Alteration of dinitrogen fixation and metabolism in cyanobacterium Anabaena PCC 7119 by phosphamidon

Environmental and F.~perimental Botany, Vol. 31, No. 4, pp. 479-488, 199 [ Printed in Great Britain.

0098 8472/91 $3.00 + 0 0 0 ~) 1991 Pergamon Press ph"

A L T E R A T I O N OF D I N I T R O G E N F I X A T I O N AND M E T A B O L I S M IN C Y A N O B A C T E R I U M A N A B A E N A PCC 7119 BY PHOSPHAMIDON E. PERONA, E. MARCO and M. I. ORUS

Departamento de Biologia, Facultad de Ciencias, Universidad Aut6noma de Madrid, 28049 Madrid, Spain

(Received 25 September 1990; acceptedin revisedform 22 March 1991) PERONA E., MARCOE. and OR0S M. I. Alteration of dinitrogenfixation and metabolism in cyanobacterium Anabaena PCC 7119 by phosphamidon. ENVIRONMENTALAND EXPERIMENTALBOTANY31, 479488, 1991. The effects ofphosphamidon (2-chloro-2-diethylcarbamoyl-l-methylvinyl dimethyl phosphate) on carbon and nitrogen metabolism ofAnabaena PCC 7119 were investigated under controlled environmental laboratory conditions. The primary action of the insecticide is dinitrogen fixation, which is negatively affected after 24 hr of treatment with 10 lLg/ml. This inhibition leads to a delayed effect on cellular composition. Photosynthetic pigments, protein, nucleic acids and carbohydrates are affected from dosages of 40 60 pg/ml. Photosynthetic O:~ evolution is significantly reduced after 72 hr and 50 pg/ml and is a consequence of the reduction on the photosynthetic pigments. These physiological alterations result in a growth reduction that can be noticed from 20 #g/ml (72 hr) or 300 #g/ml (48 hr). Microscopic examination of cultures treated with 300 #g/ml revealed vegetative cell swelling and heterocyst deterioration. However, heterocyst frequency was not altered. Anabaena PCC 7l 19 does not degrade phosphamidon.

Key words: Anabaena PCC 7119, cyanobacteria, phosphamidon, organophosphorus insecticides, dinitrogen fixation.

synthesis, chlorophyll concentration and A T P levels.!5,~5i ORGANOPHOSPHATES and carbamates are insecPhosphamidon/~) is widely used in Spain, ticides widely used in agriculture. T h e y have despite its high water solubility, which increases replaced organochlorine formulas because of their the risk of environmental impact on aquatic ecolower toxicity for non-target organisms and systems. Cyanobacteria are basic primary proshorter persistence. Extensive literature is avail- ducers in these ecosystems and N2-fixing cyanoable on the effects that are produced by organo- bacteria contribute to soil fertility. Thus, it is phosphorus insecticides on target organisms and important to know the possible interactions between phosphamidon and cyanobacteria. The on vertebrates, soil bacteria and fungi, but there are not m a n y studies on microalgae. Most avail- work of RATH and MISRA (17) suggested that this able intbrmation on organophosphorus insec- insecticide might be used as a source of nutrients ticides and algae is summarized in several reviews by the non N2-fixing cyanobacterium Oscillatoria about pesticides and micro-organisms which indi- obscura. cate the existence of effects on growth, photoT h e hypothesis to be tested in this study was 479 INTRODUCTION

480

E.

PERONA

the existence of interactions between phosphamidon and Anabaena PCC 7 119. To this purpose the ability of the cyanobacterium to remove the insecticide from the medium and the effect of the pesticide on the growth and physiology of the cells were investigated.

