Eicosanoid regulation of oosporogenesis by Lagenidium Giganteum

prostaglandins, Leukotrienes and Medicine 23: 173-178, 1986

EICOSANOID REGULATION OF OOSPOEOGENESIS BY LAGENIDIUM GIGANTEUW

James L. Eerwin, Christopher A. Simmons, and Bobert IC.Washino, Department of EntOIUdOgy, University of California, Davis, CA 95616 [reprint requests to JLK] ABSTRACT

Cell proliferation is inhibited in many biological systems by

lipid peroxides and related products derived from polyunsaturated fatty acids. Using developuentally synchronized cultures of Laqenidium yiqanteum (Oomycetes: Lagenidiales), a facultative parasite of mosquito larvae, it has been documented that oxidative lipid metabolism is necesm sary for the induction and subsequent maturation of its sexual stage, the oospore. Addition of lipoxygenase inhibitors to liquid cultures of the fungus results in the stage-specific disruption of antheridial induction, gametangial fusion, induction of meiosis and spore cell wall formation. Oosporogenesis is inhibited by these compounds at concentrations which have no discernible effect on mycelial viability or asexual reproduction. Cyclooxygenase inhibition had comparable effects using ibuprofen and to a lesser extent with indomethacin. Phenylbutamone and the salicylates affected oosporogenesis only at concentrations which decreased asexual reproduction or mycelial viability. The inhibitory effects of NDGA on oosporogenesis could be reversed using partly purified eicosanoid extracts from growth media. INTRODUCTION

Laqenidium qiqanteum (Oomycetes: Lagenidiales) is a facultative fungal parasite of mosquito larvae. Our laboratory has been using develo&zaentallysynchronized cultures of this entomopathogenic fungus to investigate lipid-mediated physiological processes involved in the induction and maturation of oospores, the sexual stage of the fungus Exogenous polyunsaturated fatty acids inhibit cell proliferat11 tion in many biological systems, with lipid peroxidation implicated in most of these processes [2-71. Preliminary investigations suggest that fatty acid oxiaative metabolites promote rather than inhibit differentiation by & giqanteum. The following report documents a role for lipoxyqenase and possibly cyclooxygenase products in oosporogenesis by this facultative mosquito parasite. ??

173

MATERIALS

AND

METHODS

Fungal culture The California strain of L. giganteum (ATCC 52675) was used in these investigations [8]. The- synchronized liquid culture system previously described [l] was used to assess the effects of stagespecific addition of lipxygenase and cyclooxygenase inhibitors on oosporogenesis. Details of this system are briefly listed in Table 1. Induction of zoosporogenesis and monitoring of viable oospore formation were done as previously described [8]. Inhibitors Cyclooxygenase and lipoxygenase inhibitors were obtained from Sigma Chemical Co., St. Louis, MO. The various compounds were added to cultures dissolved or suspended in sterile distilled water from preweighed glass vials. In order to document that the effects of cyclooxygenase and lipoxygenase inhibitors were not due to nonspecific disruption of metabolic processes or toxic side effects, an assessment of asexual reproductive capabilities of the treated cultures was made using mosquito mortality as an indirect assay [ll. Extraction of eicosanoids Culture media contained more oxidative fatty acid metabolites than the fungal mycelium as monitored by TLC according to Salmon and Flower 191. The media was acidified with formic acid (pH 3.5) and extracted twice with an equal volume of ethyl acetate. The solvent was evaporated and residual lipid was layered on a 6 ml octyl (CS) disposable extraction column (Baker). The column was washed with 12 ml water and the eicosanoids eluted with 12 ml methanol. Methanol was evaporated and the lipids resuspended in 100 ~1 absolute ethanol per 100 ml extracted media. The ethanolic solutions (100 ~1/100 ml culture media) were used in experiments in which the extracts were used in attempts to overcome lipoxygenase or cyclooxygenase inhibition. RESULTS

Lipoxygenase and cyclooxygenase inhibition of oosporogenesis Stage specific addition of five compounds known to inhibit mammalian lipoxygenases to cultures of L. giganteum blocked several discrete steps in the induction and maturation of the sexual stage of the fungus (Table 1). The developmental steps affected included induction of antheridia (32 hr and 36 hr additions), fusion of antheridia and oogonia (32 hr additions), spore cell wall formation (40 hr additions) and oospore maturation following cell wall formation (48 hr additions). At the concentrations listed in Table 1, none of the lipoxygenase inhibitors had an adverse effect on mycelial growth or asexual reproduction. When these compounds were added before 32 hr postinoculation, mycelial growth and development were inhibited to various degrees (unpubl. observ.) Of the five cyclooxygenase inhibitors evaluated, only ibuprofen

174

Effcfr

Table 1.

