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Effects of a fungicide on in vitro hemocyte viability, phagocytosis and attachment in the American oyster, Crassostrea virginica
Aquoculturr. 107 (1992) 135-140 Elsevier Science Publishers B.V., Amsterdam
135
Effects of a fungicide on in vitro hemocyte viability, phagocytosis and attachment in the American oyster, Crassostreu vir@nica Marvin R. Alvarez and Frank E. Fried1 Deparfmsn/ clBio/om.
1’nivcrsit.v of South Fiondo. Tampa. FL.. USA
ABSTRACT Alvwer, M.R. and Friedl. F.E., 1992. Effecis of a iungwide an in vitro hemoqe viability, phagocylosis and attachment in the American oyster, Crawx/reu vrrgrnica.Aqaacullure, 107: 135-140. The effect of a commercial fungicide containing Triforine as the active iwedient was tested in vilro for its effect on the ~iability;phagocytic ability and atlachmenl prop&es of hemocyles from Ihe Americatl oyster. Cra.vostrea virnlnica (Gmelin 1191). Cell viabilitv was determined by the hemocytes’ abilily~lo hydrolyze lluores&in dia&tate 10 prod& inIracellul& fluorescence.Phi&cytosis war evaluated with 2.pm fluorescent beads and cell attachment was dnennined by counting cells acmaiuing in suipnr;on in hemolymph after gentie rintrifugation onto P bead monolayer. Both cell \ iabJiry and phagccytic activity were reduced to approximately 50% ofcontrol levels wiihin a dose ranse of 0.0295 to 1.966 ug Triforine pet ml of hemolymph. Addition of the hiphest dose failed to further reduce viability or phagocytosis. Cell attacbmenl to bead surfaceswas i&rsely proportional IO the dose of fungicide. These data show lbat exposure of hemocyws to fungicide reducesthe cells’ ability 10 AI&lion m their normal capacity as immune cells.
INTRODUCTlON
The fungicide Triforine (N,N’-[ 1,4_piperazinediyl bis (2,2,2- trichloroethylidine] bis [ formamide ] ) is the active inpredient in certain commercial fungicides used to control various fungal dise2.e~ in horticultural and agricultural plants. The half life of Triforine in the soil is approximately 3 weeks and is mildly toxic to bees and tintish (Merck Index, 1989). Since it is sufficiently water soluble to be taken up systemically in plants and because it lingers in the soil, it is possible that it can enter estuarine environments in runoff. The effect of Triforine on shellfish is unknown. The hemocytes of molluscs appear to be their primary defense against invasion of the tissues by foreign particulates (Robohm, 1984). Thus, the efCorrespondence 10: MR. FL 33620-5150, USA.
0044-8486/92/$05.00
Alvarez, Department of Biology, University of South Florida, Tampa,
0 I992
Elsevier Science Publishers B.V. All rights reserved
M.R. ALVAREZ AND F.E.FRIEDL
136
fects of numerous exoaenous factors on the ability of hemocytes to bind. internalize and intracell;larly digest particulates have been examined (Fisher, 1988). In the present study we tested in vitro the effect ofa fungicide containing Triforine on the immune cells of the American oyster, Crasso~lreu virginica. The fungicide was tested for its cytotoxicity, inhibition of phagocytic uptake of particles and on adhesion of the hemocytes to particle surfaces. MATERIALS
AND
METHODS
Specimens of C. virginica were taken from a site in lower Old Tampa Bay (Alvarez et al., 1989) and kept in a circulating artificial seawater system at 20°C with salinity of 27 ppt. Hemocyte viabilityassay Hemolymph samples were taken from the pericardial cavity. Fungicide was added to the hemolvmuh to give nine different final concentrations of Trifor-
ine, ranging from &gO to 1.966 &ml of hemolymph. The cells were exposed for IO min after which 0.11 ml of a 5 mg/ml solution of fluorescein diacetate was added per ml of hemolymph andIncubated for an additional 15 min. The assay was done at room temperature. Samples of the cell suspension were placed on slides and the numbers of fluorescent and non-fluorescent cells were counted using simultaneous phase-contrast and fluorescence microscopy (Mishell et al., 1980). Five experimental replicates were run. Phagocytnsis assay
