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A prodiginine pigment toxic to embryos and larvae of Crassostrea virginica
JOURNAL
OF
INVERTEBRATE
PATHOLOGY
A Prodiginine
38, 281-293 (1981)
Pigment Toxic Crassostrea
to Embryos virginica
CAROLYN Nufiontrl
Ocuuttic
crttd
Atmospheric Center.
Administration. Milford Luhorutory.
and Larvae
of
BROWN
Notional Milford.
Marine Fisheries Srrvicr. Cottttrctictrt 06460
Northetrst
Fisherie.\
Received October 27. 1980 The red pigment produced by a marine Psetrdomonu sp. which causes abnormal development and mortality in developing embryos of the American oyster, Crusso~trru virginicu, was analyzed. A comparative study of a nonpigmented and two pigmented mutants of the red parental strain indicated that virulence was associated and varied with pigmentation. The use of sonicated cells supported lysing of the pseudomonad cells as the most probable means of pigment release. Crude pigment extracted from the red parental strain and its yellow mutant was toxic to developing oyster embryos. Neither the “pigment” extracted from the white mutant nor dimethyl sulfoxide, used for dissolving the extract, was toxic. Three pigment fractions were demonstrated by thin-layer chromatography even after purification. Studies indicate that only the fraction corresponding to R, 0.41 was necessary for virulence. The virulent pigment fraction was identified as belonging to the prodiginine group. KEY WORDS: Crrrssostreu virginicu; American oyster: Pseudomortus sp.; toxic pigment: prodiginine.
INTRODUCTION
Prodigiosin and prodiginine pigments are known to be antibacterial, antifungal, and antiprotozoan (see Williams and Heart-r, 1967; Gerber, 1975, for review). Attempts to take advantage of these potentially beneficial properties have been unsuccessful due to the toxic nature of the pigment. Thompson et al. (1956) noted a relationship between antiprotozoan activity of prodigiosin and toxicity in both rats and hamsters. Prodigiosin has been tested as a means for treating coccidioidomycosis in man, but pain and tenderness are associated with the intramuscular injections (Wier et al., 1952). Kalesperis et al. (1975) found prodigiosin to be toxic to chick embryonic development, resulting in abnormal development and death. Prodigiosin and prodiginine pigments have a wide distribution in the microbial world. Prodiginine pigments are produced by some members of the genera Streptomyces (see Gerber, 1975) and Actinomadura (see Gerber, 1973) and by Streptoverticillium ruhrireticuli (see Gerber and Stahly, 1975). Serratia marccscens has long
been known to produce prodigiosin. Lewis and Corpe (1964) reported that it also is produced by two unidentified strains of marine bacteria. Two other marine bacterial species, Vibrio psychroerythcus (see D’Aoust and Gerber, 1974) and Pseudomonas magnesiorubru (see Gandhi et al., 1976) were later found to produce this pigment. Brown (1974) showed that a red marine pseudomonad is pathogenic to laboratoryreared embryos of the hard shell clam, Mercenaria mercenaria, and the American oyster, Crassostrea virginica. The presence of this bacterium in sea water of shellfish hatcheries and laboratories is easily discernible by the pink discoloration it causes in culture containers and conditioning trays. The pigment produced by the red Pseudomonas sp. was thought to be prodigiosin or a prodiginine pigment. The bright red color of the pigment, as that of prodigiosin and prodiginine pigments, was pH dependent; it was red under acid conditions and yellow under alkaline conditions. It was capable of discoloring polyvinyl chloride as are the prodiginine pigments pro281 0022-2011/81/050281-13$01.0010 Copyright (61 1981 by Academic Precs. Inc All rights of reproduction in any form rescrxed
282
CAROLYN
duced by Streptoverticillium rubrireticuli (see Gerber and Stably, 1975). The purpose of the present study was to analyze the pigment of the red Pseudomonas sp. to determine whether it was toxic to oyster embryonic development and if the pigment belonged to the prodiginine group. MATERIALS
AND METHODS
Bacteria
The red Pseudomonas sp. was originally isolated from a moribund larval culture of the hard shell clam, Mercenaria mercenaria (see Brown, 1974). The mutants were obtained from sea water agar plates (Brown and Losee, 1978) inoculated with ultraviolet (uv) light-irradiated cells of the red pseudomonad. The procedures described by Brown (1974) were used to determine the morphological and physiological characteristics of the mutants. Sonicated
Cells
The red pseudomonad was inoculated into 7.5 ml of seawater broth and incubated overnight at 26°C. The culture was used to seed 1 liter of sea water broth in a 2.5liter Fembach flask. The culture was incubated overnight at 26°C and cells were spun down at 33008 for 30 min. The cells were sonicated, following a procedure used by Dr. R. A. Robohm of the Milford Laboratory (pers. commun.). The cells were washed twice with sterile sea water; 0.5 ml of washed cells was mixed with 3.5 ml of sterile sea water, and washed glasperlin glass beads (2 g/4 ml) were added. The cells were sonicated 3-4 min at maximum probe intensity. The broken cells were removed and diluted 1:lO with sterile sea water. A sample of the suspension was plated onto sea water agar plates to determine if all cells were broken. Pigment
Production
The Pseudomonas sp. and its mutants were grown at 26°C overnight in 7.5 ml of sea water broth. The cultures were spread
BROWN
over the surface of 1.5 liters of nutrient agar (Difco)’ made with a solution of 80.0% aged, membrane-filtered sea water and 20% distilled water in 2.5-liter Fernbach flasks. The cultures were incubated overnight at 26°C. Cells were washed off agar surfaces with sterile sea water. The cells from each culture were spun down at 33009 for 30 min. The supernatant fluid was decanted off and cells were resuspended in acetone. Acetone extraction was allowed to proceed for at least 1 hr; then the suspension was spun down at 3300g for 30 min and the supematant fluid filtered. Pigment was extracted from the filtrate according to procedures described by Williams et al. (1956). Acetone-pigment solution was mixed with petroleum ether; the acetone was removed by adding distilled water and drawing off the acetone-water phase. Petroleum ether was evaporated off in vacua at 35”C, using a rotary evaporator. Saturated solutions of the dry pigments in dimethyl sulfoxide (DMSO) were made and diluted 1:lO in sterile distilled water. Pigment
Purification
Procedures described above were used to extract pigment from the red parental strain. The pigment was purified, following a procedure reported by Wrede and Hettche (1929). The petroleum ether-pigment solution was concentrated to about 50 ml over which dry HCl gas was conveyed until the hydrochloride precipitated. The precipitate was allowed to stand and was filtered. The hydrochloride was dissolved in 100 ml of 96% ethyl alcohol while heating, was filtered, and then mixed with 5% perchloric acid until the solution became turbid. After precipitation the perchlorate was filtered, dissolved in 100 ml of warm ethyl alcohol, and followed by mixing with enough 10% sodium hydroxide until the solution turned to a yellowish-brown color. The solution was allowed to stand for 1 hr ’ Reference to trade names in this paper does not imply endorsement of commercial products by the National Marine Fisheries Service.
