Experimental Parasitology 139 (2014) 33–41

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A novel high-throughput nematicidal assay using embryo cells and larvae of Caenorhabditis elegans Yiling Lai a,b, Meichun Xiang a,⇑, Shuchun Liu a, Erwei Li a, Yongsheng Che a,c, Xingzhong Liu a,⇑ a

State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, No. 3 Park 1, Beichen West Road, Chaoyang District, Beijing 100101, China University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China c Beijing Institute of Pharmacology and Toxicology, No. 27 Taiping Road, Haidian District, Beijing 100850, China b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 C. elegans cell-based MTS assay

facilitates high-throughput cytotoxicity screening.  There is positive correlation between cell viability and nematode viability.  A two-step method based on cell assay coupled with larval assay was established.  The method can be used for highthroughput screening of potential nematicides.

a r t i c l e

i n f o

Article history: Received 17 November 2013 Received in revised form 12 February 2014 Accepted 18 February 2014 Available online 1 March 2014 Keywords: Caenorhabditis elegans Cytotoxicity High-throughput screening Nematicides

a b s t r a c t Human health safety and environmental concerns have resulted in the widespread deregistration of several agronomic important nematicides. New and safer nematicides are urgently needed. However, a highthroughput bioassay for screening potential nematicides has not been established. We developed a twostep high-throughput nematicidal screening method to combine a cell-based MTS colorimetric assay with Caenorhabditis elegans embryo cells for preliminary cytotoxicity screening (step 1) followed by in vitro larval assay for nematicidal activity (step 2). Based on three conventional nematicides’ test, high correlations were obtained between cell viability and larval viability and ‘‘r’’ values were 0.78 for Avermectin, 0.95 for Fosthiazate, and 0.65 for Formaldehyde solution. Further assays with 60 fungal secondary metabolites (extracts, fractions and pure compounds) also demonstrated the high correlation between cell viability and larval viability (r = 0.60) and between the C. elegans cell viability and the juvenile viability of soybean cyst nematode Heterodera glycines (r = 0.48) and pine wood nematode Bursaphelenchus xylophilus (r = 0.56). Six metabolites with high cytotoxicity have performed high larval mortality with a LC50 range of 6.8–500 lg/ml. These results indicate that the proposed two-step screening assay represents an efficient and labor-saving method for screening natural nematicidal products. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction ⇑ Corresponding authors. Address: Institute of Microbiology (IM), Chinese Academy of Sciences (CAS), No. 3, 1st Beichen West Road, Chaoyang District, Beijing 100101, China. Fax: +86 10 64807505 (M. Xiang). E-mail addresses: [email protected] (M. Xiang), [email protected] (X. Liu). http://dx.doi.org/10.1016/j.exppara.2014.02.012 0014-4894/Ó 2014 Elsevier Inc. All rights reserved.

Given the environmental concerns about the use of conventional chemical nematicides (Oka et al., 2000), new and safer nematicidal molecules are needed for the control of plant-parasitic

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Y. Lai et al. / Experimental Parasitology 139 (2014) 33–41

nematodes. The secondary metabolites produced by fungi have been regarded as an important source of novel natural products that might be developed into effective nematicides with low plant and human toxicity and with low persistence in the environment (Siddiqui and Mahmood, 1996). Because the number of secondary metabolites produced by fungi is very large, a reliable highthroughput screening method is needed to evaluate fungal secondary metabolites and other natural products for nematicidal activity. The ability to rapidly and correctly determine nematode status (alive or dead) is an essential feature of a high-throughput screening method for detection of nematicidal activity. Previous studies have tried various methods and techniques to distinguish paralyzed or dead nematodes from living nematodes. One method is to count the number of living and dead nematodes by directly observing the specimens with a microscope (Becker et al., 1988) or by reviewing microscopic images (Breger et al., 2007); in both approaches, nematodes that are mobile or curled are considered living and those that are immobile and straight are considered dead (Breger et al., 2007; Okoli et al., 2009). A more effective and widely used method is staining with fluorescein (Gill et al., 2003; Hunt et al., 2012) or dyes (Donald and Niblack, 1994; Jatala, 1975; Meyer et al., 1988), but these reactions often require hours to several days, depending on the type of nematodes; in addition, stains can be toxic to nematodes. In some studies, researchers have determined whether immobile nematodes are living or dead by observing the response after the nematodes are touched with a fine needle (Dong et al., 2006), which is a difficult and time-consuming procedure. Yet another method is to observe the response to NaOH (Chen and Dickson, 2000), but this requires that the nematodes be visually assessed within a very short time after treatment. None of these methods represents a rapid and reliable way to screen many compounds at the same time. Another problem is that the most of plant-parasite nematodes such as root knot nematodes and cyst nematodes are difficult to mass produce so as to obtain sufficient specimens for high-throughput screening. The bacterivorous nematode Caenorhabditis elegans has often been used as a model in toxicology studies because it produces a large number of progeny in a short time in easily maintained microplate cultures (Leung et al., 2008). Furthermore, researchers have developed a primary C. elegans cell culture system for studying cell differentiation and development (Christensen et al., 2002), and such a system could provide nematode embryo cells for cytotoxicity analyses. Because embryo cells have the potential to differentiate and develop into adult nematodes, we suspect that natural products that are toxic to embryo cells might also be harmful to nematodes. Because it is simple, rapid, convenient, and reliable, the colorimetric method based on tetrazolium salt reduction has been extensively applied in various high-throughput screening systems to quantify cell proliferation, cytotoxicity, and chemosensitivity (Mosmann, 1983). The novel tetrazolium compound 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfonyl)-2H-tetrazolium (MTS) is converted by mitochondrial dehydrogenase in metabolically active cells into an aqueous and soluble colored formazan product that is easily quantified by spectrophotometry. The amount of the colored formazan product, estimated at its maximal absorbance at 490 nm, is directly proportional to the number of living cells in a culture (Mosmann, 1983). Although the colorimetric method has been frequently used with a view toward developing a semi-automated and highthroughput assay in a wide range of cell types, including cancer cell lines (O’Toole et al., 2003; Sekhon et al., 2008), bacteria (Kuda et al., 2004), algal cultures (Capasso et al., 2003), and Leishmania promastigotes (Berg et al., 1994; Ganguly et al., 2006), we were unable to use this method in an assay with C. elegans nematodes (rather