The bottom surfaces of 25ml plastic flasks were coated with cell-free hemolymph which was poured off to provide a sticky layer. Fluorescent polystyrene beads (2 pm diameter) were suspended in membrane-filtered 0.54 M NaCl at a concentration of 2x lo6 beads/ml and 2 ml were added to the flasks and gently centrifuged. This procedure gives a relatively uniform monolayer of beads attached to the flask surface after drying. Two ml of hemolymph, containing the same doses of Triforine used in the viability tests, were added to the flasks and gently centrifuged so that all of the suspended cells contacted the attached bead monolayer at approximately the same time. The cells remained in contact with the beads for 30 min after which they were detached by trypsinization (0.4% trypsin in 0.54 M NaCI)
for 5 min. The flask opening was fitted with a filter paper wick soaked in 37% formaldehyde and the cells were vapor-fixed for 30 min. Hemocytes containing one or more intracellular beads were scored as positive for phagocytosis using fluorescence microscopy. Five rcplica~esof each test were run and 1000 cells were scored for ezh dose.
FVNGlClDfEFFECTSONOYSTBR
HEMOcYrES
137
Hemocyte attachment assay Two ml of freshly drawn pericardial hemolymph containing the same dose levels of Triforine as in the previous assays were layered over a bead monolayer. After 30 min the hemolymph was removed and the cells remaining in suspension were counted using phase-contrast microscopy. RESULTS
The effect of various Triforine concentrations on the viability of hemocytes in hemolymph are shown in Fig. 1. The control value is given as the mean percent of cells showing I:ltracellular fluorescence in the absence of the fungicide. Three hundred cells were scored for each of the doses, including controls, and five replicates were run for each dose. Thus, each point represents the mean and standard deviations for 1500 scored cells. The results show an approximately 40% reduction in hemocyte viability when exposed to Triforine as compared to the untreated controls. A leveling out effect is seen at the highest dose level. No significant difference is noted between the last two points (Pt0.01).
The effect of the fungicide on the ability of those hemocytes which survive to phagocytize particles is seen in Fig. 2. In this graph, the dose response is shown as a function of percent of control (no fungicide present). Within the dose range tested, a nearly 50% reduction in the number of cells containing internalized fluorescent beads was seen. Again, no significant difference in the mean percent of cells which took up particles at the two highest doses was seen (PcO.01). indicating a leveling out of the effect of the fungicide at the
I.
Fig. Percent hemocyte viability as a function of fungicide dose. Each point representsthe mean and standard deviation of 1500 cells.
CL900
1.600 DOSE ( w/ml
1
Fig. 2. Hemocytes comajning one or more beads as a function of Triforine dose expressedas percent ofconirols. Each point representsthe mean and standard deviation of 5rx)Ocells.
_
.
1.500--
“0
x 2
5 8
0
soom
00007 0.000
I .ooo
0.500
I .500
2.000
OOSE&/ml)
unattached
Fig. 3. Number of hemacytes per ml of hernolymph as a iunction of Trifmine dose. Line fitted by method ofleasr squares,regressioncoeflicienl=0.S25.
highest dose level. This level of reduction in phagocytic activity is consistent with the percent of I emocytes which survived exposure to the toxin, indicating that the surviving hemocytes retain their ability to take up particles. The effect of the fungicide on the hemocytes’ ability to attach io the bead surfaces is shown in Fig. 3. The number of cells/ml which remained suspended in fungicide-treated hemolymph is shown as a function of dose. The number of unattached cells increased with dose but, as in the viability and phagocytosis experiments, the number leveled out at the highest dose. Indeed,
the attachment curves.