MATERIALS

AND METHODS

The cyanobacterium Anabaena PCC 7 119 was used for this study. Batch cultures were grown in nitrogen-free medium previously described,“‘) under 90 pE/m’ set continuous illumination, at 26°C and gassed with 2.50,{, COT-enriched air. Phosphamidon was obtained from the Spanish Customs Office and was added to the culture medium at concentrations ranging from 10 to 300 pg/ml depending on the experiment. Culture density was determined at 750 nm and expressed as pg dry weight/ml with a regression line with the following equation pg/ml

= O.D. x 367.6 + 22.812.

This regression line was obtained by measuring the dry weight and the optical density of a series of dilutions of a concentrated culture. Cell counting was conducted to determine the cell con-

et al.

centration in the culture. Number of cells, filament length and heterocyst frequency were determined with a Neubauer hemocytometer. Proteins were determined by the method of LOWRYet ul.(*’ in extracts obtained by treating the samples with 1 N NaOH for 1 hr. Nucleic acids were extracted with 0.5 N perchloric acid at 70°C for 1 hr and estimated according to OGUR and RosEN.“~) The content of carbohydrates was determined by the method of DUBOIS et al. ‘+) in extracts of toluenetreated cells. For chlorophyll determination, the samples were extracted with 100% methanol for 3 min. The chlorophyll content ofthe extract was estimated according to the spectrophotometric method of MARKER.“” Phycobiliproteins were determined at 620 nm in the supernatant of a suspension of cells treated with toluene for 4 hr, according to BLUMWALI) and TEL-OR.:” Photosynthetic and respiratory exchanges were measured with a Clark-type O2 electrode (Hach Chemical Co.) .“’ Aliquots of 3 ml cell suspensions were placed in a temperature-controlled cuvette and illuminated with a quantum flux density of 300 ,nE/m’ set to determine photosynthetic Op evolution, or kept in the dark to evaluate respiratory 0s consumption. Nitrogenase activity was determined by acety-

Table 1. Eject of 50 pg/rnl of phosphamidon on the grouth oJAnabaena PCC 7 119 expressed as number _of cells per- ml of culture, on the number of cells per,filament and on the percentage of vegetative cells and heterocysts

Time (hr)

Treatment

Filament length (no. cells)

No. cell/ml x 10%

Vegetative cells (y/0)

Heterocysts (%I)

0

Control Phosphamidon

1788k2.5 1765+ 1.9

52.0 f 4.5 51.9k3.6

24

Control Phosphamidon

7647 f 9.8 6670f8.5

39.7k7.1 37.8k2.6

93.6kO.7 95.0+ 1.5

6.4kO.7 5.0* 1.5

48

Control Phosphamidon

11,194Ifr619 10,753 k 589

37.2k7.1 28.410.4*

91.1+0.2 90.1 f 0.2

8.9kO.2 9.9 f0.2

72

Control Phosphamidon

23,800k 75 11,164*926

35.4k6.2 25.4+ 1.4*

91.7f0.5 89.2&0.4*

8.3f0.5 10.8f0.4

96

Control Phosphamidon

32,745 +630 23,116)980

91.3kO.2 89.6kO.3

8.7kO.2 10.4Ifro.3*

Asterisk indicates statistically

significant

differences

with Student’s

t test: * P < 0.05.