PIGMENT
TOXIC
TO
and was then filtered. The filtrate was mixed with 30 ml of chloroform and 100 ml of distilled water. The chloroform layer, washed several times with distilled water to remove any residual alcohol, was filtered and evaporated under vacuum. A saturated solution of the pigment in DMSO was made and then diluted I:10 in sterile distilled water. Pigment
Fractions
A saturated pigment -chloroform solution was prepared from the crude and purified pigment extracts of the red pseudomonad. Fifty-microliter aliquots of the solutions were spotted on OS-mm-thick Silica Gel-G chromatoplates (Supelco). The plates were developed in benzene-ethyl acetate solution (1: 1); the different fractions were slurried in DMSO and filtered. Each dissolved fraction was diluted 1: 10 in sterile distilled water. Spawning
of Oysters
The spawning techniques of Loosanoff and Davis (1963) were modified according to the procedure described by Brown and Russo (1979). The adult oysters were spawned in lo-pm-filtered, UV-irradiated sea water. Approximately 15,000 fertilized oyster eggs were added to each 1.3-liter polypropylene beaker filled to the l-liter mark with lo-pm-filtered, UV-treated sea water. Larval cultures were sampled after 48 hr. Larvae which had developed the standard “D” shape shell were considered normal. It also was noted whether the larvae were living or dead prior to being fixed (Brown and Russo, 1979). Assays to Determine and Toxicity
Pathogenicity
Assays were conducted to determine whether the development of fertilized oyster eggs was affected by the presence of the following: bacterial broth cultures of the pink. white, and yellow mutants of the red pseudomonad; sonicated cells of the red pseudomonad: crude pigment extracts of
OYSTER
283
EMBRYOS
the pseudomonad and its white and yellow mutants; and purified extract of the pigment of the red pseudomonad. Each series of experiments was repeated four times, using quadruplicate beakers each time. Broth cultures. The red pseudomonad parental strain and the three pigment mutants were grown at 26°C for 24 hr in sea water broths. A OS-ml aliquot of cultures of each of the four bacterial strains was used to determine its effect on the development of fertilized oyster eggs. The inocula were mixed with the embryonic culture water just prior to the addition of the fertilized eggs. Sonicated cells. A lo-ml sample of the diluted suspension of broken red cells was mixed with embryonic culture water prior to the addition of the fertilized eggs. Pigment extract. Each of the diluted solutions of pigments, fractions of crude and purified extracts of the red pigment, and DMSO were mixed with culture water, using l-ml inocula, and fertilized oyster eggs added. The amount of extract used was measured spectrophotometrically at 535 nm. Characterization
of Pigment
Results obtained from thin-layer chromatography, spectrophotometry, and solubility studies for the red pigment of Pseudomonas sp. were compared with prodigiosin extracted from Serratia murcestens (ATCC 13880) to determine whether the pigment produced by the pseudomonad was a prodiginine. The same procedures described earlier for growing the pseudomonad and extracting pigment were employed for S. marcescens, except distilled water was used in preparing medium and washing cells. Thin-layer chromatography (tfc). A saturated pigment-chloroform solution was prepared for each pigment extract. Fifty microliters of each solution was spotted on chromatoplates. Plates were developed in 100 ml of a benzene-ethyl acetate solution (1:l). The spots were examined under UV
284
CAROLYN
light and sunlight, mined.
and Rf values
BROWN
deter-
Spectrophotometry. Each dried pigment extract was dissolved in 95% ethyl alcohol. The amount was adjusted so that absorbance reading was the same for each dissolved pigment at 510 nm under acid condition. Spectral readings were recorded over a range of 400-600 nm. The readings were taken under neutral, acid (pH 5), and alkaline (pH 8) conditions using 0.1 N HCl and 0.1 N NaOH to adjust pH. Solubility. Solubility was tested by adding a portion of each dried pigment extract to each of the following solvents: petroleum ether, benzene, chloroform, acetone, ethyl alcohol, methyl alcohol, amyl alcohol, dimethyl sulfoxide, sulfanilic acid, potassium hydroxide, sodium hydroxide, sea water, and distilled water. RESULTS Exposures of Developing with Bacteria
Larvae
Plates inoculated with UV-irradiated aliquots of the red pseudomonad suspended in sterile sea water yielded three types of pigment mutants. They were white, pink, and yellow. Development of fertilized oyster eggs exposed to approximately 1.4 x lo5 white mutant cells/ml of embryonic culture water was comparable to that of the untreated controls. Live-normal development of the fertilized eggs to the straight-hinge stage averaged 62.9% in cultures exposed to the white mutant and 62.2% in the untreated control beakers (Table 1). Student’s t test, however, did reveal a significant difference (P < 0.01) between the development of oyster eggs exposed either to the red parental strain or the pink mutant and that of the control cultures. Live-normal development to the straight-hinge stage averaged 18.1% in the presence of the red pseudomonad (8.4 x lo4 cells/ml) and 28.9% in beakers seeded with the pink mutant (1.4 x lo5 cells/ml). It was also noted that abnormal development was significantly higher in larval cultures exposed either to the red parental strain or pink
WI0 mm
ww --
‘0
ww --
PIGMENT
TOXIC
TO OYSTER
mutant as compared with that found in untreated controls. Conversely, total development was significantly lower in experimental cultures than in untreated controls. Table 1 also shows that the yellow mutant had a detrimental effect on development of fertilized oyster eggs. Live-normal development averaged only 19.3% in embryonic cultures exposed to the yellow mutant (1.3 x lo5 cells/ml) and 25.7% in those exposed to the red parental strain (8.6 x lo4 cells/ml). These percentages were significantly less than that found in the untreated controls, 81.1%. Averaged live-abnormal development also was significantly higher in the experimental cultures than in the untreated controls. Total development again was found to be significantly lower in larval cultures exposed to either bacterial strain than in untreated controls. Figure IA-D illustrates the morphological differences in oyster larvae after 48-hr exposure to the four bacterial strains. While the white mutant had no appreciable effect on the developing larvae (Fig. 1A), the three pigmented isolates showed varying degrees of abnormal shell formation and growth retardation which were most pronounced in the presence of the red parental strain. Larvae exposed to the pink mutant tended to be what Loosanoff and Davis larvae”; the ( 1963) termed “saddleback hinge line was concave rather than straight (Fig. IS, arrow). Larvae exposed to the pink mutant averaged 70 pm in length, compared to 75 pm in the presence of the white mutant and in untreated controls. Shell formation and growth were greatly retarded in the presence of the yellow mutant (Fig. 1C) and the red parental strain (Fig. 1D). Size averaged only about 35 pm in the presence of the red parental strain and 45 km during exposures to the yellow mutant. Exposures of Developing Sonicated Cells
Larvae
with
Bacteriological plates demonstrated that no viable cells were present after sonication. Preliminary experiments showed that
EMBRYOS
285
2.4 x lo6 sonicated cells/ml culture water resulted in only 54.9% live-normal larvae, compared to 86.9% in untreated controls. Normal development continued to decrease, while abnormal development increased as the number of sonicated cells increased. Table 2 shows that an average of only 12.7% live-normal development of fertilized eggs occurred in the presence of 4.8 x lo6 sonicated cells/ml culture water, while development in the untreated controls averaged 81.4%. Abnormal development was also significantly higher in cultures with sonicated cells than in control beakers. Table 2 also shows that total development was significantly lower in larval cultures exposed to sonicated cells (60.4%) than in control larval cultures (92.3%). Exposures of Developing with Pigments
Larr’ae
Table 3 presents the data obtained following exposures of fertilized oyster eggs to pigment extracted from the red pseudomonad (4.8 x 106 cells/ml) and its yellow (1.3 x 10’ cells/ml) and white (1.4 x lo7 cells/ml) mutants. The DMSO controls, in each study, consistently showed development comparable to that of the untreated controls. The pigment extracted from the red pseudomonad (absorbance equaled 0.220 at 535 nm) was extremely toxic, resulting in normal development of only 27.9%. Development in the untreated controls, on the other hand, was 74.9 and 75.0% in the presence of DMSO. Fertilized oyster eggs were also adversely affected by exposure to the yellow pigment (absorbance equaled 0.180 at 535 nm). Livenormal development was only 46.2% in the presence of the yellow pigment and 68.3% in the untreated controls. The “pigment” extracted from the white mutant, however, had no significant effect on the development of fertilized oyster eggs. There was a 76.2% normal development to the veliger stage when eggs were exposed to this “pigment” (absorbance equaled 0.000 at 535 nm), compared to 74.2% in the untreated controls. Percentage total develop-
286
CAROLYN
FIG. yellow
BROWN
1. Forty-eight-hour oyster larvae after exposure mutant, and (D) red parental strain. Magnification:
ment was significantly reduced by the presence of either the red or yellow pigment as compared with the control cultures. The above experiments suggest that the pigment is responsible for the retardation of normal embryonic development. To substantiate this, purified red pigment was used. Approximately 3.4 mg of the purified
to (A) 90x.