curve resembles an inverse of the viability and phagocytosis
DISCUSSION The hemocytes of molluscs appear to be their primary defense against invasion of the tissues by foreign particulates. For this reason this preliminary in vitro study was directed toward determining the effect of a water-soluble fungicide with the potential for entering estuarine environments on hemocytes of a marine bivalve. Any deleterious effects on the hemocytes increases the susceptibility of the orunisrn to phihogenic organisms. The present data show that exposure to Triforine over a 6%fold dose range is tidal to approximately one half of the hemocyte population in the circulating hemolymph. The reason for the failure of the highest dose to reduce viability to zero levels is not clear but may be related to decreased solubility of the funaicide at higher concentrations in the chemicallv comulex nlasma. Fluorescein dia&ate is commonly used as a vital stain in mammalian cell culture systems (Mishell et al., 1980). We veritied its effectiveness with bivalve hemocytes by treating hemocytes with various doses of iodoacetamide, a powerful inhibitor of glycolysis, and counting the number of fluorescent cells (Mahler and Cordes, 1966). Although these data are not shown for the sake of brevity, the respiratory inhibitor reduced the number of fluorescent hemocytes to zero, thus validating the use of this method for assessing the viability of molluscan hemocytes. Since exposure to the toxin killed only about half the hemocyte population, the question was asked do the remaining live cells retain their ability to phagocytize particles or are they damaged to the point where they are unable to cany out their primary function. Our data show that approximately half the hemocyte population retains the ability to internalize the fluorescent test particles. This proportion correlates well with the proportion of hemocytes which remain viable. Thus, it appears that cells which survive the tidal effect of the fungicide remain phagoeytically competent. Part& uptake is preceded by binding of the particle to the surface of the hemocyte, presumably through cell surface lectins (Fisher, 1990). Thus, inhibition of the binding process can prevent particle uptake even ifthe remainder of the uptake mechanism remains functional. The present data show that binding of hemocytes to the test particles is slightly inhibited by the fungicide. The shape of the inhibition curve appears almost as the reciprocal of the phagocytosis inhibition curve. Thus, it is possible that some ofthe inhibition of phagocytic uptake which was observed is due to inhibition of cell surface binding. From the present data we conclude that Triforine is somewhat cytotoxic to the immune cells of the oyster and that it inhibits slightly the binding of he-
140
M.R.AL”*aBZANDF.E.FRlEDL
mocytes to foreign particulates. However, a substantial fraction of the hemocyte population retains its ability to internalize particles. In viva experiments are needed to determine the effects ofthis substance in cczditions resembling those which might be encountered in the oyster’s natural environment.
REFERENCES Alvarez, M.R., Fried& F.E., Johnson, MS. and Hinsch, G.W., 1989. Factors affecting in vitro phagocytosis by oyster hemocytes. J. Invert. Path& 54: 233-241. Fisher. W.S.. 1988. Environmental influence on bivalve hemocvte fuaction. In: W.S. Fisher (Ediror),‘Disease Pmcesses in Marine Bivalve Molluscn. Am. Fish. Sot. Spec. Publ., 18: 225-237. Fisher, W.S., 1990. Nonself recogttitiott by lectins in semttt and mttcu~ of marine molluscs. In: International Colloquium on Patho!o# in Marine Aquaculture, Vol. 4, p. 137. Mahler, H.R. and Cordes, E.H., 1966. Biological Chemistry. Harper and ROH,New York. NY. 620 pp. Merck Index, 1989. Merck and Co., Rahway, NJ. Mishell, G.G., Shiigi, W.M., Henry, D., Chan, E.L., North, I., Gallily, R., Solmich, M., Miller, K., Marbrook, J., Parks, D. and Good, A.H., 1980. Preparation of ntouse cell suspensions. In: B.E. Mishell and W.M. Shiigi (Editors), Selected Methods in Immw-&agy. Freeman, San Francisco, CA, pp.3-27. Robohm, R.A., 1984. In vitro phagocytosis by molluscan hemocytes: a surve;~ and c.it;.we of methods. In: T.C. Cheng (Editor), Comparative Pathobiology, Vol. 6. Plenum, New York, NY, pp. 147-172.