than with nematode cell cultures) because of the large size of the nematodes (unpublished data). In this study, we first developed a high-throughput screening assay based on the MTS colorimetric assay to determine the effects of fungal extracts and pure compounds from such extracts on C. elegans embryo cells. The potential toxic extracts and compounds were further tested for their nematicidal activity by microscopic observation of treated C. elegans larvae. We demonstrate that the combination of a cell-based MTS colorimetric assay with C. elegans embryo cells for preliminary cytotoxicity screening followed by an in vitro assay with larvae represents a highly efficient method for screening natural products for nematicidal activity. 2. Materials and methods 2.1. Preparation of C. elegans embryo cells The wild type of C. elegans Bristol strain N2 was routinely cultured and maintained following the protocol described by Brenner with minor modifications (Brenner, 1974). The nematodes were fed Escherichia coli OP50 in liquid nematode growth medium (NGM) at 22 °C for 3 days until many gravid adults appeared. Embryo cells were then obtained as described by Strange et al. (2007). Briefly, the gravid adults were collected and lysed in a sodium hypochlorite solution to isolate the eggs. The eggs were then treated with a chitinase solution in an Eppendorf microtube; the microtube was gently rocked at room temperature for 80 min, at which time more than 80% of the egg shells had been digested. The embryo cells were pelleted and then resuspended in L-15 cell culture medium (Gibco, Grand Island, NY) that lacked phenol red and that contained 10% (v/v) heat-inactivated fetal bovine serum, 50 U/ml penicillin, and 50 lg/ml streptomycin. Subsequently, the cells were gently dissociated by repeatedly pipetting the cell suspension against the side of the microtube. The cell suspensions were filtered to remove remaining intact eggs, cell clumps, as well as hatched larvae. The final concentration of cells in the suspension was determined with a hemocytometer and a light microscope. 2.2. Preparation of the second-stage larvae (L2) of C. elegans C. elegans eggs that were obtained as described in the previous section were hatched overnight at 22 °C without bacteria to obtain the first-stage larvae (L1). The L1 were fed E. coli OP50. After 16– 18 h (Xie et al., 2010), the L1 had molted to L2 and were pelleted by centrifugation at 350g for 5 min and then thoroughly washed with sterile water. The suspension was adjusted to 4  103 L2/ml. 2.3. Preparation of Heterodera glycines and Bursaphelenchus xylophilus The cysts of soybean cyst nematode H. glycines were extracted from soil in the suburb of Daqing, Heilongjiang, China. The second stage juveniles (J2s) were obtained from fresh eggs by the method of Liu and Chen (2000) and were used immediately. The pine wood nematode B. xylophilus (mixed stages) were obtained from culturing medium using the Baermann funnel technique, washed by sterile water for 3 times and were used immediately (Dong et al., 2006). 2.4. Fungal secondary metabolites and conventional nematicides The inoculum was prepared by culturing each fungus (Table 1) in 50 ml liquid medium (0.4% glucose, 1% malt extract, and 0.4% yeast extract, pH 6.5) at 25 °C on a rotary shaker at 170 rpm. After

Table 1 The effects of 60 fungal secondary metabolites on C. elegans embryo cells, C. elegans L2, H. glycines and B. xylophilus. Metabolite state

Metabolite source (fungus or fungal extract)

Concentration

XC01

Crude extract Crude extract Crude extract Crude extract Crude extract Crude extract Crude extract Crude extract Crude extract Crude extract Crude extract Crude extract Crude extract Crude extract Crude extract Crude extract Crude extract Crude extract Crude extract Crude extract Fraction Fraction Fraction Fraction Fraction Fraction Fraction Fraction Fraction Fraction Fraction Fraction Fraction Fraction

Pestalotiopsis gracilis

1 mg/ml

34.4 ± 10.8

⁄⁄

Pestalotiopsis microspora

1 mg/ml

7.3 ± 2.2

⁄⁄

13.1 ± 3.5

⁄⁄

XC02 XC03 XC04 XC05 XC06 XC07 XC08 XC09 XC10 XC11 XC12 XC13 XC14 XC15 XC16 XC17 XC18 XC19 XC20 XC21 XC22 XC23 XC24 XC25 XC26 XC27 XC28 XC29 XC30 XC31 XC32 XC33 XC34

Cytospora sp.