DINITROGEN

FIXATION

INHIBITION

lene reduction”‘) in 20 ml aliquots of cell suspensions placed in 45 ml vials stoppered with a rubber stopper. The vials were pre-incubated for 10 min in a shaking water bath at 26°C and illuminated with a quantum flux density of 100 pE/m* sec. To begin the assay, 10% of the air was withdrawn and replaced with the same volume of acetylene. Thirty minutes after acetylene addition, 0.45 ml samples were taken and the ethylene content determined by injection into a Shimadzu GC8.4 gas chromatograph. The anaerobic conditions for nitrogenase determination(‘s’ were essentially as described by RIPPKA and WATERBURY.(‘~’ Assavs were oerformed in 40 ml vials each with a rubber stop’per. Prior to acetylene addition, the vials were flushed for 5 min with argon. The time course of phosphamidon degradation (initial concentration 300 lg/ml) was evaluated in cell-free control flasks and in flasks inoculated with an initial algal concentration of 50 pg/ml. At the times indicated, 10 ml samples were withdrawn and the insecticide was recovered with dichloromethene and transferred to ethyl ether. This extract was subjected to a gas chromatographic analysis according to CARLSTROM@) in a Hewlet-Packard 5840A Chromatograph with a Sy/, QFl Cromosorb G (AW-DMCS) 70/80 column and a FID detector. Data in figures are the means and standard deviations from at least three independent experiments with duplicate cultures and duplicate samples within each individual experiment. Student’s t tests were performed to determine the statistical significance of differences between control and phosphamidon-treated cultures. RESULTS Phosphamidon inhibited the growth of Anabaena PCC 7119 but the effect was noticed after different times depending on the method used to assess growth: the number of cells was reduced after 24 hr in cultures treated with 50 pg/ml (Table 1) but the dry weight of the cultures treated with 300 pg/ml was not significantly affected before 48 hr (Fig. 1A). Phosphamidon concentrations as low as 10 pg/ml affected the dry weight of Anabaena cultures after 72 hr of treatment; the inhibitory effect increased with increas-

BY PHOSPHAMIDON

481

**

3

Phosphamidon

(/Jg/ml)

FIG. 1. Effect of phosphamidon on the growth of Anabaena PCC 7119. (A) Time course of culture dry weight, (@Pa) control cultures, (O---O) 300 pg/ml phosphamidon. (B) Effect of the insecticide concentration on the dry weight at 72 hr of culture. Asterisks indicate statistically significant differences with Student’s t test: * P < 0.05. ** P < 0.01.

ing concentration of the insecticide (Fig. 1B). Routine microscopic examination revealed that the phosphamidon-induced inhibition of growth was accompanied by an increase of cellular volume of most filaments. This observation was supported by a decrease in the number of cells per filament (Table 1) and by an increase in average cell diameter (control, 3.05kO.41 pm; and 300 pg/ml phosphamidon-treated cells, 4.006 + 0.60 pm) after 72 hr of growth. In addition to this, differences were also observed in the cellular composition of the cultures treated with phosphamidon. For example, the concentration of 300 pg/ml phosphamidon in the culture medium reduced photosynthetic pigment contents (chlorophyll and phycobili-

482

E. PERONA

proteins); this effect appeared 48 hr after the beginning of treatment (Figs 2A and B). Experiments to determine the dose of phosphamidon affecting cellular composition were undertaken by treating Anabaena with a range of concentrations of the pesticide. Concentrations of 40 pg/ml or below did not affect chlorophyll content (Fig. 2C), but that of the other main nitrogenous compounds-phycobiliproteins, proteins and nucleic acids-was clearly reduced at 40 pg/ml (Figs 2D and 3). Approximate reductions of 75’& in phycobiliproteins and of 35:/,, in nucleic acids and proteins were registered in Anabaena cultures grown for 72 hr with the insecticide. The lower contents of nitrogen compounds in phosphamidon-treated cells contrasted

l

=t 5-

et al.

with an increase in the carbohydrate fraction (Fig. 3). In order to investigate the mechanism of inhibition of the insecticide, the physiological processes of Anabaena were studied (Fig. 4). Since the effects on ,&owth were clear from the lowest concentrations of insecticide assayed, a concentration of 50 pg/ml was chosen to study the eflect of phosphamidon on the time course of photosynthetic O2 evolution (Fig. 4A), respiratory O2 consumption (Fig. 4B) and nitrogenase activity (Fig. 4C). The effect of a range of concentrations between 10 and 100 pg/ml on these processes was also studied (Figs 4D, E and F). Photosynthetic O2 evolution was affected neither before 72 hr nor by concentrations below 50 pg/ml (Figs 4A and

’\

*

p

/

/’

