Two Fungicides Alter Reproduction of the Small Brown Planthopper

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Two fungicides alter reproduction of the small brown planthopper, Laodelphax striatellus by influencing gene and protein expression You Wu, Jun Ding, Bing Xu, Linlin You, Linquan Ge, Guoqing Yang, Fang Liu, David Stanley, Qisheng Song, and Jincai Wu J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00612 • Publication Date (Web): 07 Feb 2018 Downloaded from http://pubs.acs.org on February 10, 2018

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Journal of Proteome Research

Two fungicides alter reproduction of the small brown planthopper, Laodelphax striatellus by influencing gene and protein expression

You Wu a, Jun Ding a, Bing Xu a, Linlin Youa, Linquan Gea, Guoqing Yanga, Fang Liua David Stanleyb, Qisheng Songc, Jincai Wua,*

a

School of Plant Protection, Yangzhou University, Yangzhou 225009, P.R. China;

b

Biological Control of Insects Research Laboratory, USDA/Agricultural Research

Service, 1503 S. Providence Road, Columbia MO 65203; cDivision of Plant Sciences, University of Missouri, 1-31 Agriculture Building, Columbia, MO 65211, USA

* To whom correspondence should be addressed: E-mail: [email protected]; Tel: 86-0514-87979246; Fax: 86-0514-87349917

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ACS Paragon Plus Environment

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Abstract: Aside from their intended actions, fungicides can drive pest insect outbreaks, due to virtually continuous use and pest evolution. Small brown planthopper (SBPH), Laodelphax striatellus, outbreaks occurred recently in many provinces in China, with devastating rice losses.Because exposure to the fungicide jinggangmycin (JGM) increased reproduction of the brown plant hopper, Nilaparvata lugens, via its influence on fatty acid synthase, we posed the hypothesis that JGM and carbendazim (CBM) influence SBPH reproduction via their influence on enzymes involved in other aspects of lipid metabolism.Exposure to the fungicide CBM stimulated SBPH reproduction (egg-laying up by 78%) and to another fungicide, JGM, led to decreased egg-laying (down by 47.3%). These inverse effects are mediated by down-regulated expression of L-3-hydroxyacyl-Coenzyme A dehydrogenase (LCHAD) in JGM-treated females and up-regulated expression of hydroxysteroid dehydrogenase-like protein 2-like (HSD) in CBM-treated females. RNAi knockdown of, separately, LCHAD and HSD led to reduced egg-laying (down by 53.5% for dsLCHAD and by 73% for dsHSD). dsLCHAD, dsHSD and JGM treatments also led to severely reduced ovarian development in experimental SBPH, with shorted and thinned valvula and lacking egg cells in ovaries. Valvula of CBM-treated females were normal, with banana-shaped eggs in ovaries. These data strongly support our hypothesis.

Key words: Vector insect, Laodelphax striatellus, reproduction, jinggangmycin, carbendazim, LCHAD, HSD

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1 Introduction The major agricultural chemicals include insecticides, fungicides, herbicides, and fertilizers. Over the past decades these have been misused in several ways and in agroecosystems, they are major drivers of some of the global ecological and agroecological problems, such as serious environmental pollution, species extinctions and pest outbreaks or resurgence. The negative effects of insecticides are well documented1-9 and have led to a very large research enterprise aimed to create novel insect management technologies that help mitigate the deleterious effects. Similarly, herbicides are the most overused agricultural chemicals, which has led to weed resistance. The potential influences of fungicide use and overuse have not been thoroughly investigated, possibly due to their low immediate environmental toxicity. Jinggangmycin (JGM; C20H35O13N) has been used for decades in rice cropping systems to control the fungal plant disease rice sheath blight (RSB) Rhizoctonia solani.10 We recently reported that JGM exerts an entirely unexpected influence on rice agroecosystems. JGM stimulates reproduction of the brown planthopper (BPH), Nilaparvata lugens Stål. BPHs are serious rice pests and the idea that a fungicide, developed and used in China to protect rice cropping systems can positively influence populations of a pest insect has enormous implications for sustainable agriculture. We consider, for example, the possibility of RSB resistance to JGM (which has not occurred) leading to increased JGM applications which may boost BPH reproduction to the point of population outbreaks. BPHs are members of the plant hopper guild, a group of sympatric species operating closely similar niches. JGM exerts the paradoxical influence of suppressing reproduction in the white-backed planthopper (WBPH), Sogatella furcifera Horvath.11 The significance of this finding is the effects of JGM on other members of the plant hopper guild cannot be predicted without dedicated research into each species. The small brown planthopper (SBPH), Laodelphax striatella Fallén, is another member of the guild. The SBPH is a pest of three major crops, rice, wheat and maize. Its distribution and host ranges are wider than BPH and WBPH. SBPHs exert direct feeding damage and indirect damage by transmitting plant viral diseases, such as rice 3