Of upcxy*cn.se

.na c,“U”*,(lsulrr

L.,.._”_.__ _ __. ;

~“~

_

Oo~pore Yield (x10-6)/2 Viability’ Addirion Tim (h after lnoiulation)c

32

40

36

40

3.6’0.4/U1+7 ab,t inhlbiLore nordihydroguaiarcric acid

0.02 0.025 0.05 ?? ~licylhydrox~micacid 1.0 1.25 propglg~ll~tc 0.05 1.0 a-aaphchol 0.05 0.1 c*culetio 1.0 Cyclooxygenasc inhibitors ikrprof en 1.o indomamin 0.5 1.0 pbenylbutuone 1 .o 1.5 2.0 aspirin 0.25 0.4 1.o 1.5 2.0 ?? alicylaLe 0.25 0.5 1 .o 1.5

0.33tO.21/6+6 0.02*0.02/0 o/o

0.67+0.26/16?6 0.07to.O2/3L-

O.O7K~07/27 2

3.9*0.5/02t2

0.02?0.02/0

4.1+0.2/32+10 3.3'0.1138'3

0.4*0.2/14'14 0.14~.06/0 0.1YJ.1/0

o/o

o/o

O.O3~.03/22Cl2

__ 3.39.7/20+16 3.4D.513r3

3.4%3.2/27f2 1.220.1114fl

0.03'0.03/40+25 0.03t0.02/0 2.Sto.l/39fo 010

-

2.7io.4/7+4

3.7~.4/11~10

0.36+0.04/o

3.5'0.417f2

0.3M.2/4*3

3.7$0.2/0.3'0.6 3.9*0.2/65+2 3.at0.3/3t3e

6.6%i.O3/0 010

3.6iO.4/5b~16 O.WO.bllc)~d

__ 3.9t0.4/1bflb

TOXiC

2SK).1/4bt7 1.3~.2/53~ Toxic 2.0M.5132+3 Toxic

b.lS.b/8Ots Toxic -

3.5+0.2/68t3 i.2to.ali7*i7d __

4.3%.1148*3

3.br0.3152'3

3.5+0.4/71t6

3.l+o.el36+8

3.9t0.2/78U TOXiC __

?OXiC

. Oosporc yield per 100 ml of medium+ 6 ml zoospore suspension (mean f std. dev.). All media wrc teated rhrce tin*. Perceota6ebviability deter&U& ~81% previouslydemcribcd ~rphologic~l charactcristlcsIS]. Added .t rhe wrioua Liaea suepcodrdor dissolved in ?? tctile dclonirrd mter. c Stageix of fungal development .s outlined in detail clsevherc 111: 32 h - Hycclial growth nearly caplctc. Hyphal ccptation ca. 85% complete. 36 h - Initietfonof aaLheridia k6ins. 38 h - Fusion of anrheridia with ongonla bcgine and meloais is initiated. 40 h - Primordial oorpores (withoof cell walls) form dis:rctc sphrres of cytoplerm. 48 h Pcrriotcnt #pore cell wall* are prc.ent. 46 h - 5 daya - OoQmre rsLuraLion continues. d Toxic to 20-901of the Zun6al myceliu. Toxic denotes laO% mortality. * Reduction of ?? ~ex\ul reproduction .s monitored by sentinel larval mortality by 20-100X IL~AL~PC to control*.

art13+o or

n lesser

maturation

viability methacin ionophore

indomethacin

oospore

constantly

formation

without

reduced

adversely

the

induction

affecting

mycelial

or asexual reproduction (Table 1). The results with indoare somewhat suspect since this compound acts as a calcium in some biological systems [lo]. Disruption of calcium

metabolism Reversal

extent of

has of

a profound

lipoxygenase

effect and

on

all

stages

cyclooxygenase

Partly purified eicosanoid extracts pared and their effect on oosporogenesis

of

oosporogenesis.

inhibition from

culture

media

were

pre-

in cultures inhibited by NDGA and, to a lesser extent, ibuprofen, were evaluated. Stage specific addition of extracts isolated from cultures at different developmental stages could overcome some of the effects of NDGA on oosporogenesis depending upon the time after inoculation that they were added (Table 2). When cultures were inhibited at 32 hr post-

175

Table 2.