white
mutant,
(B) pink
mutant,
(C)
red pigment was mixed with the culture water in each experimental beaker. Bacteriological studies showed that about 8.7 x log cells were needed to obtain this amount of pigment. As shown in Table 4, livenormal development of oyster eggs to the straight-hinge stage was significantly different between the untreated controls (72.6%)
PIGMENT
PERCENTAGE
TOXIC
287
TO OYSTER EMBRYOS
TABLE 2 OF FERTILIZED OYSTER Ec;c;s EXPOSED TD SONICATED CELLS OF RED Psrudomorzu.s sp. DEVELOPMENT
Treatment Sonicated cells (4.8 x lO”/ml culture water) Number of replicates Live-normal (4 2 SE”) Dead-normal (.? 2 SE) Live-abnormal (4 + SE) Dead-abnormal (X i SE) Total (.U t- SE)
16 12.7 t l4.1* 4.4 t 5.2 34.2 t 17.2* 9.0 t 5.8* 60.4 i- 22.8*
Control 16
c t k
8.2 0.8
92.3 t
4.2
81.4 1.6 8.6 0.7
8.3 IO.4
” Standard error at 99% confidence interval. * Significantly different from control (P < 0.01).
and the cultures with purified red pigment (36.6%). There was no significant difference between the untreated controls and the DMSO controls (71.4%). The averaged percentages for dead-normal and deadabnormal also were higher in the presence of the purified red pigment than in the control culture. As had been demonstrated in the preceding exposures, the presence of the purified pigment resulted in a significant decrease in total development. Toxicity of Various Fractions of the Red Pigment
A comparative chromatographic study of the crude and purified red pigment was conducted and the fraction(s) responsible for the toxic effect determined. Thin-layer chromatography of the crude red pigment (Fig. 2) revealed five spots: yellow (R, 0.971, pink (I?, 0.68), orange (Rl 0..55), pink (I?, 0.41), and purple (R, 0.00). After recovery from the chromatograms, only the orange (Rr 0.55) and pink (R, 0.41) fractions were able to affect adversely the development of fertilized oyster eggs. After purification, only three of the five spots were still present on tic plates: orange (Rf 0.55), pink (Rf 0.40), and purple (R/ 0.00). As shown in Table 5. the orange and pink fractions continued to be toxic. There was a significant difference between live-normal development in the presence of either the pink
fraction (58.8%) or the orange fraction (49.3%) and the controls (71.3%). Liveabnormal development was greater in the presence of either one of the two fractions than in the control beakers. Total development, however, was significantly lower only in the presence of the orange fraction. Pigment Characterization
Comparisons made between prodigiosin extracted from Serratia marcescens and the pigment extracted from the red Pseudomonas sp. indicate that the latter also belongs to the group called prodigiosinlike or prodiginine. Figure 2 illustrates the five spots that developed from the two crude pigment extracts on the plates. The R, value for each spot developed from the pigment of the red pseudomonad was identical to the corresponding spot developed from prodigiosin. The folIowing spots, in ascending order, were visible: purple (Rf 0.00). pink (Rrl 0.41). orange (R, 0.55). pink (RI 0.68), and yellow (R, 0.97). The spots corresponding to Rf 0.41 and R, 0.55 were the major ones. Three of the five spots, purple (RI O.OO), pink (R, 0.40), and orange (Rf 0.55), also were visible when purified preparations were spotted on 0.5-mm thick Silica Gel-G plates (Supelco). Only the purple spot and a pinkish-orange spot (R, 0.20) were developed, however, on 0.25-mm Silica Gel 6OF-254 plates (E. Merck).
288
CAROLYN
BROWN
The maximum absorbance was at 535 nm under neutral and acid conditions (Fig. 3A and B, respectively) but shifted to 470 nm under alkaline condition for pigment extracted from both Pseudomonas sp. and S. marcescens (Fig. 3C). The minimum absorbance at 600 nm was the same for the two pigments. A shoulder was visible at about 510 nm under acid and neutral conditions. Figure 3A and B shows that there was also a shoulder at 580 nm; under alkaline condition, this shoulder started to peak (Fig. 3C). The peak at 580 nm, however, was more prominent for prodigiosin than for the red pigment. The pigment extracts of S. marcescens and Pseudomonas sp. were identical in their ability to be dissolved in various solvents. The dried pigment extracts were insoluble in sea water and distilled water. Both were moderately soluble in sulfanilic acid, potassium hydroxide, and sodium hydroxide. The two pigments were extremely soluble in all the organic solvents tested. DISCUSSION
www mm-
*ww ---
www m-m
Data collected from the spectrophotometric, chromatographic, and solubility studies indicate that the pigment produced by the red Pseudomonas sp. belongs to the group called prodiginine. The maximum spectrophotometric peak of the red pigment in acidified ethanol fell within the range for prodigiosin and prodiginine pigments. Gerber (1973) reported that prodiginine pigments, in acidified ethanol, have a maximum absorption range from 542 to 525 nm. Prodigiosin extracted from Serratia marcescens has a maximum absorption at 537 nm under the same conditions. Figure 2 shows that the maximum absorption, in the present study, was about 535 nm for both prodigiosin and the red pigment. Lewis and Corpe (1964) also indicated that prodigiosin extracted from S. marcescens has a maximum peak at 535 nm under acid and neutral conditions. The shift of the maximum peak from 535 to 470 nm under alkaline conditions is also in agreement
PIGMENT
PERCENTAGE
TOXIC
DEVELOPMENT PURIFIED
TO
OYSTER
TABLE
4
289
EMBRYOS
OYSTER Ecus RED PSEUDOMONAD
OF FERTILIZED PIGMENT
OF
EXPOSED
Treatment Purified red pigment Number of replicates Live-normal (4 r SE”) Dead-normal (.i 2 SE) Live-abnormal (4 + SE) Dead-abnormal (h 2 SE) Total (E t- SE)
36.6 1.2 4.9 2.7 51.4
DMSO
16 + 17.9* k 7.0* k 3.2 t 2.1* t 19.6*
71.4 2.8 1.9 1.7 78.8
16 + 11.6 ‘- 2.8 t 1.6 i_ 1.3 + 9.0
‘TO
____-Control 12.6 2.2 2.8 1.2 78.8
16 -+ 11.7 i- 2.0 + 1.7 -t 1.2 i 9.6
-.
-
w Standard error at 99% confidence interval. * Significantly different from control (P < 0.01).
with the findings of Lewis and Corpe (1964). Differences existed between the present study and the one conducted by Lewis and Corpe; minimum absorption is at 420 nm and the shoulder at 580 nm is not visible in the latter study.
FK. 2. Thin-layer chromatographic separation of prodigiosin from S. mttrrrscens and the red pigment from the shellfish pathogenic pseudomonad. Stationary phase: Silica Gel-G. OS-mm thick. Solvent: Benzene-ethyl acetate solution (1:I).