1 mg/ml

Cell viability (%, mean ± SD)

Difference with controla

L2 viability (%, mean ± SD)

Difference with controlb

Difference between cell viability and L2 viabilityc

H. glycines viability (%, mean ± SD)

B. xylophilus viability (%, mean ± SD)

71.9 ± 2.0

⁄⁄

⁄⁄

47.1 ± 3.8

81.5 ± 4.6

36.0 ± 1.0

⁄⁄

⁄⁄

52.3 ± 4.4

93.2 ± 3.3

42.0 ± 3.7

⁄⁄

⁄⁄

59.6 ± 6.8

78.9 ± 8.4

70.6 ± 5.4

⁄⁄

⁄⁄

64.2 ± 3.9

88.0 ± 7.0

Psathyrella gracilis

1 mg/ml

26.9 ± 2.4

⁄⁄

Beauveria bassiana

1 mg/ml

17.7 ± 4.7

⁄⁄

24.2 ± 2.9

⁄⁄

55.6 ± 0.3

82.1 ± 6.1

Pestalotiopsis adusta

1 mg/ml

23.8 ± 5.9

⁄⁄

37.7 ± 6.3

⁄⁄

46.9 ± 0.5

87.4 ± 12.8

37.6 ± 6.8

⁄⁄

42.0 ± 7.9

65.4 ± 2.9

Trichoderma velutinum

1 mg/ml

33.7 ± 3.9

⁄⁄

Pestalotiopsis besseyi

1 mg/ml

19.5 ± 7.1

⁄⁄

1.2 ± 1.4

⁄⁄

⁄

33.8 ± 6.5

46.0 ± 7.8

Pestalotiopsis paeoniae

1 mg/ml

36.5 ± 1.1

⁄⁄

27.8 ± 3.3

⁄⁄

⁄

21.5 ± 3.0

69.7 ± 2.9

Preussia africana

1 mg/ml

97.5 ± 4.6

33.5 ± 3.9

102.8 ± 2.9

⁄

37.8 ± 0.5

72.8 ± 7.2

93.3 ± 5.2

Leptosphaeria sp.

1 mg/ml

21.9 ± 1.7

⁄⁄

16.1 ± 1.4

⁄⁄

Stilbella sp.

1 mg/ml

14.4 ± 4.7

⁄⁄

51.5 ± 3.0

⁄⁄

⁄⁄

31.5 ± 4.3

55.2 ± 10.4

Pholiota sp.

1 mg/ml

23.4 ± 2.2

⁄⁄

62.4 ± 2.2

⁄⁄

⁄⁄

38.2 ± 6.0

57.0 ± 6.9

42.7 ± 4.7

⁄⁄

28.0 ± 4.3

32.9 ± 6.9

Tolypocladium inflatum

1 mg/ml

55.1 ± 6.7

⁄⁄

Pestalotiopsis microspora

1 mg/ml

43.2 ± 5.8

⁄⁄

10.7 ± 5.1

⁄⁄

⁄⁄

21.2 ± 4.4

37.5 ± 9.3

Pestalotiopsis yunnanensis

1 mg/ml

47.9 ± 9.0

⁄⁄

79.1 ± 4.4

⁄

⁄⁄

32.9 ± 3.5

75.2 ± 16.7

Pestalotiopsis fici

1 mg/ml

90.7 ± 9.8

96.1 ± 1.0

⁄⁄

33.0 ± 2.7

102.9 ± 1.6

7.9 ± 1.4

⁄⁄

⁄⁄

62.1 ± 5.8

9.6 ± 2.9

Purpureocillium lilacinum

1 mg/ml

26.6 ± 4.4

⁄⁄

Scopulariopsis chartarum

1 mg/ml

16.0 ± 13.6

⁄⁄

39.5 ± 4.2

⁄⁄

⁄⁄

35.1 ± 4.7

48.3 ± 12.2

Paecilomyces farinosus

1 mg/ml

7.2 ± 2.6

⁄⁄

25.2 ± 3.5

⁄⁄

⁄⁄

45.5 ± 6.3

72.8 ± 8.6

3.6 ± 0.7 4.8 ± 1.7 10.1 ± 1.3 13.5 ± 2.7 18.7 ± 2.2 15.8 ± 1.1 26.2 ± 0.9 18.5 ± 2.0 12.5 ± 0.8 10.6 ± 2.9 24.1 ± 1.8 11.6 ± 1.1 58.2 ± 15.5 22.7 ± 1.3

⁄⁄

42.8 ± 4.2 8.3 ± 3.2 76.3 ± 2.2 53.5 ± 5.2 33.0 ± 3.7 4.4 ± 3.6 19.4 ± 6.9 0.0 ± 0.0 36.2 ± 1.3 34.6 ± 4.1 33.4 ± 8.15 48.6 ± 3.47 93.5 ± 8.4 76.8 ± 12.7

⁄⁄

⁄⁄

23.5 ± 4.2 18.9 ± 4.2 41.0 ± 1.6 34.4 ± 4.2 32.0 ± 4.6 23.3 ± 3.0 9.6 ± 3.3 11.3 ± 1.4 20.1 ± 4.5 32.8 ± 2.8 45.7 ± 2.2 37.5 ± 2.6 48.9 ± 0.2 28.0 ± 2.1

48.6 ± 2.9 53.0 ± 18.2 80.9 ± 8.0 75.0 ± 7.1 52.7 ± 8.5 95.9 ± 10.4 0.0 ± 0.0 0.0 ± 0.0 59.6 ± 7.9 69.0 ± 8.7 63.0 ± 2.1 76.6 ± 2.8 99.6 ± 2.0 68.7 ± 11.9

XC18 XC18 XC18 XC18 XC18 XC18 XC18 XC18 XC14 XC14 XC14 XC14 XC14 XC14

1 mg/ml 1 mg/ml 1 mg/ml 1 mg/ml 1 mg/ml 1 mg/ml 1 mg/ml 1 mg/ml 1 mg/ml 1 mg/ml 1 mg/ml 1 mg/ml 1 mg/ml 1 mg/ml

