Cytotoxicity against insect cells of entomopathogenic fungi

of the genera Hypocrella (anamorph Aschersonia) : possibleagents for biological control

Patricia WATTS1*, Prasat KITTAKOOP1, Sukitaya VEERANONDHA1, Supakit WANASITH1,

Rossukon THONGWICHIAN2,3, Pattama SAISAHA2,3, Suthichai INTAMAS1

and Nigel L. HYWEL-JONES1

1National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Paholyothin Road, Khlong 1,Khlong Luang, Pathum Thani 12120, Thailand.2Junior Science Talent Project, Thailand.3Department of Chemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.E-mail : patwatts@biotec.or.th

Received 26 June 2002; accepted 30 March 2003.

Extracts from entomopathogenic fungi of the genus Hypocrella (7 species) and its anamorph Aschersonia (11 species)

were screened for cytotoxicity to Sf9 and C6/36 insect cells and L929, BHK(21)C13 and HepG2 mammalian cells.Cytotoxic extracts to insect cells (ID50’sf10 mg mlx1) but not to mammalian cells (ID50’so10 mg mlx1) conformedto the criteria of the project and were considered ‘ lead’ extracts for further investigation. ‘Leads’ were found in

two of the Hypocrella species : H. discoidea, and H. tamurai and in three of the Aschersonia species : A. samoensis,A. badia, and A. tamurai. Bioassay-guided fractionation of the cell extract of the fungus A. samoensis BCC 1393 ledto the identification of two known anthraquinone dimers, (+)rugulosin (1) and skyrin (2) which showed selectivetoxicity towards insect cells. (+)Rugulosin (1) and skyrin (2) exhibited strong cytotoxic activity against the insect

cell line Sf9 with respective ID50 values of 1.2 and 9.6 mg mlx1, but showed weak activity toward mammaliancells. This first report of (+)rugulosin (1) and skyrin (2) in A. samoensis is confirmed and demonstrated inanother four strains of A. samoensis isolated in Thailand. The preferential cytotoxicity against Sf9 insect cells

gives evidence that these insect-pathogenic fungi of the Hypocrella/Aschersonia group might be useful as an agentfor pest control.

INTRODUCTION

The ascomycete genus Hypocrella has a pantropicaldistribution and is specific to ‘scale insects ’ (Coccidaeand Aleyrodidae ; Homoptera) (Hywel-Jones & Evans1993). It consists of ca 30 species with usually brightlycoloured stromata (yellow, pink, orange, and scarletespecially). Significantly, the genus has long been con-sidered to have a sole anamorph, Aschersonia (Petch1921). Recently, there have been reports of otheranamorphs (Hywel-Jones & Samuels 1998). While theteleomorph genus Hypocrella now has more than onereported anamorph,Aschersonia is known only to be as-sociated with Hypocrella. A long-running programmesurveyingHypocrella and Aschersonia in natural forestsin Thailand has resulted in a detailed survey of theHypocrella and Aschersonia species in the country

(Evans & Hywel-Jones 1990, Hywel-Jones & Evans1993, Hywel-Jones & Samuels 1998).

Historically, Aschersonia was considered for usein biocontrol (e.g. Fawcett 1908, Petch 1921). Morerecently, much work has been done on the use ofAschersonia for control of scale insects in the protectedcrops environment (Meekes 2001). There have alsobeen increasing reports of the insecticidal propertiesof metabolites from insect fungi (Roberts 1969, Suzukiet al. 1977, Claydon 1978, Kanaoka et al. 1978,Claydon, Grove & Pople 1979, Claydon & Grove 1982,Liu, Boucias &McCoy 1995, Krasnoff &Gibson 1996).However, to date most work on Aschersonia has dealtwith A. aleyrodis for biocontrol.

Since very few Aschersonia spp. and still fewerHypocrella spp. have been investigated in culture, theopportunity was taken to screen extracts of manyHypocrella and Aschersonia species against insect andmammalian cell lines to look for evidence of cytotox-icity. In the present study we investigated extracts from* Corresponding author.

Mycol. Res. 107 (5): 581–586 (May 2003). f The British Mycological Society 581

DOI: 10.1017/S0953756203007846 Printed in the United Kingdom.

11 Aschersonia species and seven Hypocrella speciesfor cytotoxic effects and compared the cytotoxicity ofsixHypocrella/Aschersonia teleomorph/anamorph con-nections. The BIOTEC Culture Collection houses over265 isolates from these two genera, and these were usedin this in-house screen.