Chromatographic study showed the same five spots for crude extracts of prodigiosin and the red pigment, and the same three spots for the purified pigments. Gandhi et al. (1976) reported that prodigiosin extracted from Pseudomonus magnesiorubra runs as a single spot on silica gel plates developed in benzene-ethyl acetate. This discrepancy may have been due to the use of different commercial plates in the two studies. Different results were obtained in the present study by using two different brands of silica gel plates. Discrepancies can also be due to differences in the age of the cultures, length of storage, method of extraction, and handling of the pigments (Lewis and Corpe, 1964). Gerber (1973) stated that prodigiosin-like pigments are soluble in nonpolar solvents and in polar solvents, such as acetone and chloroform. Kalesperis et al. (1975) reported that prodigiosin is also soluble in dimethyl sulfoxide. The present study showed that the extracted red pigment and prodigiosin were very soluble in the organic solvents tested. Both pigments were insoluble in sea water and distilled water. The agreement in soiubility for the two pigment extracts supports the findings of the spectrophotometric and chromatographic studies, i.e., the red pigment is prodiginine. The shellfish pathogenic pseudomonad differed from other prodigiosin-producing, marine pseudomonads. e.g., Pseudomonas
290
CAROLYN
BROWN
TABLE PERCENTAGE
DEVELOPMENT
OF FERTILIZED
5
OYSTER
OF PURIFIED
RED
EGGS
EXPOSED
TO Two
COMPONENTS
PIGMENT
Treatment Control Number of replicates Live-normal (X + SE“) Dead-normal (X -r- SE) Live-abnormal (X t SE) Dead-abnormal (I _’ SE) Total (X 2 SE)
71.3 2.6 4.8 0.4 79.1
16 + 5 t k 2
3.7 1.2 3.6 0.4 4.9
DMSO 72.5 2.1 4.3 0.7 79.5
16 ” + zt t f
4.0 0.8 2.9 0.6 4.8
Pink (& 0.40) 58.8 2.6 10.8 0.4 72.6
16 2 7.8” 2 1.4 + 6.8* _’ 0.5 k 6.2
Orange (R, 0.55) 49.3 2.6 12.9 1.0 65.8
16 I 12.7* k 1.2 5 7.3* Ik 1.3 c 7.0*
” Standard error at 99% confidence interval. * Significantly different from control (P < 0.01).
magnesiorubra, in that it has a lower salt
tolerance and was unable to reduce nitrates to nitrites or produce acid in galactose, maltose, or mannitol. The pathogen also differed from the two marine isolates reported by Lewis and Corpe (1964); it produced acid in sucrose rather than in trehalose and grew in media prepared with distilled water supplemented with only sodium chloride and magnesium sulfate. Lewis and Corpe (1964) found that one of the bacterial isolates in their study requires potassium, in addition to sodium and magnesium ions, for growth. The authors reported that calcium ions are required for pigment production. The red shellfish pathogen also required calcium ions for pigment production. Stiles and Longwell (1973) studied irregularities of meiosis and early cleavage in laboratory-spawned American oysters. These irregularities offer some explanation as to why, under artificial conditions, total development of fertilized oyster eggs rarely equals 100% when calculating actual percentage development. Because some eggs invariably break down soon after fertilization, they cannot be detected when larval samples are counted. In the present study, total development in the untreated controls ranged from 76.9 to 92.3%; while livenormal development ranged from 62.2 to 81.4%. The results showed that both livenormal and total developments were significantly lower in cultures exposed to the
red parental strain or its pigment than in untreated control cultures. Exposures to the white mutant, its “pigment,” or DMSO had no such effect; development was basically the same as in the untreated controls. These results demonstrate that the pigment produced by the red parental strain is indeed toxic to oyster embryonic development. The suggestion that the pigmented isolates contain varying amounts of the toxic components and the white mutant is missing them entirely is supported by the results of the thin-layer chromatography studies. Material extracted from the white mutant produced no visible spots on tic plates. The pink mutant contained four of the five spots found in the red pseudomonad; it lacked the spot corresponding to Rf 0.67. Two spots with Rf values corresponding to 0.41 and 0.97 were found in the pigment extracted from the yellow mutant. The data suggested that to be virulent a strain or mutant must contain a pigment fraction having an Rf value of 0.41. Although dimethyl sulfoxide, at the concentration used (1:10), was nontoxic to developing oyster embryos, its use had two serious drawbacks: (1) it could not explain how a non-water-soluble pigment is released to affect oyster embryonic development, and (2) as a penetrant carrier (Jacob et al., 1964), the possibility existed that it enhanced absorption of pigment across the cell membrane of the developing embryos,
PIGMENT
TOXIC
TO
OYSTER
291
EMBRYOS
30$ ;t; 2 5 2
2b
_----_ lo-
WAVELENGTH
FIG. 3. Spectrophotometric conditions: (A) neutral (l:lO),
(NM1
WAVELENGTH
lNM1
WAVELENGTH
(NM1
curves of prodigiosin and the red pigment (B) acid (l:lO), and (C) alkaline (1:5).
in ethanol
under
the following
292
CAROLYN
thus causing the adverse effect. The use of sonicated cells more naturally demonstrated what happens. Taylor and Williams (1959) reported that mechanical disruption of Serratia marcescens renders prodigiosin “water-soluble”; the pigment is bound to particulate matter. It was noted that in infected larval cultures red pseudomonad cells could not be recovered from larvae or culture water 24 hr after onset of the disease. The phenomenon suggests that bacterial cells begin to die before any overt evidence of a disease occurs. Particulatebound pigment probably was released when cells of the red pseudomonad were lysed. Particulate matter tended to accumulate around the edges of the larval culture containers at the air-water intersurface; thus, this area was discolored. An earlier study (Brown, 1974) showed that more than lo3 actively dividing cells/ml culture water have to be added to elicit a toxic effect. The present study, using sonicated cells, showed that the number of cells actually required was about 4.8 x IO6 cells/ml, which was equivalent to adding 3.4 yg pigment/ml of culture water. The mechanism involved by which this pigment is toxic has not been determined. Boryu (1957) proposed that prodigiosin is toxic due to its ability to penetrate the cell membrane. Perhaps the pigment alters the lipoida1 layer. In older larvae, the prodiginine pigment produced by the Pseudomonas sp. appeared to accumulate in the mantle region. ACKNOWLEDGMENTS The editorial assistance and typing of this manuscript by Miss Rita S. Riccio are greatly appreciated. The author also is deeply grateful to Drs. Sung Y. Feng, Antonio H. Romano, and Lawrence R. Penner for their constructive criticism during this study.
REFERENCES BORYU, S. I. 1957. On the mechanism of antibiotic action of Bucillus prodigiosum. Microbiology, 26, 462-465. BROWN, C. 1974. A pigment-producing pseudomonad which discolors culture containers of embryos of a bivalve mollusk. Chesupeuke Sci., 15, 17-21.
BROWN
BROWN, C., AND LOSEE. E. 1978. Observations on natural and induced epizootics of vibriosis in Crcrssostreu virginicu larvae. J. Invertebr. Puthol.. 31. 41-47.
BROWN. C., AND Russo, D. .I. 1979. Ultraviolet light disinfection of shellfish hatchery sea water. 1. Elimination of five pathogenic bacteria. Aquucullure. 17, 17-23. D’AOUST, J. Y., AND GERBER, N. N. 1974. Isolation and purification of prodigiosin from Vibrio psychroerythrus.
J. Bacterial.,
118,
756-7.57.
GANDHI, N. M., PATELL. J. R., GANDHI, .I.. DE SOUZA, N. J.. AND KOHL. H. 1976. Prodigiosin metabolites of a marine Pseudomonus species. Mar. Biol.,
34,
223-221.
GERBER, N. N. 1973. Minor prodiginine pigments from Actinomuduru mudurue and Actinomuduru pelletieri.
J. Heterocycl.
Chem.,
10, 925-929.
GERBER, N. N. 1975. Prodigiosin-like Rev.
Microbial.,
pigments.
Crit.
13, 469-485.
GERBER, N. N., AND STAHLY, D. P. 1975. Prodiginine (prodigiosin-like) pigments from Streptoverticillium rubrireticuli. an organism that causes pink staining of polyvinyl chloride. Appl. Microbiof.. 30, 807-810.
JACOB, S. W., BISCHEL, M.. AND HERSCHLER, R. 5. 1964. Dimethyl sulfoxide (DMSO): A new concept in pharmaco-therapy. Curr. Ther. Res. Clin. Exp.. 6, 134-135. Kalesperis, G. S.. PRAHLAD, K. V., AND LYNCH, D. L. 1975. Toxigenic studies with the antibiotic pigments from Serrutiu murcescens. Cunud. J. Microbiol.,
21, 213-220.
