing and immunity may be limited in their ability to fight disease during active regeneration of wounds (Malham et al., 2003). Nevertheless, this experiment indicated that injection with PBS had little effect on the scallop immune system (Fig. 4), this may be due to differences in experimental animals, bacterial species or sampling point. In addition, the data showed that cell-free hemolymph may have a greater effect on elimination of invasive bacteria than hemocyte, Effects of exposure and injection on variations in enzyme activity were clearly different, which may be due to difference in treatment times and/or injuries caused during administration of injections. Furthermore, scallops exposed to E. faecalis showed higher digestive enzyme activity and lower ACP activity than those treated with E. coli. Therefore, differential retention of E. faecalis and E. coli in scallop could be related to the degree of inhibition of digestive enzyme activities and immune responses in hemolymph. And on the other hand, it can also be deduced that long-term exposure to non-pathogenic bacteria may have a negative effect on digestion, immunity and even growth of the scallop, by upsetting the balance of natural microflora and/or enzyme expression in the digestive tract. However, further research on the interactions between the diversity of natural microflora and development of mollusks and the relationship between exposure to common fecal bacteria and disease outbreaks in bivalves should be paid more attentions. Understanding the immune response in bivalves to invasive infections at cellular and molecular levels offers a theoretical base for exploring molluscan probiotics. Also, due to enhancing the nonspecific defense mechanisms of the host and improving the quality of the aquatic environment, studies on probiotics have received increased attention (Raa, 1996; Li et al., 2007; Bachère et al., 1995; Balcázar et al., 2006). As stated above, the interaction between microbiota, including probiotics, and the host is not limited to the intestinal tract. Probiotic bacteria are not only active on the gills or skin of the host, but also in its ambient environment (Verschuere et al., 2000).
Acknowledgements This work was funded by grants from Special Scientific Research Fund of Agricultural Public Welfare Profession of China (nyhyzx07047) and National High Technology Research and Development Program of China (2006AA100304). The authors thank Wang Wei and Wang Shihuan of Zhangzidao Fisheries Company and Xia
Yuanzheng of Dalian University of Technology for the use of experimental materials and instruments.
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