135
Effects of a fungicide on in vitro hemocyte viability, phagocytosis and attachment in the American oyster, Crassostreu vir@nica Marvin R. Alvarez and Frank E. Fried1 Deparfmsn/ clBio/om.
1’nivcrsit.v of South Fiondo. Tampa. FL.. USA
ABSTRACT Alvwer, M.R. and Friedl. F.E., 1992. Effecis of a iungwide an in vitro hemoqe viability, phagocylosis and attachment in the American oyster, Crawx/reu vrrgrnica.Aqaacullure, 107: 135-140. The effect of a commercial fungicide containing Triforine as the active iwedient was tested in vilro for its effect on the ~iability;phagocytic ability and atlachmenl prop&es of hemocyles from Ihe Americatl oyster. Cra.vostrea virnlnica (Gmelin 1191). Cell viabilitv was determined by the hemocytes’ abilily~lo hydrolyze lluores&in dia&tate 10 prod& inIracellul& fluorescence.Phi&cytosis war evaluated with 2.pm fluorescent beads and cell attachment was dnennined by counting cells acmaiuing in suipnr;on in hemolymph after gentie rintrifugation onto P bead monolayer. Both cell \ iabJiry and phagccytic activity were reduced to approximately 50% ofcontrol levels wiihin a dose ranse of 0.0295 to 1.966 ug Triforine pet ml of hemolymph. Addition of the hiphest dose failed to further reduce viability or phagocytosis. Cell attacbmenl to bead surfaceswas i&rsely proportional IO the dose of fungicide. These data show lbat exposure of hemocyws to fungicide reducesthe cells’ ability 10 AI&lion m their normal capacity as immune cells.
INTRODUCTlON
The fungicide Triforine (N,N’-[ 1,4_piperazinediyl bis (2,2,2- trichloroethylidine] bis [ formamide ] ) is the active inpredient in certain commercial fungicides used to control various fungal dise2.e~ in horticultural and agricultural plants. The half life of Triforine in the soil is approximately 3 weeks and is mildly toxic to bees and tintish (Merck Index, 1989). Since it is sufficiently water soluble to be taken up systemically in plants and because it lingers in the soil, it is possible that it can enter estuarine environments in runoff. The effect of Triforine on shellfish is unknown. The hemocytes of molluscs appear to be their primary defense against invasion of the tissues by foreign particulates (Robohm, 1984). Thus, the efCorrespondence 10: MR. FL 33620-5150, USA.
0044-8486/92/$05.00
Alvarez, Department of Biology, University of South Florida, Tampa,
0 I992
Elsevier Science Publishers B.V. All rights reserved
M.R. ALVAREZ AND F.E.FRIEDL
136
fects of numerous exoaenous factors on the ability of hemocytes to bind. internalize and intracell;larly digest particulates have been examined (Fisher, 1988). In the present study we tested in vitro the effect ofa fungicide containing Triforine on the immune cells of the American oyster, Crasso~lreu virginica. The fungicide was tested for its cytotoxicity, inhibition of phagocytic uptake of particles and on adhesion of the hemocytes to particle surfaces. MATERIALS
AND
METHODS
Specimens of C. virginica were taken from a site in lower Old Tampa Bay (Alvarez et al., 1989) and kept in a circulating artificial seawater system at 20°C with salinity of 27 ppt. Hemocyte viabilityassay Hemolymph samples were taken from the pericardial cavity. Fungicide was added to the hemolvmuh to give nine different final concentrations of Trifor-
ine, ranging from &gO to 1.966 &ml of hemolymph. The cells were exposed for IO min after which 0.11 ml of a 5 mg/ml solution of fluorescein diacetate was added per ml of hemolymph andIncubated for an additional 15 min. The assay was done at room temperature. Samples of the cell suspension were placed on slides and the numbers of fluorescent and non-fluorescent cells were counted using simultaneous phase-contrast and fluorescence microscopy (Mishell et al., 1980). Five experimental replicates were run. Phagocytnsis assay
The bottom surfaces of 25ml plastic flasks were coated with cell-free hemolymph which was poured off to provide a sticky layer. Fluorescent polystyrene beads (2 pm diameter) were suspended in membrane-filtered 0.54 M NaCl at a concentration of 2x lo6 beads/ml and 2 ml were added to the flasks and gently centrifuged. This procedure gives a relatively uniform monolayer of beads attached to the flask surface after drying. Two ml of hemolymph, containing the same doses of Triforine used in the viability tests, were added to the flasks and gently centrifuged so that all of the suspended cells contacted the attached bead monolayer at approximately the same time. The cells remained in contact with the beads for 30 min after which they were detached by trypsinization (0.4% trypsin in 0.54 M NaCI)
for 5 min. The flask opening was fitted with a filter paper wick soaked in 37% formaldehyde and the cells were vapor-fixed for 30 min. Hemocytes containing one or more intracellular beads were scored as positive for phagocytosis using fluorescence microscopy. Five rcplica~esof each test were run and 1000 cells were scored for ezh dose.