LEWIS, S. M., AND CORPE, W. A. 1964. Prodigiosinproducing bacteria from marine sources. Appt. Microbiol., 12, 13- 17. LOOSANOFF, V. L., AND DAVIS, H. C. 1963. Rearing of bivalve mollusks. In “Advances in Marine Biology” (F. S. Russell, ed.). Vol. 1. pp. l- 136. Academic Press. London. STILES, S. S., AND LONGWELL, A. C. 1973. Fertilization, meiosis and cleavage in eggs from large mass spawnings of Crussostrea virginicu Gmelin, the commercial American oyster. Curyologia, 26, 253-262.
TAYLOR, W. W.. Particulate-bound (Chromobucterium
AND WILLIAMS, pigment of Serrutiu prodigiosum).
R. P. 1959.
murcescens Experientiu,
15.
143-144. THOMPSON, P. E., MCCARTHY, D.A., BAYLES, A., REINERTSON, J. W., AND COOK. A. R. 1956. Comparative effects of various antibiotics against Endumoebu histolyticu in vitro and in experimental animals. Antibiot. Chemother., 6, 337-350. WIER. R. H.. EC;EBERC;, R. 0.. LACK, A. R.. AND LEIBY, G. M. 1952. A clinical trial of prodigiosin in disseminated coccidioidomycosis. Amer. J. Med. Sci., 224, WILLIAMS.
70-76. R. P.,
GREEN.
J. A.,
AND
RAPPOPORT.
PIGMENT
TOXIC
D. A. 1956. Studies on pigmentation of Serruticr r~~~ce.sce)>.s.1. Spectral and paper chromatographic properties of prodigiosin. J. Bucteriol.. 71, 115-120. WII I LAMS, R. P., AND HEARN, W. R. 1967. Prodigiosin. 1!1 “Antibiotics” (D. Gottlieb and P. D.
TO OYSTER EMBRYOS
293
Shaw, eds.), Vol. II, pp. 410-432. Springer-Verlag. Berlin/Heidelberg/New York. WREDE. F.. AND HE~TCHE. 0. 1929. iiber das Prt,digiosin. den roten Farbstoff des Buci//rr.\ pry,Jipiosu.s. (1. Mitteil.). Ber. Derrr. C/,e,n. (;(J.L. 62. 2678-2685.
OF
INVERTEBRATE
PATHOLOGY
A Prodiginine
38, 281-293 (1981)
Pigment Toxic Crassostrea
to Embryos virginica
CAROLYN Nufiontrl
Ocuuttic
crttd
Atmospheric Center.
Administration. Milford Luhorutory.
and Larvae
of
BROWN
Notional Milford.
Marine Fisheries Srrvicr. Cottttrctictrt 06460
Northetrst
Fisherie.\
Received October 27. 1980 The red pigment produced by a marine Psetrdomonu sp. which causes abnormal development and mortality in developing embryos of the American oyster, Crusso~trru virginicu, was analyzed. A comparative study of a nonpigmented and two pigmented mutants of the red parental strain indicated that virulence was associated and varied with pigmentation. The use of sonicated cells supported lysing of the pseudomonad cells as the most probable means of pigment release. Crude pigment extracted from the red parental strain and its yellow mutant was toxic to developing oyster embryos. Neither the “pigment” extracted from the white mutant nor dimethyl sulfoxide, used for dissolving the extract, was toxic. Three pigment fractions were demonstrated by thin-layer chromatography even after purification. Studies indicate that only the fraction corresponding to R, 0.41 was necessary for virulence. The virulent pigment fraction was identified as belonging to the prodiginine group. KEY WORDS: Crrrssostreu virginicu; American oyster: Pseudomortus sp.; toxic pigment: prodiginine.
INTRODUCTION
Prodigiosin and prodiginine pigments are known to be antibacterial, antifungal, and antiprotozoan (see Williams and Heart-r, 1967; Gerber, 1975, for review). Attempts to take advantage of these potentially beneficial properties have been unsuccessful due to the toxic nature of the pigment. Thompson et al. (1956) noted a relationship between antiprotozoan activity of prodigiosin and toxicity in both rats and hamsters. Prodigiosin has been tested as a means for treating coccidioidomycosis in man, but pain and tenderness are associated with the intramuscular injections (Wier et al., 1952). Kalesperis et al. (1975) found prodigiosin to be toxic to chick embryonic development, resulting in abnormal development and death. Prodigiosin and prodiginine pigments have a wide distribution in the microbial world. Prodiginine pigments are produced by some members of the genera Streptomyces (see Gerber, 1975) and Actinomadura (see Gerber, 1973) and by Streptoverticillium ruhrireticuli (see Gerber and Stahly, 1975). Serratia marccscens has long
been known to produce prodigiosin. Lewis and Corpe (1964) reported that it also is produced by two unidentified strains of marine bacteria. Two other marine bacterial species, Vibrio psychroerythcus (see D’Aoust and Gerber, 1974) and Pseudomonas magnesiorubru (see Gandhi et al., 1976) were later found to produce this pigment. Brown (1974) showed that a red marine pseudomonad is pathogenic to laboratoryreared embryos of the hard shell clam, Mercenaria mercenaria, and the American oyster, Crassostrea virginica. The presence of this bacterium in sea water of shellfish hatcheries and laboratories is easily discernible by the pink discoloration it causes in culture containers and conditioning trays. The pigment produced by the red Pseudomonas sp. was thought to be prodigiosin or a prodiginine pigment. The bright red color of the pigment, as that of prodigiosin and prodiginine pigments, was pH dependent; it was red under acid conditions and yellow under alkaline conditions. It was capable of discoloring polyvinyl chloride as are the prodiginine pigments pro281 0022-2011/81/050281-13$01.0010 Copyright (61 1981 by Academic Precs. Inc All rights of reproduction in any form rescrxed
282
CAROLYN
duced by Streptoverticillium rubrireticuli (see Gerber and Stably, 1975). The purpose of the present study was to analyze the pigment of the red Pseudomonas sp. to determine whether it was toxic to oyster embryonic development and if the pigment belonged to the prodiginine group. MATERIALS
AND METHODS
Bacteria
The red Pseudomonas sp. was originally isolated from a moribund larval culture of the hard shell clam, Mercenaria mercenaria (see Brown, 1974). The mutants were obtained from sea water agar plates (Brown and Losee, 1978) inoculated with ultraviolet (uv) light-irradiated cells of the red pseudomonad. The procedures described by Brown (1974) were used to determine the morphological and physiological characteristics of the mutants. Sonicated
Cells
The red pseudomonad was inoculated into 7.5 ml of seawater broth and incubated overnight at 26°C. The culture was used to seed 1 liter of sea water broth in a 2.5liter Fembach flask. The culture was incubated overnight at 26°C and cells were spun down at 33008 for 30 min. The cells were sonicated, following a procedure used by Dr. R. A. Robohm of the Milford Laboratory (pers. commun.). The cells were washed twice with sterile sea water; 0.5 ml of washed cells was mixed with 3.5 ml of sterile sea water, and washed glasperlin glass beads (2 g/4 ml) were added. The cells were sonicated 3-4 min at maximum probe intensity. The broken cells were removed and diluted 1:lO with sterile sea water. A sample of the suspension was plated onto sea water agar plates to determine if all cells were broken. Pigment
Production
The Pseudomonas sp. and its mutants were grown at 26°C overnight in 7.5 ml of sea water broth. The cultures were spread
BROWN
over the surface of 1.5 liters of nutrient agar (Difco)’ made with a solution of 80.0% aged, membrane-filtered sea water and 20% distilled water in 2.5-liter Fernbach flasks. The cultures were incubated overnight at 26°C. Cells were washed off agar surfaces with sterile sea water. The cells from each culture were spun down at 33009 for 30 min. The supernatant fluid was decanted off and cells were resuspended in acetone. Acetone extraction was allowed to proceed for at least 1 hr; then the suspension was spun down at 3300g for 30 min and the supematant fluid filtered. Pigment was extracted from the filtrate according to procedures described by Williams et al. (1956). Acetone-pigment solution was mixed with petroleum ether; the acetone was removed by adding distilled water and drawing off the acetone-water phase. Petroleum ether was evaporated off in vacua at 35”C, using a rotary evaporator. Saturated solutions of the dry pigments in dimethyl sulfoxide (DMSO) were made and diluted 1:lO in sterile distilled water. Pigment
Purification
Procedures described above were used to extract pigment from the red parental strain. The pigment was purified, following a procedure reported by Wrede and Hettche (1929). The petroleum ether-pigment solution was concentrated to about 50 ml over which dry HCl gas was conveyed until the hydrochloride precipitated. The precipitate was allowed to stand and was filtered. The hydrochloride was dissolved in 100 ml of 96% ethyl alcohol while heating, was filtered, and then mixed with 5% perchloric acid until the solution became turbid. After precipitation the perchlorate was filtered, dissolved in 100 ml of warm ethyl alcohol, and followed by mixing with enough 10% sodium hydroxide until the solution turned to a yellowish-brown color. The solution was allowed to stand for 1 hr ’ Reference to trade names in this paper does not imply endorsement of commercial products by the National Marine Fisheries Service.