MATERIALS AND METHODS

Cultivation of insect fungi and preparation offungus extract

Hypocrella andAschersonia isolates were obtained fromthe BIOTECCulture Collection. Cultures were receivedgrowing on 9 cm Petri plates of Potato Dextrose Agar(PDA). These formed the inoculum. Mycelium was cutfrom these plates and used to inoculate Potato DextroseBroth (PDB, 50 ml). This was cultivated for seven daysat 22–25 xC. A 10% inoculum was then added to 25 mlof the selected growth media: PDB, which has a highpotential to produce metabolites ; M102, a high glucosecontent, fast growth medium; and MM, a minimumessential growth medium, completely defined with yeastextract. The cultures were cultivated at 22–25 x, understatic conditions. The harvested cultures were freeze-dried and extracted with organic solvents (CH2Cl2 :MeOH, 1:1), this was allowed to evaporate and thesample was dissolved in dimethyl sulphoxide (DMSO)at a concentration of 20 mg mlx1.

Isolation of (+)rugulosin (1) and skyrin (2)

For the isolation of anthraquinones the mycelia of thefungus were separated from the culture broth by fil-tration and extracted sequentially with MeOH (400 ml)and CH2Cl2 (400 ml). Both MeOH and CH2Cl2 extractswere pooled and dried to yield a crude extract.The crude extract was subjected to Sephadex LH-20(100 g, 2.5r60 cm) column chromatography, elutedwith MeOH (43 fractions, 75 ml each). Fraction 8 wasre-chromatographed on Sephadex LH-20 (MeOH aseluent) to yield (+)rugulosin (1) (104 mg), while frac-tions 30–35 were combined and re-chromatographedon Sephadex LH-20 (eluted with MeOH) to affordskyrin (2) (25 mg).

Instruments for characterization of compounds andHPLC conditions

1H, 13C, DEPTs, 1H-1H COSY, HMQC (optimized for1JHC=145 Hz) and HMBC (optimized for nJHC=8.0 Hz) spectra were recorded on a Bruker DRX 400,operating at 400.1 MHz for proton and 100.6 MHz forcarbon. The ESITOFMS spectra were obtained from aMicromass LCT mass spectrometer. Optical rotationwas measured on a Jasco DIP370 polarimeter. A C18

column was used for HPLC; reversed phase and mobilephase was a mixture of acetonitrile and water (45:55) ;UV detector was 254 nm; with a flow rate of

1 ml minx1. Under these HPLC conditions, standardcompounds, (+)rugulosin (1) and skyrin (2) exhibitedretention times at 12.6 and 44.8 min, respectively.

Cell culture

Sf9 cells (ovarian cells of the fall armyworm, Spodop-tera frugiperda ; European Collection of Animal CellCulture, ECACC 89070101) were cultured in TC100with 10% foetal calf serum (FCS) and 0.35 g lx1

sodium bicarbonate; C6/36 cells (unspecified cells ofmosquito larvae, Aedes albopictus ; ECACC 8905175)were cultured in MEM, 10% FCS and 1.1 g lx1 sodiumbicarbonate; L929 cells (mouse connective tissue,ECACC 85011425) were cultured in DMEM,10% FCSand 3.75 g lx1 sodium bicarbonate; BHK(21)C13 cells(baby hamster kidney cells, ECACC 85011433) werecultured in GMEM 5% FCS and 2.75 g lx1 sodiumbicarbonate; and HepG2 cells (human, hepatoma,American Type Culture Collection, ATCC HB-8065)were cultured in MEM, 10% FCS and 2.2 g lx1 sodiumbicarbonate. The insect cells were incubated at 28 x.The mammalian cells were incubated at 37 x with 5%CO2, fully humidified. All cells were incubated for 48 hto allow attachment and initiation of growth beforeapplying samples. The stock cells were cultured anti-biotic-free and monitored for mycoplasma. Antibiotics,penicillin and streptomycin, were added to test samples.Cells were plated in 96-well plates at an optimumconcentration (cells/well) in 200 ml/well of medium.Optimum cell seeding density for each cell line was de-termined: 4r103 cells/well for Sf9, 1.6r104 cells/wellfor C6/36 cells ; 5r102 cells/well for L929 cells ; 1r103

cells/well for BHK(21)C13 cells ; and, 1r103 cells/wellfor HepG2 cells.