FVNGlClDfEFFECTSONOYSTBR
HEMOcYrES
137
Hemocyte attachment assay Two ml of freshly drawn pericardial hemolymph containing the same dose levels of Triforine as in the previous assays were layered over a bead monolayer. After 30 min the hemolymph was removed and the cells remaining in suspension were counted using phase-contrast microscopy. RESULTS
The effect of various Triforine concentrations on the viability of hemocytes in hemolymph are shown in Fig. 1. The control value is given as the mean percent of cells showing I:ltracellular fluorescence in the absence of the fungicide. Three hundred cells were scored for each of the doses, including controls, and five replicates were run for each dose. Thus, each point represents the mean and standard deviations for 1500 scored cells. The results show an approximately 40% reduction in hemocyte viability when exposed to Triforine as compared to the untreated controls. A leveling out effect is seen at the highest dose level. No significant difference is noted between the last two points (Pt0.01).
The effect of the fungicide on the ability of those hemocytes which survive to phagocytize particles is seen in Fig. 2. In this graph, the dose response is shown as a function of percent of control (no fungicide present). Within the dose range tested, a nearly 50% reduction in the number of cells containing internalized fluorescent beads was seen. Again, no significant difference in the mean percent of cells which took up particles at the two highest doses was seen (PcO.01). indicating a leveling out of the effect of the fungicide at the
I.
Fig. Percent hemocyte viability as a function of fungicide dose. Each point representsthe mean and standard deviation of 1500 cells.
CL900
1.600 DOSE ( w/ml
1
Fig. 2. Hemocytes comajning one or more beads as a function of Triforine dose expressedas percent ofconirols. Each point representsthe mean and standard deviation of 5rx)Ocells.
_
.
1.500--
“0
x 2
5 8
0
soom
00007 0.000
I .ooo
0.500
I .500
2.000
OOSE&/ml)
unattached
Fig. 3. Number of hemacytes per ml of hernolymph as a iunction of Trifmine dose. Line fitted by method ofleasr squares,regressioncoeflicienl=0.S25.
highest dose level. This level of reduction in phagocytic activity is consistent with the percent of I emocytes which survived exposure to the toxin, indicating that the surviving hemocytes retain their ability to take up particles. The effect of the fungicide on the hemocytes’ ability to attach io the bead surfaces is shown in Fig. 3. The number of cells/ml which remained suspended in fungicide-treated hemolymph is shown as a function of dose. The number of unattached cells increased with dose but, as in the viability and phagocytosis experiments, the number leveled out at the highest dose. Indeed,
the attachment curves.