⁄⁄ ⁄⁄ ⁄⁄ ⁄⁄ ⁄⁄ ⁄⁄ ⁄⁄ ⁄⁄ ⁄⁄ ⁄⁄ ⁄⁄ ⁄ ⁄⁄

⁄⁄ ⁄⁄

⁄⁄

⁄⁄

⁄⁄

⁄⁄

⁄⁄

⁄⁄

⁄⁄

⁄⁄ ⁄⁄

⁄⁄

⁄⁄

⁄⁄

⁄⁄

⁄⁄

⁄⁄ ⁄⁄

⁄⁄ ⁄

⁄

⁄⁄

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Y. Lai et al. / Experimental Parasitology 139 (2014) 33–41

Metabolite code

36

Table 1 (continued) Metabolite state

Metabolite source (fungus or fungal extract)

Concentration

Cell viability (%, mean ± SD)

Difference with controla

L2 viability (%, mean ± SD)

Difference with controlb

Difference between cell viability and L2 viabilityc

H. glycines viability (%, mean ± SD)

B. xylophilus viability (%, mean ± SD)

XC35 XC36 XC37 XC38 XC39 XC40 XC41 XC42 XC43 XC44 XC45 XC46 XC47 XC48 XC49 XC50 XC51 XC52 XC53 XC54 XC55 XC56 XC57 XC58 XC59 XC60 DMSO Control

Fraction Fraction Fraction Fraction Fraction Fraction Compound Compound Compound Compound Compound Compound Compound Compound Compound Compound Compound Compound Compound Compound Compound Compound Compound Compound Compound Compound

XC07 XC07 XC07 XC07 XC07 XC07 XC14/XC18 XC18 XC18 XC07/XC18 XC16 XC08 XC16 XC15/XC16 XC08 XC19 XC16 XC15 XC15 XC14 XC14 XC14 XC14 Pestalotiopsis zonata Humicola fuscoatra Gymnoascus sp.

1 mg/ml 1 mg/ml 1 mg/ml 1 mg/ml 1 mg/ml 1 mg/ml 200 lM 200 lM 200 lM 200 lM 200 lM 200 lM 200 lM 200 lM 200 lM 200 lM 200 lM 200 lM 200 lM 200 lM 200 lM 200 lM 200 lM 200 lM 200 lM 200 lM 1% (v/v)

77.5 ± 0.7 20.1 ± 0.9 117.8 ± 5.1 101.3 ± 2.7 21.4 ± 5.5 66.2 ± 1.0 97.4 ± 1.6 71.8 ± 1.5 97.4 ± 2.7 69.7 ± 3.7 42.9 ± 0.4 39.7 ± 0.9 92.5 ± 2.0 100.8 ± 3.2 37.4 ± 1.2 37.6 ± 1.7 72.5 ± 4.6 96.9 ± 7.5 87.3 ± 7.6 67.0 ± 4.5 91.4 ± 3.3 64.0 ± 6.1 30.1 ± 3.4 90.1 ± 2.5 49.0 ± 1.5 82.7 ± 10.1 104.3 ± 9.2 100.0 ± 9.3

⁄⁄

32.0 ± 6.6 39.2 ± 0.9 95.6 ± 3.2 94.3 ± 3.2 18.8 ± 3.5 84.4 ± 1.2 97.9 ± 1.6 82.4 ± 4.3 94.5 ± 2.3 93.1 ± 2.2 92.7 ± 3.2 92.6 ± 2.0 68.2 ± 6.1 47.5 ± 4.6 6.2 ± 4.4 90.1 ± 2.0 35.8 ± 4.4 52.5 ± 2.3 97.7 ± 2.0 95.3 ± 2.2 92.3 ± 1.5 97.0 ± 0.81 97.0 ± 3.23 96.3 ± 2.3 0.0 ± 0.0 93.1 ± 1.6 96.8 ± 1.0 100.0 ± 1.1

⁄⁄

⁄⁄

25.1 ± 6.9 21.5 ± 2.2 30.5 ± 4.8 36.9 ± 3.3 26.1 ± 1.0 44.7 ± 5.9 87.4 ± 4.6 74.3 ± 3.9 91.7 ± 10.0 70.9 ± 3.1 69.2 ± 4.9 82.2 ± 5.5 79.1 ± 3.9 77.2 ± 5.1 44.8 ± 8.8 62.1 ± 4.5 76.1 ± 6.7 64.5 ± 4.3 78.1 ± 7.1 74.4 ± 6.6 81.7 ± 2.8 64.2 ± 5.7 52.2 ± 5.4 42.6 ± 7.0 23.5 ± 3.1 67.2 ± 11.9 100.0 ± 2.3 100.0 ± 6.5

72.2 ± 7.4 38.3 ± 4.5 100.7 ± 0.9 102.6 ± 0.3 32.7 ± 7.9 103.7 ± 1.4 102.2 ± 1.1 100.7 ± 0.8 103.7 ± 0.5 103.6 ± 0.3 102.6 ± 1.0 101.9 ± 1.8 101.5 ± 0.9 102.1 ± 2.1 49.5 ± 11.3 103.0 ± 1.6 63.8 ± 4.7 102.5 ± 0.5 102.8 ± 2.0 102.6 ± 1.5 96.6 ± 5.3 102.2 ± 1.6 101.5 ± 2.1 103.3 ± 1.2 43.0 ± 7.3 104.0 ± 0.1 92.2 ± 9.7 100.0 ± 1.6

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a Significant differences (as indicated by Student’s unpaired t-test) between cell viability in wells containing the secondary metabolites vs. cell viability in wells containing the control are indicated by one asterisk (P < 0.05) or two asterisks (P < 0.01). b Significant differences (as indicated by Student’s unpaired t-test) between L2 viability in wells containing the secondary metabolites vs. L2 viability in wells containing the control are indicated by one asterisk (P < 0.05) or two asterisks (P < 0.01). c Significant differences between cell viability and L2 viability (as indicated by Student’s unpaired t-test) are indicated by one asterisk (P < 0.05) or two asterisks (P < 0.01).