A 5

I

I

24

z

I

I

72

48

I

36 Tlme(hr)

** f

l

l

I

24

I

48

0 I

72

96

++’200

G

E

*

251

I

I

I

20

40

60

80

I

100

‘&

I ‘300

I 20

Phosphamidon(

1 40 fig/

, 60

I 80

I 100

ml)

FIG. 2. Effect of‘phosphamidon on the cellular content of photosynthetic pigments of Anabaena PCC 7119. (A, B) Time course of the content of chlorophyll (A) and phycobiliprotein (B), (~~~~~-@) control cultures, (O---O) 300 pg/ml phosphamidon. (C, D) Effect of a gradient of concentration of insecticide on the content of chlorophyll (C) and phycobiliprotein (D) at 72 hr of culture. Asterisks indicate statistically significant differences with Student’s t test:

DINI’I’ROGEN

FIXATION

20 40 60 80 PHOSPHAMIDONWgld

INHIBITION

100 )

FIG. 3. Effect ofinsecticide concentration on the major organic fractions at 72 hr of culture. (O--O) Proteins, (O---O) nucleic acids, (0. . . .O) carbohydrates. Asterisks indicate statistically significant differences with Student’s I test: * P < 0.05, ** P < 0.01.

D). A clear response of respiratory O2 consumption to phosphamidon treatment was not found (Figs 4B and E). However, there was a remarkable effect of the insecticide on N, fixation from the first 24 hr at the lowest concentrations tested ( 10 pg/ml) (Figs 4C and F) .

Table 2. E#ect of50

BY PHOSPHAMIDON

Nitrogen fixation takes place in most cyanobacteria in specialized cells called heterocysts. We examined their frequency in the culture to test whether or not the impairment of this process could be related to a negative effect on the differentiation of such cells. In fact, our results indicated that their frequency slightly increased in phosphamidon-treated cultures (Table 1). However, microscopic examination revealed changes in appearance of the treated heterocysts (Fig. 5), which may explain their inefficiency and the resulting low values of nitrogen fixation. Since normal heterocysts provide the anaerobic environment needed for the activity of the O,-sensitive nitrogenase, the damaged aspect of phosphamidon-treated heterocysts suggested that the reduced nitrogenase activity might have resulted from a defective protection to 0, diffusion. The hypothesis that the effect of phosphamidon was caused by increased 0, diffusion inside the heterocysts was tested by measuring the nitrogenase activity under anaerobic conditions of growth and/or assay. The hypothesis was not supported because the inhibitory action of the insecticide on nitrogenase was not prevented by the exclusion of O2 during the experiments (Table 2). Finally, the possible role of Anabaena in degrading phosphamidon was studied. Figure 6 shows the time course of the insecticide concentration in cell-free and cell-inoculated culture media. Our results show that this insecticide was not removed by the cyanobacterium during the time assayed.

pgjml of phosphamidon on nitrogenase activity at 24 hr of treatment in aerobic and anaerobic conditions of culture and assayed

Aerated 24 hr with air + 2% CO,

Treatment

Assayed aerobic

Control Phosphamidon Percentage of nitrogenase

320f 18 230f 15* 72+ 5

Data expressed Asterisks indicate ** P < 0.01.

483

activity

as nmol ethylene/mg statistically

significant

dry weight differences

Assayed anaerobic

24 hr in anaerobiosis Assayed anaerobic

202+11 149+ 2* 74+ 5 x hr and as percentage with Student’s t test:

280+ 16 97f 15** 34& 5 of activity. * P < 0.05,

E. PHOTOSYNTHESIS

PERONA

et al.