Cytotoxicity assays

All fungus extracts were initially screened with a %Survival Assay. For this assay a single high concen-tration (100 mg mlx1) dose of each extract was assessedwith the MTT Assay (see below) for cytotoxicity to twoinsect cell lines : Sf9 and C6/36. Cytotoxins were de-fined as any fungus extract that resulted in 10% or lesscell survival. All fungus extracts that were toxic to eitherof the insect cell lines were further assessed to deter-mine the ID50. Finally, any extract with an ID50f10 mg mlx1, for either of the insect cell lines was as-sessed to determine its ID50 against three mammaliancell lines (L929, BHK(21)C13, and HepG2); theextracts that were not toxic to mammalian cell lines(ID50>10 mg mlx1) satisfied the two criteria that ident-ified them as ‘ lead’ compounds for further biopesticideinvestigation. Briefly, the test samples (20 mg mlx1)were diluted 1:200 (for screening test) and 1:100 (forID50 assay) in growth medium giving a final concen-tration of 0.5 or 1% DMSO, respectively. The extractsfor the ID50 assay were serially diluted in growthmedium at a ratio of 1:2, giving concentrations of

Cytotoxicity of Hypocrella (anamorph Aschersonia) 582

200, 100, 50, 25, 12.5, 6.25, 3.125, and 1.56 mg mlx1.The cells were seeded and after 48 h the growth mediumwas aspirated from the wells and replaced with thesample at the various concentrations. The cells wereexposed to the sample for 24 h. Medium with toxinwas then aspirated and replaced with fresh medium,and the cultures were incubated for a further 24 h.The cytotoxicity of the samples was quantified with amodified 3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyltetra-zolium bromide (MTT) colorimetric assay (Plumb,Milroy & Kaye 1989), which measures the metabolicconversion in the mitochondria of tetrazolium (yellow)to formazan (blue). 50 ml of MTT in PBS (2 mg mlx1

for all cell lines except L929, which required 5 mg mlx1)was added to the medium in each well. The cells wereincubated for 4 h and then the medium with MTT wasaspirated from the wells, the formazan was solubilizedwith DMSO and stabilized with 25 ml of Sorensen’sglycine buffer, pH 10.5. The optical density was readwith a plate reader at 570 nm. The average of four wellswas used to determine the mean of each point. The firstand the last columns contained medium only and wereused as the blank. The data were analysed with theSoftMax program (Molecular Devices) to determinethe dose required to inhibit 50% of the metaboliccompetence (ID50) of the cells for each sample. Two con-trols were set up, one with growth medium and thesecond with 1% DMSO in the medium, the resultsshowed no difference between them (data not shown).Under these conditions, mitomycin C exhibited cyto-toxicity to L929 cells with an ID50 of 0.8–2 mg mlx1

(Watts et al. 1996), this value influenced our choice ofthe cytotoxicity cut-off point of 10 mg mlx1. The sep-arated extracts of mycelia and culture broth were as-sayed with the same MTT assay to determine the ID50.

RESULTS

The extracts from insect fungi of the genera Hypocrellaand Aschersonia were screened for cytotoxicity to insectcells and mammalian cells (Tables 1–2). Eight species ofHypocrella, 11 species of the anamorph genus Ascher-sonia, and some unidentified isolates from both genera,were surveyed. Cytotoxic extracts came from threeof the seven Hypocrella species: H. discoidea, H. raci-borski, and H. tamurai. Of these, H. discoidea (3 of 5isolates) and H. tamurai (1 of 2 isolates) were toxic toinsect cells (ID50’sf10 mg mlx1) but not to mammaliancells (ID50’so10 mg mlx1). In the Aschersonia group,extracts from isolates in six species were cytotoxic in thescreening assay:A. badia,A. hypocreoidea,A. oxystoma,A. samoensis, A. tamurai, A. tubulata, and one unidenti-fied isolate. Isolates from three of these species weretoxic to the insect cells but not to the mammalian cells :A. badia (1), A. tamurai (2), and A. samoensis (4).Quantification of cytotoxicity with the insect and mam-malian cell lines for these ‘ lead’ cytotoxic insect fungusextracts is given in Table 3. Of two insect cell linestested, cytotoxicity was limited to Sf9 cells. The culture

conditions also affected expression of the cytotoxinswith toxic extracts only being derived from fungi cul-tured in PDB and MM media (Table 3).

To study the chemicals responsible for the cyto-toxicity, we used isolate A. samoensis BCC 1393 asa model. Bioassay-guided fractionation of the cell ex-tract of the fungus A. samoensis BCC 1393 led to theidentification of two known anthraquinone dimers,(+)rugulosin (1) and skyrin (2) (Fig. 1). Gel filtrationon Sephadex LH-20 of the cell extract of the fungusA. samoensis BCC 1393 furnished dimeric anthra-quinones, (+)rugulosin (1) and skyrin (2), as major pro-ducts. (+)Rugulosin (1) ([a]30D+276 x, 0.35 g 100 mlx1,

Table 1. The Aschersonia and Hypocrella species in the survey with

summary of % Survival screening with insect cells and ID50

results for insect and mammalian cells.