Reversal of NDGA inhibition of oosporogenesis using culture medium extracts.

Treatmentb

Gospore yield (x~O'~)/% Viabilitya

Control 32 h inhibition/ 32 h addition control 32 h extract 38 h extract 40 h extract 48 h extract 36 h inhibition/ 36 h addition control 36 h extract 42 h inhibition/ 42 h addition control 36 h extract 48 h inhibition/ 48 h addition control 36 h extract

3.3+0.4/82+9 -

0.035/0 1.9/0.3 0.8/9 0.1/o 0.04/o

0.07/l 2.6/3

0.2/4 3.1/7

3.9/11 3.5/5

a See Table 1, footnote a. Each figure is the mean of two experiments. Control is mean + std. pv. of 4 cultures run concurrent with the different treatments. NDGA was added at 0.025 mM except for the 48 h inhibitions which used 0.05 mM. Extracts were added in 100 ml absolute EtOH using the eicosanoid extract from a similar volume of culture media collected at different times following inoculation. See Table 1, footnote c for developmental stages corresponding to various times after medium inoculation. inoculation, medium extracts of 32 hr-old cultures increased oosporogenesis initiation from less than 1% to ca. 60% of the control value. There was progressively less inhibition reversal from 38, 40 and 48 hr culture extracts (Table 2). The 32 hr extracts allowed completion of all develogmsntal steps through the formation of a persistent oospore wall, but in no instance was the viability of oospores sigificantly increased. These results suggest that stage-specific production of different lipoxygenase products occurs during the initiation and subsequent maturation of & giganteum oospores. DISCUSSION From these initial observations it appears that 2. giaa.nteugl produces eicosanoids which regulate the induction and maturation of oospores. A specific role for lipoxygenase products is evident clueto

176

the specificity of their effects on se-1 reproduction without apparent effect on asexual reproduction or mycelial viability. The role of cyclooxygenase products is less clear due to the deleterious effects of especially the salicylates and phenylbutasone on mycelium and asexual reproduction before inhibition of the various stages of oosporogenesis is evident. This could be due to a multiple role for cyclooxygenase products in vegetative growth as well aa sexual and asexual reproduction; alternately it may reflect the sensitivity of &. giwm to the toxic side effects of these compounds. The results using indomethacin must be considered suspect in the absence of further information because of its possible action as a calcium ionophore [lo] which would independently disrupt oosporogenesis. Ibuprofen is the only other cyclooxyqenase inhibitor evaluated which consistently exhibited specific inhibition of sexual reproduction and a role for cyclooxygenase products in oosporogenesis cannot bs discounted. A single report recently suggested such a role for cyclooxygenase products in regulating growth and reproduction by several oomycetous fungi closely related to k. gianteum In that study growth inhibition of the watermold Achlya [Ill caroliniana (Comycetes: Saproleqniales) by indomethacin was relieved by 40% following addition of PGF,o to culture media at 2 &ml. Further references to eicosanoids in fungi have not been found. ??

Research on eicosanoids in biology have largely centered on mamalian systems (12-141. More recently roles for prostaqlandins and/or leukotrienes have been documented for other animal systems, e.g. atimulation of Schistosoma mansoni cercarial penetration 115,161 and regulation of reproduction and a number of other developmental processes in insects [17,18]. It appears likely that as other non-mammalian biological systems are examined, further examples of eicosanoid occurrence will be documented. ACKNOWLEDGEMENTS We are grateful to the following people for providing initial guidance in our isolation of eicosanoids from &. giqanteum: E. J. Corey, M. Rigaud and V. Ziboh. REFERFWCES 1.

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