PIGMENT
TOXIC
TO
and was then filtered. The filtrate was mixed with 30 ml of chloroform and 100 ml of distilled water. The chloroform layer, washed several times with distilled water to remove any residual alcohol, was filtered and evaporated under vacuum. A saturated solution of the pigment in DMSO was made and then diluted I:10 in sterile distilled water. Pigment
Fractions
A saturated pigment -chloroform solution was prepared from the crude and purified pigment extracts of the red pseudomonad. Fifty-microliter aliquots of the solutions were spotted on OS-mm-thick Silica Gel-G chromatoplates (Supelco). The plates were developed in benzene-ethyl acetate solution (1: 1); the different fractions were slurried in DMSO and filtered. Each dissolved fraction was diluted 1: 10 in sterile distilled water. Spawning
of Oysters
The spawning techniques of Loosanoff and Davis (1963) were modified according to the procedure described by Brown and Russo (1979). The adult oysters were spawned in lo-pm-filtered, UV-irradiated sea water. Approximately 15,000 fertilized oyster eggs were added to each 1.3-liter polypropylene beaker filled to the l-liter mark with lo-pm-filtered, UV-treated sea water. Larval cultures were sampled after 48 hr. Larvae which had developed the standard “D” shape shell were considered normal. It also was noted whether the larvae were living or dead prior to being fixed (Brown and Russo, 1979). Assays to Determine and Toxicity
Pathogenicity
Assays were conducted to determine whether the development of fertilized oyster eggs was affected by the presence of the following: bacterial broth cultures of the pink. white, and yellow mutants of the red pseudomonad; sonicated cells of the red pseudomonad: crude pigment extracts of
OYSTER
283
EMBRYOS
the pseudomonad and its white and yellow mutants; and purified extract of the pigment of the red pseudomonad. Each series of experiments was repeated four times, using quadruplicate beakers each time. Broth cultures. The red pseudomonad parental strain and the three pigment mutants were grown at 26°C for 24 hr in sea water broths. A OS-ml aliquot of cultures of each of the four bacterial strains was used to determine its effect on the development of fertilized oyster eggs. The inocula were mixed with the embryonic culture water just prior to the addition of the fertilized eggs. Sonicated cells. A lo-ml sample of the diluted suspension of broken red cells was mixed with embryonic culture water prior to the addition of the fertilized eggs. Pigment extract. Each of the diluted solutions of pigments, fractions of crude and purified extracts of the red pigment, and DMSO were mixed with culture water, using l-ml inocula, and fertilized oyster eggs added. The amount of extract used was measured spectrophotometrically at 535 nm. Characterization
of Pigment
Results obtained from thin-layer chromatography, spectrophotometry, and solubility studies for the red pigment of Pseudomonas sp. were compared with prodigiosin extracted from Serratia murcestens (ATCC 13880) to determine whether the pigment produced by the pseudomonad was a prodiginine. The same procedures described earlier for growing the pseudomonad and extracting pigment were employed for S. marcescens, except distilled water was used in preparing medium and washing cells. Thin-layer chromatography (tfc). A saturated pigment-chloroform solution was prepared for each pigment extract. Fifty microliters of each solution was spotted on chromatoplates. Plates were developed in 100 ml of a benzene-ethyl acetate solution (1:l). The spots were examined under UV
284
CAROLYN
light and sunlight, mined.
and Rf values
BROWN
deter-
Spectrophotometry. Each dried pigment extract was dissolved in 95% ethyl alcohol. The amount was adjusted so that absorbance reading was the same for each dissolved pigment at 510 nm under acid condition. Spectral readings were recorded over a range of 400-600 nm. The readings were taken under neutral, acid (pH 5), and alkaline (pH 8) conditions using 0.1 N HCl and 0.1 N NaOH to adjust pH. Solubility. Solubility was tested by adding a portion of each dried pigment extract to each of the following solvents: petroleum ether, benzene, chloroform, acetone, ethyl alcohol, methyl alcohol, amyl alcohol, dimethyl sulfoxide, sulfanilic acid, potassium hydroxide, sodium hydroxide, sea water, and distilled water. RESULTS Exposures of Developing with Bacteria
Larvae
Plates inoculated with UV-irradiated aliquots of the red pseudomonad suspended in sterile sea water yielded three types of pigment mutants. They were white, pink, and yellow. Development of fertilized oyster eggs exposed to approximately 1.4 x lo5 white mutant cells/ml of embryonic culture water was comparable to that of the untreated controls. Live-normal development of the fertilized eggs to the straight-hinge stage averaged 62.9% in cultures exposed to the white mutant and 62.2% in the untreated control beakers (Table 1). Student’s t test, however, did reveal a significant difference (P < 0.01) between the development of oyster eggs exposed either to the red parental strain or the pink mutant and that of the control cultures. Live-normal development to the straight-hinge stage averaged 18.1% in the presence of the red pseudomonad (8.4 x lo4 cells/ml) and 28.9% in beakers seeded with the pink mutant (1.4 x lo5 cells/ml). It was also noted that abnormal development was significantly higher in larval cultures exposed either to the red parental strain or pink
WI0 mm
ww --
‘0
ww --
PIGMENT
TOXIC
TO OYSTER
mutant as compared with that found in untreated controls. Conversely, total development was significantly lower in experimental cultures than in untreated controls. Table 1 also shows that the yellow mutant had a detrimental effect on development of fertilized oyster eggs. Live-normal development averaged only 19.3% in embryonic cultures exposed to the yellow mutant (1.3 x lo5 cells/ml) and 25.7% in those exposed to the red parental strain (8.6 x lo4 cells/ml). These percentages were significantly less than that found in the untreated controls, 81.1%. Averaged live-abnormal development also was significantly higher in the experimental cultures than in the untreated controls. Total development again was found to be significantly lower in larval cultures exposed to either bacterial strain than in untreated controls. Figure IA-D illustrates the morphological differences in oyster larvae after 48-hr exposure to the four bacterial strains. While the white mutant had no appreciable effect on the developing larvae (Fig. 1A), the three pigmented isolates showed varying degrees of abnormal shell formation and growth retardation which were most pronounced in the presence of the red parental strain. Larvae exposed to the pink mutant tended to be what Loosanoff and Davis larvae”; the ( 1963) termed “saddleback hinge line was concave rather than straight (Fig. IS, arrow). Larvae exposed to the pink mutant averaged 70 pm in length, compared to 75 pm in the presence of the white mutant and in untreated controls. Shell formation and growth were greatly retarded in the presence of the yellow mutant (Fig. 1C) and the red parental strain (Fig. 1D). Size averaged only about 35 pm in the presence of the red parental strain and 45 km during exposures to the yellow mutant. Exposures of Developing Sonicated Cells
Larvae
with
Bacteriological plates demonstrated that no viable cells were present after sonication. Preliminary experiments showed that
EMBRYOS
285
2.4 x lo6 sonicated cells/ml culture water resulted in only 54.9% live-normal larvae, compared to 86.9% in untreated controls. Normal development continued to decrease, while abnormal development increased as the number of sonicated cells increased. Table 2 shows that an average of only 12.7% live-normal development of fertilized eggs occurred in the presence of 4.8 x lo6 sonicated cells/ml culture water, while development in the untreated controls averaged 81.4%. Abnormal development was also significantly higher in cultures with sonicated cells than in control beakers. Table 2 also shows that total development was significantly lower in larval cultures exposed to sonicated cells (60.4%) than in control larval cultures (92.3%). Exposures of Developing with Pigments