Y. Lai et al. / Experimental Parasitology 139 (2014) 33–41

Metabolite code

Y. Lai et al. / Experimental Parasitology 139 (2014) 33–41

five days, spore concentration of the liquid culture was adjusted to spore/cell suspension of 1  106/ml with sterilized, distilled H2O. Aliquot of 5.0 ml of the spore inoculum was inoculated into Fernbach flask (500 ml) containing 80 g of rice and incubated at 25 °C for 20 days. The fungal metabolites were extracted from fermented rice with 500 ml EtOAc, and the crude extract was dried under reduced pressure by evaporating organic solvent. The fractions were prepared by fractionating the dried extract using silica gel vacuum liquid chromatography (VLC) with petroleum ether–EtOAc gradient elution. The pure compounds were purified from the fraction by semi-preparative RP HPLC. The fungal secondary metabolites including 20 crude extracts from 18 different fungal strains, 20 fractions from 3 crude extracts, 20 pure compounds (17 from 7 crude extracts, and 3 from other fungal extracts) (Table 1), and conventional nematicide Avermectin (Institute for the Control of Agrochemicals, Ministry of Agriculture, China) were prepared in 1% DMSO, which as well as nematicides Fosthiazate (Ishihara Sangyo Kaisha, Ltd) and Formaldehyde solution (Sinopharm Chemical Reagent Beijing Co., Ltd) were diluted with L-15 cell medium for cell-MTS assay or with sterile water for larval assay to different concentrations (1 mg/ml for crude extracts and fractions, 200 lM for pure compounds, 0.08–40 lg/ml for Avermectin, 10–2500 lg/ ml for Fosthiazate, and 0.02–1.25‰ v/v for Formaldehyde solution). 2.5. Standard curve of absorbance vs. cell density in the MTS assay Embryo cells were suspended and diluted in L-15 cell culture medium and added to a 96-well tissue plate in numbers ranging from 6.25  104 to 2  106/100 ll/well. A 20-ll volume of MTS reagent (CellTiter 96Ò AQueous One Solution Cell Proliferation Assay, Promega) was then added to each well, and the plates were incubated at 37 °C for 4 h (Sekhon et al., 2008). Absorbance was measured at 490 nm with an ELISA reader (Bio Rad Model 680). An MTS standard curve for determining linearity range was prepared by plotting absorbance against the number of embryo cells added per well.

37

25 °C for 48 h, the numbers of living and dead nematodes were determined by microscopic observation and physical stimuli with a fine needle: nematodes that were curled or moving were considered living, and those that were straight and immobile under the stimuli were considered dead (Breger et al., 2007; Dong et al., 2006; Okoli et al., 2009). The mean values of living nematodes were converted to a percentage that was corrected for survival in the control by the following formula: corrected percentage of living nematodes = percentage living nematodes under chemical treatment/percentage living nematodes in the control. 2.8. Determination of LC50 of potential nematicidal fungal metabolites The fungal secondary metabolites (three crude extracts, one fraction, and two pure compounds) that caused >80% mortality of C. elegans L2 in the in vitro assay (see previous section) were dissolved in 1% DMSO solution and diluted to different concentrations (final concentrations ranged from 0.2 to 1.0 mg/ml for the crude extracts and fraction, and from 12.5 to 200 lM for the pure compounds). The metabolites were added to 96-well tissue plates (50 ll per well) containing 200 L2 in 50 ll of water per well. Sterile water was used as the control, and the plates were incubated at 25 °C. After 48 h, L2 viability was assessed as described earlier. The mean values of living L2 were converted to percentages (corrected to account for control viability), and the corrected percentages were used to determine LC50 values, as described earlier. 2.9. Statistical analysis Statistical differences in the end points between the blank control and treated groups were determined using the unpaired twotailed Student’s t-test (Sekhon et al., 2008). A P value of less than 0.05 was considered to be significant. 3. Results 3.1. Determination of the linear range of MTS assay

2.6. Colorimetric MTS-based cytotoxicity assay Embryo cells in L-15 cell culture medium were added to 96-well tissue plates (1  106 cells in 50 ll of medium per well). A 50-ll volume of a chemical solution in 1% DMSO (as described earlier) was then added to each well. The L-15 cell culture medium was used as the control. The plates were then kept at 25 °C for 24 h before 20 ll of MTS was added to each well. The plates were then kept at 37 °C for 4 h before absorbance was measured at 490 nm. The percentage of cell viability was calculated as follows: (ODtODn)/(ODc ODb)  100%, where ODt = specific absorbance of chemical-treated cells (at each chemical concentration); ODn = specific absorbance of the chemical (at each concentration) without cells; ODc = specific absorbance of untreated cells; ODb = background absorbance of L-15 cell culture medium with MTS. Accordingly, the LC50 for each conventional nematicide, i.e., the concentration of nematicide that decreased cell viability by 50% was graphically extrapolated by plotting the percentage of viability against the respective nematicide concentration (Ganguly et al., 2006). 2.7. In vitro assay for nematicidal activities Nematode suspension (C. elegans L2s/H. glycines J2s/B. xylophilus) was added to 96-well tissue plates (100 nematodes in 50 ll of water per well). A 50-ll volume of a chemical solution in 1% DMSO (as described earlier) was then added to each well. Sterile water was used as the control. After the plates were kept at