RESPIRATION

NITROGENASE i

A

I 24

I

B

I

1 48

72

I

I

I

I

1

20

40

60

80

100

1

I

24 Timc4Thr

I

)72

g6

I

I

I

I

20

40

60

80

Phosphamidon(

I

100

*

C

1

I

I

I

1

I

24

40

72

96

I

I

I

I

20

40

60

80

I

100

fig/ml)

FIG. 4. Effect ofphosphamidon

on physiological processes. (A-C) Time course ofphotosynthetic 0, evolution (A), respiratory 0, consumption (B) and nitrogenase activity (C), (0-e) control cultures, (O---O) 50 pg/ml phosphamidon. (DF) Effect of the concentration on photosynthetic 0, evolution at 96 hr of culture (D), respiratory 0, consumption at 96 hr (E) and nitrogenase activity at 24 hr of culture (F). Asterisks indicate statistically significant differences with Student’s t test: * P < 0.05, ** P < 0.01.

DISCUSSION There is a limited number of studies on the effects of organophosphorus insecticides on cyanobacteria; these studies do not permit the drawing of conclusions because the effects are variable depending on the kind of insecticide and on the micro-organism as well as on the experimental conditions of each study.(7) A previous report on the interaction between phosphamidon and microalgae suggested a stimulating effect of the insecticide on the growth of the non-Ns-fixing cyanobacterium Oscillatoria obscura, suggesting that this chemical might be acting as a source of nutrients for the alga.(‘7) However, our results with Anabaena PCC 7 119 do

support that hypothesis since they indicate that this alga does not remove the insecticide in the test culture. As far as other organophosphorous insecticides are concerned, MECHARAJ et al.(“) reported that Phormidium tenue metabolized monocrotophos and quinalphos. RAO and LAL(‘~) found that malathion was accumulated but not metabolized by Anabaena ARM 310 and Aulossira fertilissima. MARCO”) observed that trichlorfon is neither metabolized nor taken up by the six different cyanobacteria assayed. Therefore the metabolism of organophosphorus insecticides by cyanobacteria seems to vary with the chemical tested. The results of the resent research indicate a strong action of phosphamidon on N, fixation in not

DINI’I’ROGEN

FIG. 5. Effect

FIXATION

INHIBITION

BY

PHOSPHAMIDON

485

of 300 ppm of phosphamidon on the morphology of Anabaena PCC 7119 at 72 hr of culture. (A) Control cells, (B) phosphamidon-treated cells. Arrows indicate heterocysts.

DINITROGEN

FIXATION

INHIBITION

BY

PHOSPHAMIDON

487

but the relationship between both effects was not established since different experimental procedures and times of treatment were employed to assess the effect of the insecticides on growth and on dinitrogen fixation. Results similar to those presented in the present work were obtained with trichlorfon by 0~6s et a1.(‘4) working with Anabaena PCC 7 119 and by MARCO@) working with Gloeothece PCC 6501, Nostoc UAM 205 and Chlorogloeopsis PCC 6912: treatment with trichlorfon inhibited N, fixation leading to a decrease in nitrogenous compounds and a reduction in growth. There was a decrease in the photosynthetic activity, estimated as O2 evolution, of dnabaena cells treated with phosphamidon. This inhibition seemed to be a secondary effect of the insecticide, and may be related to a decrease in photosynthetic pigments since it appeared only after 48 hr of treatment and at insecticide concentrations higher than those that affect nitrogen fixation. Our interpretation is in agreement with the review of LAL and DHANARAJ.“’ We can conclude that phosphamidon exerts a toxic action on Anabaena PCC 7119 and that this cyanobacterium does not remove the insecticide from the culture medium. Our data support the hypothesis that the primary action of phosphamidon is the inhibition of dinitrogen fixation.

fertilissima,

DAYS

FIG. 6. Time course of phosphamidon concentration in culture media (l --a), cell-free control flasks, (O- ----0) Anabaena PCC 7119 inoculated flasks. Statistical analysis by Student’s t test indicates no statistically significant differences between control and inoculated flasks.