Larr’ae
Table 3 presents the data obtained following exposures of fertilized oyster eggs to pigment extracted from the red pseudomonad (4.8 x 106 cells/ml) and its yellow (1.3 x 10’ cells/ml) and white (1.4 x lo7 cells/ml) mutants. The DMSO controls, in each study, consistently showed development comparable to that of the untreated controls. The pigment extracted from the red pseudomonad (absorbance equaled 0.220 at 535 nm) was extremely toxic, resulting in normal development of only 27.9%. Development in the untreated controls, on the other hand, was 74.9 and 75.0% in the presence of DMSO. Fertilized oyster eggs were also adversely affected by exposure to the yellow pigment (absorbance equaled 0.180 at 535 nm). Livenormal development was only 46.2% in the presence of the yellow pigment and 68.3% in the untreated controls. The “pigment” extracted from the white mutant, however, had no significant effect on the development of fertilized oyster eggs. There was a 76.2% normal development to the veliger stage when eggs were exposed to this “pigment” (absorbance equaled 0.000 at 535 nm), compared to 74.2% in the untreated controls. Percentage total develop-
286
CAROLYN
FIG. yellow
BROWN
1. Forty-eight-hour oyster larvae after exposure mutant, and (D) red parental strain. Magnification:
ment was significantly reduced by the presence of either the red or yellow pigment as compared with the control cultures. The above experiments suggest that the pigment is responsible for the retardation of normal embryonic development. To substantiate this, purified red pigment was used. Approximately 3.4 mg of the purified
to (A) 90x.
white
mutant,
(B) pink
mutant,
(C)
red pigment was mixed with the culture water in each experimental beaker. Bacteriological studies showed that about 8.7 x log cells were needed to obtain this amount of pigment. As shown in Table 4, livenormal development of oyster eggs to the straight-hinge stage was significantly different between the untreated controls (72.6%)
PIGMENT
PERCENTAGE
TOXIC
287
TO OYSTER EMBRYOS
TABLE 2 OF FERTILIZED OYSTER Ec;c;s EXPOSED TD SONICATED CELLS OF RED Psrudomorzu.s sp. DEVELOPMENT
Treatment Sonicated cells (4.8 x lO”/ml culture water) Number of replicates Live-normal (4 2 SE”) Dead-normal (.? 2 SE) Live-abnormal (4 + SE) Dead-abnormal (X i SE) Total (.U t- SE)
16 12.7 t l4.1* 4.4 t 5.2 34.2 t 17.2* 9.0 t 5.8* 60.4 i- 22.8*
Control 16
c t k
8.2 0.8
92.3 t
4.2
81.4 1.6 8.6 0.7
8.3 IO.4
” Standard error at 99% confidence interval. * Significantly different from control (P < 0.01).
and the cultures with purified red pigment (36.6%). There was no significant difference between the untreated controls and the DMSO controls (71.4%). The averaged percentages for dead-normal and deadabnormal also were higher in the presence of the purified red pigment than in the control culture. As had been demonstrated in the preceding exposures, the presence of the purified pigment resulted in a significant decrease in total development. Toxicity of Various Fractions of the Red Pigment
A comparative chromatographic study of the crude and purified red pigment was conducted and the fraction(s) responsible for the toxic effect determined. Thin-layer chromatography of the crude red pigment (Fig. 2) revealed five spots: yellow (R, 0.971, pink (I?, 0.68), orange (Rl 0..55), pink (I?, 0.41), and purple (R, 0.00). After recovery from the chromatograms, only the orange (Rr 0.55) and pink (R, 0.41) fractions were able to affect adversely the development of fertilized oyster eggs. After purification, only three of the five spots were still present on tic plates: orange (Rf 0.55), pink (Rf 0.40), and purple (R/ 0.00). As shown in Table 5. the orange and pink fractions continued to be toxic. There was a significant difference between live-normal development in the presence of either the pink
fraction (58.8%) or the orange fraction (49.3%) and the controls (71.3%). Liveabnormal development was greater in the presence of either one of the two fractions than in the control beakers. Total development, however, was significantly lower only in the presence of the orange fraction. Pigment Characterization
Comparisons made between prodigiosin extracted from Serratia marcescens and the pigment extracted from the red Pseudomonas sp. indicate that the latter also belongs to the group called prodigiosinlike or prodiginine. Figure 2 illustrates the five spots that developed from the two crude pigment extracts on the plates. The R, value for each spot developed from the pigment of the red pseudomonad was identical to the corresponding spot developed from prodigiosin. The folIowing spots, in ascending order, were visible: purple (Rf 0.00). pink (Rrl 0.41). orange (R, 0.55). pink (RI 0.68), and yellow (R, 0.97). The spots corresponding to Rf 0.41 and R, 0.55 were the major ones. Three of the five spots, purple (RI O.OO), pink (R, 0.40), and orange (Rf 0.55), also were visible when purified preparations were spotted on 0.5-mm thick Silica Gel-G plates (Supelco). Only the purple spot and a pinkish-orange spot (R, 0.20) were developed, however, on 0.25-mm Silica Gel 6OF-254 plates (E. Merck).
288
CAROLYN
BROWN
The maximum absorbance was at 535 nm under neutral and acid conditions (Fig. 3A and B, respectively) but shifted to 470 nm under alkaline condition for pigment extracted from both Pseudomonas sp. and S. marcescens (Fig. 3C). The minimum absorbance at 600 nm was the same for the two pigments. A shoulder was visible at about 510 nm under acid and neutral conditions. Figure 3A and B shows that there was also a shoulder at 580 nm; under alkaline condition, this shoulder started to peak (Fig. 3C). The peak at 580 nm, however, was more prominent for prodigiosin than for the red pigment. The pigment extracts of S. marcescens and Pseudomonas sp. were identical in their ability to be dissolved in various solvents. The dried pigment extracts were insoluble in sea water and distilled water. Both were moderately soluble in sulfanilic acid, potassium hydroxide, and sodium hydroxide. The two pigments were extremely soluble in all the organic solvents tested. DISCUSSION
www mm-
*ww ---
www m-m
Data collected from the spectrophotometric, chromatographic, and solubility studies indicate that the pigment produced by the red Pseudomonas sp. belongs to the group called prodiginine. The maximum spectrophotometric peak of the red pigment in acidified ethanol fell within the range for prodigiosin and prodiginine pigments. Gerber (1973) reported that prodiginine pigments, in acidified ethanol, have a maximum absorption range from 542 to 525 nm. Prodigiosin extracted from Serratia marcescens has a maximum absorption at 537 nm under the same conditions. Figure 2 shows that the maximum absorption, in the present study, was about 535 nm for both prodigiosin and the red pigment. Lewis and Corpe (1964) also indicated that prodigiosin extracted from S. marcescens has a maximum peak at 535 nm under acid and neutral conditions. The shift of the maximum peak from 535 to 470 nm under alkaline conditions is also in agreement
PIGMENT
PERCENTAGE
TOXIC
DEVELOPMENT PURIFIED
TO
OYSTER
TABLE
4
289
EMBRYOS
OYSTER Ecus RED PSEUDOMONAD
OF FERTILIZED PIGMENT
OF
EXPOSED
Treatment Purified red pigment Number of replicates Live-normal (4 r SE”) Dead-normal (.i 2 SE) Live-abnormal (4 + SE) Dead-abnormal (h 2 SE) Total (E t- SE)
36.6 1.2 4.9 2.7 51.4
DMSO
16 + 17.9* k 7.0* k 3.2 t 2.1* t 19.6*
71.4 2.8 1.9 1.7 78.8
16 + 11.6 ‘- 2.8 t 1.6 i_ 1.3 + 9.0
‘TO
____-Control 12.6 2.2 2.8 1.2 78.8
16 -+ 11.7 i- 2.0 + 1.7 -t 1.2 i 9.6
-.