Specific absorbance was measured for increasing densities of living C. elegans embryo cells (6.25  105–2.00  107 cells/ml, 100 ll/well). Absorbance at 490 nm was positively correlated with cell density (Fig. 1). The correlation was stronger with the lower range of cell density (from 6.25  104 to 1  106 cells per well; Fig. 1B) than with the complete range of cell density (Fig. 1A). The absorbance signal from 6.25  104 cells was more than 3 standard deviation (SD) greater than the background absorbance in wells containing MTS reagent and L-15 culture medium without cells (i.e., at cell concentrations greater than 104/ml, the signal from cells was much stronger than the signal from background). Because the MTS assay was intended to be used to measure a decrease in viability following a cytotoxic treatment, a cell density of about 1  106 cells per well (which was in the cell density range where the correlation was strong, as indicated in Fig. 1B) was selected as the density for assays with candidate toxic metabolites. 3.2. Cytotoxicity and nematicidal activity of conventional nematicides As nematicide concentration increased, both the viability of the C. elegans embryo cells and the viability of L2 decreased (Fig. 2). In addition, the viabilities had similar dose-dependent manner in the MTS assay and the larval assay, i.e., the toxicity results obtained with cells were correlated with those obtained with larvae (r = 0.78 for Avermectin, r = 0.95 for Fosthiazate, and r = 0.65 for Formaldehyde solution; all P values less than 0.05). Correlations also existed between the cell viability and viability of plant para-

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Y. Lai et al. / Experimental Parasitology 139 (2014) 33–41

Fig. 1. Effect of cell density on absorbance at 490 nm measured using the CellTiter 96Ò AQueous One Solution Cell Proliferation Assay. From 0 to 2  106 C. elegans embryo cells in 100 ll of L-15 culture medium were added to the wells of a 96-well plate. MTS solution was then added at 20 ll/well. After 4 h at 37 °C, the absorbance at 490 nm was recorded with an ELISA plate reader (Bio-Rad 680). Each point represents the mean ± SD of three replicates. Values for all cell densities are shown in (A) and for samples less than 1  106 cells per well are shown in (B). The background absorbance detected at zero cells per well was subtracted from these values.

sitic nematodes (Fig. 2). For H. glycines, ‘‘r’’ values were 0.91 for Avermectin, 0.95 for Fosthiazate, and 0.83 for Formaldehyde solution (P < 0.01). For B. xylophilus, ‘‘r’’ values were 0.92 for Avermectin, 0.96 for Fosthiazate, and 0.70 for Formaldehyde solution (P < 0.05). These results indicated that the tested nematicides with nematode toxicity also performed similar effects on cells at the same concentration. Based on the dose-response curve for each nematicide, the estimated LC50 values for cells were 25 lg/ml for Avermectin, 308 lg/ml for Fosthiazate and 0.08‰ (v/v) for Formaldehyde solution; the estimated LC50 values for C. elegans L2 were 2.5 lg/ml for Avermectin, 136 lg/ml for Fosthiazate and 0.35‰ (v/v) for Formaldehyde solution; the estimated LC50 values for H. glycines were 5 lg/ml for Avermectin, 200 lg/ml for Fosthiazate and 0.13‰ (v/v) for Formaldehyde solution; the estimated LC50 values for B. xylophilus were 5 lg/ml for Avermectin, 124 lg/ml for Fosthiazate and 0.54‰ (v/v) for Formaldehyde solution (Fig. 2). 3.3. Cytotoxicity of fungal metabolites The effects of 20 crude extracts (XC1–XC20, 1 mg/ml), 20 fractions (XC21–XC40, 1 mg/ml), and 20 pure compounds (XC41– XC60, 200 lM) on embryo cells were estimated using the MTS assay. The cytotoxicities of the 60 fungal secondary metabolites were classified into three grades according to the cell viabilities: high cytotoxicity (50%) (Table 1). Seven crude extracts and 10 fractions at 1 mg/ml exhibited high toxicity; 10 extracts, 5 fractions at 1 mg/ ml, and 6 compounds at 200 lM exhibited medium toxicity; and 3 extracts, 5 fractions, and 14 compounds exhibited low cytotoxicity (Table 1). Absorbance increased after treatment with fraction XC37, indicating that this fraction might cause cell activation or proliferation. 3.4. Nematicidal effects of fungal metabolites

Fig. 2. Viability of C. elegans embryo cells, C. elegans L2, H. glycines and B. xylophilus as affected by concentrations of the nematicides Avermectin (A), Fosthiazate (B), and Formaldehyde solution (C). Embryo cells (1  106 cells per 50 ll per well) or nematodes (100 per 50 ll per well) were treated with concentrations of these nematicides (50 ll per well) at 25 °C for 24–48 h. In the cell assay, 20 ll of MTS solution was added to per well, and after 4 h at 37 °C the absorbance at 490 nm was recorded using an ELISA plate reader (Bio-Rad 680). In the nematode assay, the living and dead nematodes were counted under invert microscope. Cell viability and nematode viability in wells with nematicide was determined by comparison with viability in cells or nematodes with the control. Each point represents the mean ± SD of three replicates. The correlation coefficient (r) represents the relationship between cell viability and nematode viability.