Anabaena PCC 7119, as it was affected within a short time after treatment and from the lowest dose of insecticide assayed. This inhibition was not mediated by a negative effect on heterocyst differentiation but could be related to the structural disorganization of these specialized cells that carry out nitrogen fixation and supply the reduced nitrogen required for growth. Similar results have been obtained in Cylindrospermurn with five carbamates that affected N, fixation without changing the frequency of heterocysts.““’ However, the organophosphorus insecticides fenithrotion and chlorpyrifos inhibited this process as well as affecting the frequency in Anabaena and Aulo.rsira.‘7’ Anabaena has been grown in culture media without nitrogen. Under these conditions, N2 fixation is essential for growth because it is the only source of reduced N for the synthesis of proteins, nucleic acids and photosynthetic pigments. This scenario offers a possible explanation for why an inhibition of nitrogenase activity brings about a decrease of growth of Anabaena when insecticide is present in culture. Monocrotophos and quinalphos have been reported to affect dinitrogen fixation without inhibiting the growth of Nostoc cultures.(“’ LAL et a1.‘7’ found that fenitrothion and chlorpyrifos interfered with nitrogen fixation and with the growth of Anabaena ARM 310 and Aulossira

Acknowledgement -This research was accomplished financial support from the Spanish Department Sciences and Education.

with of

REFERENCES (Bayer 15 922, L 1. BAYER A. G. (1965) Dipterex 13/59). Farvenfabriken Bayer AG Leverkusen. E. (1982) Osmo2. BLUMWALD E. and TEL-OR regulation and cell composition in salt-adaptation of Nostoc muscorum. Arch. Microbial. 132, 168-l 72. 3. CARLSTROM A. A. (1972) Gas chromatography determination of phosphamidon insecticide in formulations. J. AOAC 55, 1331-1334. 4. DUBOIS M., GILLIES R. A., HAMILTON J. K., RUBERS P. A. and SMITH F. (1956) Calorimetric method for determination of sugar and related substances. Analyt. Chem. 28, 350-356. 5. LAL R. (1982) Accumulation, metabolism and effects of organophosphorus insecticides on microorganisms. Adv. af$l. Microbial. 28, 149-200.

488

E.

PERONA

6. LAL R. and DHANARAJ P. S. ( 1985) Cellular aspects of microbe-insecticide interactions. Znt. Rev. Cytol. 96, 239-262. 7. LAL S., SAXENA D. M. and LAL R. (1987) EfIects of DDT, fenitrothion and chlorpyrifos on growth, photosynthesis and nitrogen fixation in Anabaena (Arm 3 10) and Aulosira fertilissima. A
et al. 13. OGUR M. and ROSEN G. (1950) The nucleic acids of plant tissues. I. The extraction and estimation of desoxypentose nucleic acid and pentose nucleic acid. Arch. Biochem. 25, 262-276. 14. 0~6~ M. I., MARCO E. and MARTINEZ F. (1990) Effect of trichlorfon on N,-fixing cyanobacterium Anabaena PCC 7119. Arch. Envir. Contam. Toxicol.

19,297-301. and pesticides. 15. PADHY R. N. (1985) Cyanobacteria Residue Rev. 95, l-44. 16. RAO L. and LAL R. (1987) Uptake and metabolism of insecticides by blue-green algae Anabaena and Aulossira fertilissima. Microbios Lett. 36, 143% 147. 17. RATH S. and MISRA B. N. (1981) Effect of “Dimecron- 100” an organophosphorus insecticide on the growth of Osrillatoria obscura, Bruht et Biswas. Camp. Physiol. Ecol. 6, 137-140. 18. RIPPKA R. and STANIER R. Y. (1978) The effects of anaerobiosis on nitrogenase synthesis and heterocyst development by Nostocacean cyanobacteria. J. gen. Microbial. 105, 83-94. 19. RIPPKA R. and WATERBURY J. B. (1977) Anaerobic nitrogenase synthesis in non-heterocystous cyanobacteria. FEMS Lett. 2, 83-86. 20. STEWART W. D. P., FITZGERALD G. P. and BURRIS R. H. (1968) Acetylene reduction by nitrogenfixing blue-green algae. Arch. Microbial. 62, 336348.