Species

Iso-

lates

% Survival

(Dose:

100 mg mlx1)

insect cells

(<10%)

ID50

Insect cells

(<10 mg mlx1)

Mammalian

cells

(>10 mg mlx1)

Aschersonia

badia 14 1 1 1

coffeae 3 0

confluens 1 0

hypocreoidea 4 0 0

marginata 3 0

oxystoma 6 2 0

paraphysata 1 0

placenta 27 2 0

samoensis 24 14 4 4

tamurai 8 3 3 2

tubulata 17 8 0

spp. 11 1 0

Totals 119 32 8 7

Hypocrella

discoidea 9 5 3 3

javanicus 1

mollii 1 0

raciborski 14 2 0

reineckiana 4 0

schizostachyi 1 0

tamurai 2 2 1 1

tubulata 5 0

spp. 2 0

Totals 39 9 4 4

Table 2. Comparison of the teleomorph/anamorph isolates from

Hypocrella and Aschersonia.

Hypocrella (teleomorphs) Aschersonia (anamorphs)

(cytotoxic/

total) Leads

(cytotoxic/

total) Leads

H. discoidea (5/9) 3 A. samoensis (14/24) 4

H. tamurai (2/2) 1 A. tamurai (3/8) 2

H. tubulata (0/5) A. tubulata (8/17)

H. mollii (0/1) A. confluens (0/1)

H. reineckiana (0/4) A. marginata (0/3)

H. raciborski (2/14) A. placenta (2/27)

Teleomorph

unknown

A. badia (1/14) 1

P. Watts and others 583

CHCl3) was obtained as yellow needles, and its 1H and13C NMR spectral data were in accordance with thosepublished in the literature (Shibata et al. 1968, San-kawa et al. 1968, Kobayashi et al. 1968, Toma et al.1975). Skyrin (2) was obtained as a red amorphoussolid; its 1H and 13C NMR data agreed to that reported

in the literature (Toma et al. 1975). Purified (+)rugu-losin (1) and skyrin (2) exhibited strong cytotoxicactivity against the insect cell line Sf9 with respectiveID50 values of 1.2 and 9.6 mg mlx1, but showed weakactivity towards the mosquito (C6/36), human hepa-toma (HepG2) and mouse (L929) cell lines (Table 4).

Five other Aschersonia samoensis isolates wereexamined for the presence or absence of (+)rugulosin(1) and skyrin (2) (Table 5). As before, a cytotoxic re-sponse was only demonstrated against Sf9 cells and notagainst C6/36 cells (data not shown). (+)Rugulosin (1)and skyrin (2) were demonstrated in the mycelia ofthree cytotoxic extracts, but not in the extracts fromtwo cultures that were not cytotoxic (Table 5).

DISCUSSION

Species ofHypocrella and their Aschersonia anamorphsproved to be cytotoxic to Sf9 insect cell lines but notto C6/36 insect cell lines or mammalian cell lines.That these fungi were cytotoxic reflects partially theteleomorph/anamorph relationship of the species:H. discoidea is the teleomorph of A. samoensis, andH. tamurai that of A. tamurai (Table 2). This is notunexpected since the teleomorphs revert to the asexualphase of reproduction in culture.

All the cytotoxic extracts from A. samoensis myceliawere only cytotoxic to Sf9 cells and not to C6/36 cells,corroborating the specificity demonstrated by thepurified (+)rugulosin (1) and skyrin (2) and consistentwith whole-culture extract data. The presence of thetoxins in the mycelia supports the idea that these arenon-secreted toxins. The presence of (+)rugulosin (1)and skyrin (2) previously unreported in A. samoensishas now been demonstrated in four strains of A. samo-ensis isolated in Thailand.

(+)Rugulosin (1) and skyrin (2) are known metab-olites produced by several fungi including those of

Table 3. Quantification of toxicity of ‘ lead’ compounds.