An experiment was conducted to determine whether the toxicity of the 20 crude extracts, 20 fractions, and 20 pure compounds to C. elegans embryo cells in cell-based MTS assay (see previous section) was correlated with their toxicity to C. elegans L2 as well as plant parasitic nematodes (H. glycines and B. xylophilus); the concentrations used in both assays were the same. The effects of the 60 metabolites on cell viability were positively related to their effects on L2 viability (r = 0.60, P < 0.01, Fig. 3A). The numbers of secondary metabolites causing high, medium, and low toxicity of cells and L2 are shown in Table 2. For example, among the 17 metabolites that exhibited high toxicity to embryo cells, four exhibited high, ten exhibited medium, and three exhibited low toxicity to L2 (Table 2). In total, 26 of the 38 metabolites that significantly

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In addition, the cytotoxicity of the 60 metabolites also correlated to their effects on H. glycines viability (r = 0.48, P < 0.01, Fig. 3B), and B. xylophilus viability (r = 0.56, P < 0.01, Fig. 3C), respectively. This indicated the potential toxic metabolites screened by the cell-MTS assay also performed toxic to plant parasitic nematodes. 3.5. Determination of LC50 of potentially nematicidal metabolites For those secondary metabolites that were toxic to both C. elegans embryo cells and L2, dose-response curves were plotted to determine the dose that resulted in 50% survival of L2. Six metabolites were selected, including three crude extracts (XC08, XC15, and XC18), one fraction (XC39), and two pure compounds (XC49 and XC59). Based on the dose-response curves (Fig. 4), the LC50 values were estimated to be 20.8 lg/ml for XC08, 31.3 lg/ml for XC15, 125 lg/ml for XC18, 500 lg/ml for XC39, 90 lM (23.9 lg/ml) for XC49, and 25 lM (6.8 lg/ml) for XC59. These relatively low LC50 values indicate that the six fungal metabolites have potential for nematode control. 4. Discussion A high-throughput screen for nematicidal natural products requires a reliable method to distinguish living and dead nematodes. However, most traditional methods for determining whether nematodes are dead (e.g., staining (Donald and Niblack, 1994; Jatala, 1975; Meyer et al., 1988), probing (Dong et al., 2006), and NaOHstimulus (Chen and Dickson, 2000)) are subjective and time-consuming. Therefore, we devised a C. elegans embryo cell-based MTS assay as a first step and a larval assay as a second step to enable high-throughput in vitro screening of natural product libraries for nematicidal activity. A key feature of this combined strategy is that the first step is highly efficient and eliminates ineffective metabolites. Moreover, the complete screen identifies the effects of candidate compounds on both embryo cells and whole nematodes.

Fig. 3. Relationship between the viability of C. elegans embryo cells (as determined in the MTS assay) and of C. elegans L2 (A) or H. glycines (B) or B. xylophilus (C) in assays with 60 fungal secondary metabolites. Each point is the mean of three replicates.

Table 2 Distribution of the numbers of 60 fungal secondary metabolites causing three categories of cell viability vs. L2 viability (50%). Cell viability

50% Total

L2 viability

Total

50%

4 7 0 11

10 5 4 19

3 9 18 30

17 21 22 60

reduced embryo cell viability also reduced L2 viability. Similarly, 18 of 22 metabolites with low cytotoxicity in the MTS assay also had low toxicity against L2; the other four metabolites had medium toxicity against L2. Therefore, those secondary metabolites having weak cytotoxicity could be excluded from the L2 assay, although some nematicidal molecules would potentially be discarded.

Fig. 4. Viability of C. elegans L2 as affected by concentrations of six fungal secondary metabolites: three crude extracts and one fraction (A) and two pure compounds (B). L2 (200 per 50 ll per well) were treated with the metabolites (50 ll per well) at 25 °C. After 48 h, the living and dead nematodes were counted. Nematode viability was determined by comparison with the control. Each point represents the mean ± SD of four replicates. In all cases, viability was significantly less (P < 0.01) in wells with secondary metabolites than in well with the control according to unpaired Student’s t-tests.