curve resembles an inverse of the viability and phagocytosis
DISCUSSION The hemocytes of molluscs appear to be their primary defense against invasion of the tissues by foreign particulates. For this reason this preliminary in vitro study was directed toward determining the effect of a water-soluble fungicide with the potential for entering estuarine environments on hemocytes of a marine bivalve. Any deleterious effects on the hemocytes increases the susceptibility of the orunisrn to phihogenic organisms. The present data show that exposure to Triforine over a 6%fold dose range is tidal to approximately one half of the hemocyte population in the circulating hemolymph. The reason for the failure of the highest dose to reduce viability to zero levels is not clear but may be related to decreased solubility of the funaicide at higher concentrations in the chemicallv comulex nlasma. Fluorescein dia&ate is commonly used as a vital stain in mammalian cell culture systems (Mishell et al., 1980). We veritied its effectiveness with bivalve hemocytes by treating hemocytes with various doses of iodoacetamide, a powerful inhibitor of glycolysis, and counting the number of fluorescent cells (Mahler and Cordes, 1966). Although these data are not shown for the sake of brevity, the respiratory inhibitor reduced the number of fluorescent hemocytes to zero, thus validating the use of this method for assessing the viability of molluscan hemocytes. Since exposure to the toxin killed only about half the hemocyte population, the question was asked do the remaining live cells retain their ability to phagocytize particles or are they damaged to the point where they are unable to cany out their primary function. Our data show that approximately half the hemocyte population retains the ability to internalize the fluorescent test particles. This proportion correlates well with the proportion of hemocytes which remain viable. Thus, it appears that cells which survive the tidal effect of the fungicide remain phagoeytically competent. Part& uptake is preceded by binding of the particle to the surface of the hemocyte, presumably through cell surface lectins (Fisher, 1990). Thus, inhibition of the binding process can prevent particle uptake even ifthe remainder of the uptake mechanism remains functional. The present data show that binding of hemocytes to the test particles is slightly inhibited by the fungicide. The shape of the inhibition curve appears almost as the reciprocal of the phagocytosis inhibition curve. Thus, it is possible that some ofthe inhibition of phagocytic uptake which was observed is due to inhibition of cell surface binding. From the present data we conclude that Triforine is somewhat cytotoxic to the immune cells of the oyster and that it inhibits slightly the binding of he-
140
M.R.AL”*aBZANDF.E.FRlEDL
mocytes to foreign particulates. However, a substantial fraction of the hemocyte population retains its ability to internalize particles. In viva experiments are needed to determine the effects ofthis substance in cczditions resembling those which might be encountered in the oyster’s natural environment.
REFERENCES Alvarez, M.R., Fried& F.E., Johnson, MS. and Hinsch, G.W., 1989. Factors affecting in vitro phagocytosis by oyster hemocytes. J. Invert. Path& 54: 233-241. Fisher. W.S.. 1988. Environmental influence on bivalve hemocvte fuaction. In: W.S. Fisher (Ediror),‘Disease Pmcesses in Marine Bivalve Molluscn. Am. Fish. Sot. Spec. Publ., 18: 225-237. Fisher, W.S., 1990. Nonself recogttitiott by lectins in semttt and mttcu~ of marine molluscs. In: International Colloquium on Patho!o# in Marine Aquaculture, Vol. 4, p. 137. Mahler, H.R. and Cordes, E.H., 1966. Biological Chemistry. Harper and ROH,New York. NY. 620 pp. Merck Index, 1989. Merck and Co., Rahway, NJ. Mishell, G.G., Shiigi, W.M., Henry, D., Chan, E.L., North, I., Gallily, R., Solmich, M., Miller, K., Marbrook, J., Parks, D. and Good, A.H., 1980. Preparation of ntouse cell suspensions. In: B.E. Mishell and W.M. Shiigi (Editors), Selected Methods in Immw-&agy. Freeman, San Francisco, CA, pp.3-27. Robohm, R.A., 1984. In vitro phagocytosis by molluscan hemocytes: a surve;~ and c.it;.we of methods. In: T.C. Cheng (Editor), Comparative Pathobiology, Vol. 6. Plenum, New York, NY, pp. 147-172.