-
w Standard error at 99% confidence interval. * Significantly different from control (P < 0.01).
with the findings of Lewis and Corpe (1964). Differences existed between the present study and the one conducted by Lewis and Corpe; minimum absorption is at 420 nm and the shoulder at 580 nm is not visible in the latter study.
FK. 2. Thin-layer chromatographic separation of prodigiosin from S. mttrrrscens and the red pigment from the shellfish pathogenic pseudomonad. Stationary phase: Silica Gel-G. OS-mm thick. Solvent: Benzene-ethyl acetate solution (1:I).
Chromatographic study showed the same five spots for crude extracts of prodigiosin and the red pigment, and the same three spots for the purified pigments. Gandhi et al. (1976) reported that prodigiosin extracted from Pseudomonus magnesiorubra runs as a single spot on silica gel plates developed in benzene-ethyl acetate. This discrepancy may have been due to the use of different commercial plates in the two studies. Different results were obtained in the present study by using two different brands of silica gel plates. Discrepancies can also be due to differences in the age of the cultures, length of storage, method of extraction, and handling of the pigments (Lewis and Corpe, 1964). Gerber (1973) stated that prodigiosin-like pigments are soluble in nonpolar solvents and in polar solvents, such as acetone and chloroform. Kalesperis et al. (1975) reported that prodigiosin is also soluble in dimethyl sulfoxide. The present study showed that the extracted red pigment and prodigiosin were very soluble in the organic solvents tested. Both pigments were insoluble in sea water and distilled water. The agreement in soiubility for the two pigment extracts supports the findings of the spectrophotometric and chromatographic studies, i.e., the red pigment is prodiginine. The shellfish pathogenic pseudomonad differed from other prodigiosin-producing, marine pseudomonads. e.g., Pseudomonas
290
CAROLYN
BROWN
TABLE PERCENTAGE
DEVELOPMENT
OF FERTILIZED
5
OYSTER
OF PURIFIED
RED
EGGS
EXPOSED
TO Two
COMPONENTS
PIGMENT
Treatment Control Number of replicates Live-normal (X + SE“) Dead-normal (X -r- SE) Live-abnormal (X t SE) Dead-abnormal (I _’ SE) Total (X 2 SE)
71.3 2.6 4.8 0.4 79.1
16 + 5 t k 2
3.7 1.2 3.6 0.4 4.9
DMSO 72.5 2.1 4.3 0.7 79.5
16 ” + zt t f
4.0 0.8 2.9 0.6 4.8
Pink (& 0.40) 58.8 2.6 10.8 0.4 72.6
16 2 7.8” 2 1.4 + 6.8* _’ 0.5 k 6.2
Orange (R, 0.55) 49.3 2.6 12.9 1.0 65.8
16 I 12.7* k 1.2 5 7.3* Ik 1.3 c 7.0*
” Standard error at 99% confidence interval. * Significantly different from control (P < 0.01).
magnesiorubra, in that it has a lower salt
tolerance and was unable to reduce nitrates to nitrites or produce acid in galactose, maltose, or mannitol. The pathogen also differed from the two marine isolates reported by Lewis and Corpe (1964); it produced acid in sucrose rather than in trehalose and grew in media prepared with distilled water supplemented with only sodium chloride and magnesium sulfate. Lewis and Corpe (1964) found that one of the bacterial isolates in their study requires potassium, in addition to sodium and magnesium ions, for growth. The authors reported that calcium ions are required for pigment production. The red shellfish pathogen also required calcium ions for pigment production. Stiles and Longwell (1973) studied irregularities of meiosis and early cleavage in laboratory-spawned American oysters. These irregularities offer some explanation as to why, under artificial conditions, total development of fertilized oyster eggs rarely equals 100% when calculating actual percentage development. Because some eggs invariably break down soon after fertilization, they cannot be detected when larval samples are counted. In the present study, total development in the untreated controls ranged from 76.9 to 92.3%; while livenormal development ranged from 62.2 to 81.4%. The results showed that both livenormal and total developments were significantly lower in cultures exposed to the
red parental strain or its pigment than in untreated control cultures. Exposures to the white mutant, its “pigment,” or DMSO had no such effect; development was basically the same as in the untreated controls. These results demonstrate that the pigment produced by the red parental strain is indeed toxic to oyster embryonic development. The suggestion that the pigmented isolates contain varying amounts of the toxic components and the white mutant is missing them entirely is supported by the results of the thin-layer chromatography studies. Material extracted from the white mutant produced no visible spots on tic plates. The pink mutant contained four of the five spots found in the red pseudomonad; it lacked the spot corresponding to Rf 0.67. Two spots with Rf values corresponding to 0.41 and 0.97 were found in the pigment extracted from the yellow mutant. The data suggested that to be virulent a strain or mutant must contain a pigment fraction having an Rf value of 0.41. Although dimethyl sulfoxide, at the concentration used (1:10), was nontoxic to developing oyster embryos, its use had two serious drawbacks: (1) it could not explain how a non-water-soluble pigment is released to affect oyster embryonic development, and (2) as a penetrant carrier (Jacob et al., 1964), the possibility existed that it enhanced absorption of pigment across the cell membrane of the developing embryos,
PIGMENT
TOXIC
TO
OYSTER
291
EMBRYOS
30$ ;t; 2 5 2
2b
_----_ lo-
WAVELENGTH
FIG. 3. Spectrophotometric conditions: (A) neutral (l:lO),
(NM1
WAVELENGTH
lNM1
WAVELENGTH
(NM1
curves of prodigiosin and the red pigment (B) acid (l:lO), and (C) alkaline (1:5).
in ethanol
under
the following
292
CAROLYN
thus causing the adverse effect. The use of sonicated cells more naturally demonstrated what happens. Taylor and Williams (1959) reported that mechanical disruption of Serratia marcescens renders prodigiosin “water-soluble”; the pigment is bound to particulate matter. It was noted that in infected larval cultures red pseudomonad cells could not be recovered from larvae or culture water 24 hr after onset of the disease. The phenomenon suggests that bacterial cells begin to die before any overt evidence of a disease occurs. Particulatebound pigment probably was released when cells of the red pseudomonad were lysed. Particulate matter tended to accumulate around the edges of the larval culture containers at the air-water intersurface; thus, this area was discolored. An earlier study (Brown, 1974) showed that more than lo3 actively dividing cells/ml culture water have to be added to elicit a toxic effect. The present study, using sonicated cells, showed that the number of cells actually required was about 4.8 x IO6 cells/ml, which was equivalent to adding 3.4 yg pigment/ml of culture water. The mechanism involved by which this pigment is toxic has not been determined. Boryu (1957) proposed that prodigiosin is toxic due to its ability to penetrate the cell membrane. Perhaps the pigment alters the lipoida1 layer. In older larvae, the prodiginine pigment produced by the Pseudomonas sp. appeared to accumulate in the mantle region. ACKNOWLEDGMENTS The editorial assistance and typing of this manuscript by Miss Rita S. Riccio are greatly appreciated. The author also is deeply grateful to Drs. Sung Y. Feng, Antonio H. Romano, and Lawrence R. Penner for their constructive criticism during this study.
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