Species

Growth

medium

ID50 (mg mlx1)¡S.E. (n=3)

Insect cells Mammalian cells

Sf9 C6/36 L929 BHK HepG2

Aschersonia badia BCC 1846 MM 10¡0.8 >200 >200 >200 180¡5

A. samoensis BCC 1616 PDB 4¡2 80¡3 48¡2 21¡0.4 64¡17

MM 3¡2 80.9¡3.3 >200 22¡0.3 94¡4

A. samoensis BCC 1749 PDB 10¡2 >200 >200 76¡4 >200

A. samoensis BCC 1605 PDB 6¡1 90¡3 27¡2 42¡3 63¡15

A. samoensis BCC 1393 PDB 3¡0.4 >200 153¡14 50¡5.1 65¡11

A. tamurai BCC 1767 MM 9¡0.1 87¡4 >200 41¡1.7 46¡4

A. tamurai BCC 1464 PDB 7¡0.1 >200 159¡0.2 60¡2.2 70¡3.9

MM 3¡0.2 22.4¡1.7 98¡11.7 15¡3.2 21¡0.9

Hypocrella discoidea BCC 2203 MM 6¡0.2 >200 >200 107¡7 >200

H. discoidea BCC 1606 PDB 5¡1 169¡28 104¡3 36¡2 69¡5.3

H. discoidea BCC 2097 PDB 5¡1 92¡2 50¡3 30¡1 78¡9.3

H. tamurai BCC 1680 PDB 3¡0.3 >200 32¡0.3 37¡3 33¡1.6

MM 2¡0.04 >200 40¡3 20¡2 25¡1

OH

O

O

O

1, (+)rugulosin

H

HOH

HOH

H

2, skyrin

OH OHO

O

OH

HO

OH OH

O

O

O

O

Fig. 1. Chemical structures of (+)rugulosin (1) and skyrin (2).

Cytotoxicity of Hypocrella (anamorph Aschersonia) 584

Penicillium (Shibata et al. 1968, Sankawa et al. 1968,Kobayashi et al. 1968, Toma et al. 1975, Bouhet et al.1976), pyrenomycetes (Carey & Nair 1975), basidio-mycetes (Melvyn, Alberto & McKenzie 1988, Melvyn& Alberto 1991), endophytes (Calhoun et al. 1992), andlichenized fungi (Huneck & Schreiber 1974). However,to our knowledge, there have been no reports on thepresence of these anthraquinone dimers in insect-pathogenic fungi. (+)Rugulosin (1) and skyrin (2) werepreviously reported to possess toxic and mutagenicactivities (Takashi et al. 1975, Krivobok et al. 1992).Skyrin (2) and its analogues have been claimed for thetreatment of Diabetes mellitus (West et al. 1994) while(+)rugulosin (1) was found to have insecticidal activity(Dobias, Betina & Nemec 1980).

The culture conditions of the fungus also affected thecytotoxicity. Our past experience has shown that fungicultured in PDB often express high levels of metab-olites. TheMMmedium is a minimum, defined mediumand the fungi cultured in this medium grew slowly andwere likely to be under stress. It is, however, oftensuggested that the expression of toxic metabolites in-creases in slow growth, stressful conditions, rather thanin a fast growth (high glucose) environment where theemphasis is on cell division.

Prior to this work, the only other report of skyrinfrom the Hypocreales was from the fungicolous Hypo-myces trichothecoides (Carey & Nair 1975). We can findno record of rugulosin having been reported from theHypocreales making this the first record. The genusHypocrella has been the subject of recent molecularstudies (Artjariyasripong 1999, Artjariyasripong et al.2001, Lutthisungneon, Spatafora & Hywel-Jones, un-publ.). Artjariyasripong et al. (2001) confirmed that

Hypocrella is a ‘recent ’ genus within the Clavicipita-ceae. Hypocrella can be divided into those species thatproduce and discharge whole ascospores (Hywel-Jones& Evans 1993) and those which discharge part-spores.Most of the positive isolates were from Hypocrellaisolates (or their Aschersonia anamorphs) with wholeascospores : 18 of 32 of Aschersonia, and 7 of 9 ofHypocrella. Significantly, all the ‘ leads ’ were fromthe ‘whole-ascospore ’ group (Table 2).

Safe biopesticides should be toxic only to insects andnot other organisms such as mammals. The 11 ‘ lead’extracts found in this survey satisfied both criteria.Species specificity would be an additional safety feature.The preferential cytotoxicity against Sf9 insect cellsgives further evidence that these insect-pathogenicfungi of theHypocrella/Aschersonia groupmight be use-ful as agents for pest control. In the present work, wefound that purified (+)rugulosin (1) and skyrin (2) ex-hibited strong cytotoxic activity against the insect cellline Sf9 but showed weak activity towards the mosquito(C6/36), human hepatoma (HepG2) and mouse (L929)cell lines (Table 4). It should be noted that L929 cellsare regarded as a standard mammalian test cell line(USP24).

ACKNOWLEDGEMENTS

We are indebted to the Biodiversity Research and Training Program

(BRT) and BIOTEC/NSTDA for financial support. We are grateful

to the Fermentation Technology Laboratory for mass cultivation of

the fungi.

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Corresponding Editor: N. Magan

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