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Primary cultures of C. elegans embryo cells have been previously used as an in vitro model system to identify factors involved in morphogenesis and development, but our study is the first to use C. elegans embryo cells as targets for screening potential nematicides. One important advantage of using C. elegans embryo cells in screen for nematicides is that large numbers are easily obtained. The first screening step (the MTS assay with embryo cells) determines cell viability after exposure to test metabolites. MTS is a tetrazolium salt that is reduced to water-soluble colorimetric formazan mainly by mitochondrial succinate dehydrogenase (Mosmann, 1983), and the color intensity of the formazan dye, which is measured by an automated, high-throughput ELISA reader, is correlated with the number of viable cells. As a consequence, the MTS assay is widely used to study cell proliferation (Kuda et al., 2004), cytotoxicity (Sekhon et al., 2008), and chemosensitivity (O’Toole et al., 2003). In our preliminary study, absorbance with C. elegans larvae produced from MTS assay was not proportional to the number of living larvae (unpublished data); however, there is a significant linear regression between living C. elegans embryo cells and the absorbance from MTS assay (Fig. 1). Avermectin, Fosthiazate and Formaldehyde solution are important, representative and common used insecticides and nematicides in potato, corn, soybean, peanut and cotton and were chosen for cytotoxic test in this study. The macrocyclic lactone Avermectin and the organophosphate Fosthiazate are potent neurotoxins to impair movement or paralyze nematodes. However, these nematicides also performed cytotoxic effect on mammalian ovary cells or neuroblastoma cells (Kokoz et al., 1999; Slotkin and Seidler, 2007; Veronesi and Ehrich, 1993) and showed dose-response manner on embryo cells in this study, indicating that the MTS assay with embryo cells could be used to measure a decrease in viability following a cytotoxic treatment. Combined with 96-well microplates and an ELISA plate reader, the embryo cell-based MTS assay facilitates high-throughput screening of natural products for cytotoxicity. Moreover, positive correlation between cell viability and nematode viability including C. elegans L2, H. glycines and B. xylophilus under the effects of these three nematicides revealed that cytotoxicity could be used as an indicator of nematicidal activity. As noted, an important advantage of the MTS assay with embryo cells is that it rapidly identifies ineffective fungal metabolites because cytotoxicity generally reflects larval mortality, i.e., compounds that kill nematode cells usually kill entire nematodes. In the MTS assay with embryo cells, a cytotoxicity level of 50% at a given concentration (1 mg/ml for fungal crude extracts and fractions, and 200 lM for pure compounds) was considered as the screening standard to eliminate weak secondary metabolites, in part because the LC50 value is a recognized standard. In the larval assay, a survival level of 20% was suggested as a screening standard to further identify secondary metabolites with potential nematicidal activity. Because the isolated embryo cells can differentiate into various cell types that form the newly hatched larvae (Strange et al., 2007), fungal metabolites that are ineffective in the cell assay are likely to be ineffective in the larval assay. Indeed, cell viability was positively correlated with larval viability. In addition, the MTS assay with embryo cells could be used to trace the bioactive components of mixtures. For example, the isolated components from crude extract XC07 included four fractions with low toxicity (XC35, XC37, XC38, and XC40) and two with high toxicity (XC36 and XC39). In other words, the cytotoxicity of XC07 could be attributed to the fractions XC36 and XC39. It is also possible, however, that bioactivity is lost during purification; for example, the pure compounds XC41–XC44, which were isolated from crude extract XC18, were much less cytotoxic than XC18. This suggests that cytotoxicity may sometimes involve synergy between components or even antagonism between components. Regardless, the cell-based MTS assay can determine which

component is responsible for the cytotoxicity and can subsequently guide the purification of that component. The two-step sequential screening for the identification of potential nematicides also has some limitations. Unlike single cells, whole nematodes have an immune system that can protect against toxic chemicals. As a result, secondary metabolites that are toxic to cells might not be toxic to larvae. Therefore, it is unlikely that the first step in the new method will eliminate all secondary metabolites that have low toxicity against larvae, i.e., the cell-based MTS assay cannot be used alone to identify metabolites with nematicidal activity. As noted, some potential nematicidal metabolites might be discarded in the cell-based MTS assay, the reason of which might be their different effects on cells and nematodes. Metabolites that kill or paralyzed nematodes by acting on neuron cells or muscle cells will likely not result in cell death in the cell-based MTS assay. C. elegans cells were less sensitive than nematodes under the effect of potent neurotoxins Avermectin and Fosthiazate (higher LC50 value on cells than those on nematodes). However, in previous studies those nematicides also performed cytotoxic effect on mammalian ovary cells or neuroblastoma cells (Kokoz et al., 1999; Maran et al., 2010; Slotkin and Seidler, 2007; Veronesi and Ehrich, 1993) and in our study the cytotoxicity and mortality were correlated. Therefore, although a small number of metabolites were missed in the cell assay, the majority potential nematicidal metabolites were maintained in the screening. As these screened potential metabolites will finally be applied to kill harmful plant parasitic nematodes, and some nematode species such as pine wood nematode B. xylophilus can be easily cultured by fungus on large scale, the use of cell cultures from plant parasitic nematodes for screening will be considered in the future study. Another limitation is that examining and counting living and dead larvae is a time-consuming, rate-limiting step. An automated, perhaps colorimetric method to assess and count living and dead nematodes would greatly increase the screening throughput. 5. Conclusion We have developed a novel two-step screening method for identifying fungal secondary metabolites with nematicidal activity. The method combines a high-throughput C. elegans embryo cellbased MTS assay for cytotoxicity and a C. elegans larval mortality assay. The cell-based MTS assay enables the rapid elimination of ineffective metabolites and thereby facilitates the efficient screening for nematicidal activity. Acknowledgments We would like to thank B.A. Jaffee for his valuable comments and suggestions on article editing, thank Prof. Heng Jian of China Agricultural University for providing pine wood nematode B. xylophilus, and thank Institute for the Control of Agrochemicals, Ministry of Agriculture, China for providing Avermectin. This research was supported by the Special Fund for Agro-scientific Research in the Public Interest of China (No. 201103018) and 973 Program (2013CB127506). References Becker, J.O., Zavaleta-Mejia, E., Colbert, S.F., Schroth, M.N., Weinhold, A.R., Hancock, J.G., Van Gundy, S.D., 1988. Effects of rhizobacteria on root-knot nematodes and gall formation. Phytopathology 78, 1466–1469. Berg, K., Zhai, L., Chen, M., Kharazmi, A., Owen, T.C., 1994. The use of a water-soluble formazan complex to quantitate the cell number and mitochondrial function of Leishmania major promastigotes. Parasitol. Res. 80, 235–239. Breger, J., Fuchs, B.B., Aperis, G., Moy, T.I., Ausubel, F.M., Mylonakis, E., 2007. Antifungal chemical compounds identified using a C. elegans pathogenicity assay. PLoS Pathog. 3, e18. Brenner, S., 1974. The genetics of Caenorhabditis elegans. Genetics 77, 71–94.

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