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striped virus disease (RSV disease), black streaked dwarf, and maize rough disease virus.12-17 Viral disease epidemics of rice, wheat and maize have threatened rice production and food safety globally. In China, losses of 20-30% have been recorded in japonica rice cropping systems in 2001, 2003, 2004, and 2005 in Jiangsu, Zhejiang, Shandong, Anhui, and Henan provinces.18-19 SBPH populations in wheat can migrate to rice fields, where they transmit rice virus diseases

19

and RSV epidemics in rice

fields are closely related to SBPH population sizes in wheat. The concern is that induced SBPH resurgence may lead to local ecological disasters with far reaching consequences for world food and nutrition security. JGM is commonly applied two or three times during rice growth with leaf spray at the middle and late stages of rice growth when RSB occurs. RSB is responsible for substantial crop losses, which are partly alleviated by JGM. It is also used for controlling sharp eyespot of wheat Rhizoctonia cerealis Vander Hoeven and rice false smut Ustilaginoide avirens (Cooke). 20 Carbendazim (CBM; C9H9N3O2) is a broad-spectrum fungicide widely used for controlling various crop diseases. For example, it is used to control fusarium head blight Fusarium graminearum Schw in wheat and to control rice blast Pyricularia grisea (Cooke), RSB, and rice phyllosticta leaf blight Phyllosticta oryzicola Hara.20 In quantitative terms, CBM is the most heavily applied fungicide in Chinese and possibly global agriculture. Research into its potential, albeit unexpected, effects on insect pests is necessary. Because JGM increased reproduction of BPHs via its influence on fatty acid synthase, we posed the hypothesis that JGM and CBM influence SBPH reproduction via their influence on enzymes involved in other aspects of lipid metabolism. Here, we report on the outcomes of experiments designed to test our hypothesis.

2. Materials and Methods 2.1. Insects, rice variety and fungicides Experimental SBPHs were collected from natural populations on the experimental farm of Yangzhou University, Yangzhou, China. The population was maintained on a 4


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susceptible japonica rice variety, Wuyuejing 3, in an insectary under standard conditions of 25 ± 1 °C,16L:8D photoperiod, rh = 70-80%. Rice seeds were sown in cement tanks at the Yangzhou University experimental farm. Rice plants with six leaves were transplanted into plastic pots (D = 35 cm, H = 50 cm) at four hills/pot and four seedlings/hill. Technical grade CBM (98% a.i) and JGM (13.5%) were purchased from Shanghai Kaman Biological Science and Technique Co. Ltd., Shanghai, China) and Zhejing Qianjiang Biochemical Co. Ltd., Tonlu, Zhejiang, China), respectively.

2.2 Experiment Three hundred third-instar nymphs were released onto each potted plant at the tillering stage. Rice plants were sprayed with either JGM diluted with tap water and 10 % emulsifier (3205-C, Nanjing Taihua Chemical Co. Ltd., Jiangsu) or CBM (dissolved with hydrochloric acid and 10 % emulsifier) at 200 ppm 12 h after insect release. Control plants were sprayed with tap water containing 10% emulsifier. Each treatment was replicated three times (using 3 rice pots). The treated and control plants were covered with cages (80 mesh sieve) and placed in a greenhouse at ambient temperature (mean ~25-28 °C and 16L:8D). Ten nymphs were collected from the treated or control plants at 48 h post-treatment, and were placed in a glass jar (D = 10 cm, H = 12 cm) with untreated plants. They were maintained in a biological culture box under standard conditions. After adult emergence, a single male and female pair was placed in a glass jar containing untreated rice stems for egg-laying. If the male partner died, new males were added until the female died. The stems were changed daily before egg-laying and changed every 3 days after onset of egg-laying. The number of eggs laid/female was counted under a microscope. Eggs were scraped from the leaf sheath using a pin. Each treatment and control experiments were replicated over twenty times.

2.3. Female body and length measurements Individual female (n = 10), 2 days post-emergence (PE) was weighed with a SOPTOP electronic balance (Model: FA1004, 1/10,000g sensitivity) and the length of 5



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their valvula was measured under a microscope.

2.4. Proteomic analysis We conducted iTRAQ proteomic analysis of treated females and compared differential protein expressions of two female groups: JGM-treated females (JGM-TF) vs controls (CF), and CBM-treated females (CBM-TF) vs CF. Proteomic analysis of all samples in two batches were conducted by BGI Co. Ltd., Shenzhen, China. For each treatment or control experiment in the first batches, two

biologically

independent groups of 50 unmated females were processed for proteomic analysis at 2 days post foliar exposure to CBM fungicide; for that in the second batches, three biologically independent groups of 50 unmated females were processed at 2 days post foliar exposure to JGM fungicide. The quantitative proteomics strategy consists of three steps. Briefly, i) protein extraction, digestion and iTRAQ labelling. The iTRAQ labelling pattern was as follows:

In JGM-treated, JGM-TF1 (117 tag),

JGM-TF2(119 tag), JGM-TF3 (121 tag), CF1 (113 tag), CF2 (114 tag), CF3 (116 tag); In CBM-treated, CBM-TF1(116 tag), CBM-TF2 (121 tag), CF1 (114 tag), CF2 (115 tag); ii) LC-MS/MS analysis; iii) proteomic analysis. The converted MS/MS data were searched by Mascot (Matrix Science, London, UK; version 2.3.02) against a protein database that was translated from transcriptome of L. striatellus, containing 27023 unigens (the database was provided by Prof. Fang Liu of Rice Pest Research Team, Yangzhou university). The workflow of IQuant basically comprised of the following steps: protein identification, tag impurity and data normalization, missing value imputation (to avoid biasing estimation, a missing reporter is imputed as the lowest observed values in each sample in IQuant, and the imputed values could be basically evaluated by checking an output file), protein ratio calculation, statistical analysis, and result presentation. All the proteins with a false discovery rate (FDR) less than 1% will be confirmed by a further analysis including GO, COG and Pathway analysis. The fold changes of >1.2 with Q-values 10-fold in viruliferous SBPHs.37 Our data shows this protein was down-regulated by about 90% after exposure to JGM. The biological significance of the protein remains unknown, although it also occurs in other insects, the B-biotype whitefly, Bemisia tabaci,38 and in the beetle Cylas formicarius.39 Overall, our analyses of proteins and selected genes indicate that JGM and CBM influence SBPH fecundity through very fundamental mechanisms, by altering expression of genes and proteins. Our broad interpretation, as discussed elsewhere, is that members of the plant hopper guild have evolved a syndrome of adaptations that make them a dangerous element of agroecosystems.40

SUPPORTING INFORMATION 14


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The

following

files

are

available

free

of

charge

at

ACS

website

http://pubs.acs.org: SUPPLEMENTARY TEXT 1: PROTEOMIC

ANALYSIS OF

TREATED

FEMALES SUPPORTING INFORMATION: PROTEOME INFORMATION Table S1: UP-REGULATED PROTEINS OF JGM vs CONTROL Table S2: DOWN-REGULATED PROTEINS OF JGM vs CONTROL Table S3: UP-REGULATED PROTEINS OF CBM vs CONTROL Table S4: DOWN-REGULATED PROTEINS OF CBM vs CONTROL

Acknowledgements This research was partially funded by the National Nature Science Foundation of China (31571999 and 31371938) and the Ph.D. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicap.

There is no Conflict of Interest Statement References

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Table 1. Primers used in this research. Primers

Primer sequence

Product length

For quantitative real-time PCR QLCHAD-F 5’-CCCATCACTTGCAAGGACAC-3’ QLCHAD-R 5’-AGTGGGTTGTCTGGGAACTT-3’ QHSD-F 5’-CTTGACTGGCACTCTGGAGA-3’ QHSD-R 5’-CGACATGCCATACTTTGCCA-3’ Actin-F 5’-TGGACTTCGAGCAGGAAATGG-3’ Actin-R 5’-ACGTCGCACTTCAGATCGAG-3’ ForLCHAD and HSD dsRNA synthesis LCHAD-F 5’-GCTGCTCAAAGTGGACACAA-3’ LCHAD-R 5’-TTTGCTCCACTCCATCACCT-3’ HSD-F 5’-CTTGACTGGCACTCTGGAGA-3’ HSD-R 5’-TATGCGTCGATTTGGGCTTG-3’ For GFP dsRNA synthesis GFP-F 5’-AAGGGCGAGGAGCTGTTCACCG-3’ GFP-R 5’-CAGCAGGACCATGTGATCGCGC-3’

20


235bp 210bp 200bp

470bp 473bp

688bp

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Table 2. JGM up-regulated and CBM down-regulated proteins Protein_IDa

Protein

Descriptionb

Coverage

CL1313.Cont ig1_MIX2

Unigene1813 8_MIX2

Unigene2195 9_MIX2

3 2033 PREDICTED: alpha,alpha-trehalose-phosphat e synthase [UDP-forming] A-like isoform 2 [Acyrthos iphonpisum]

glutathione s-transferase D2 [Sogatella furcifera]

RH14316p melanogaster]

[Drosophila

Unique c

Unique d

Fold e

Peptide

Spectrum

change

18.9

11

19

0.81

19.9

12

50

1.2

82.9

14

52

0.7

56.5

2

85

1.25

21.4

6

8

0.72

34.6

8

66

1.31

a: Primary protein ID of group. b:

Protein description.

c: The number of amino acids in the unique peptide identified by this protein, as a percentage of the total protein amino acid. d: Identified unique peptide number of protein. e: Identified unique spectrum number of protein.

21



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Table 3 JGM down-regulated andCBM up-regulated proteins. JGM data are represented blue font Protein_ID

Description

CL1951.Contig1_

3

MIX2

lugens]

Protein

Unique

Coverage

gi|478647659|gb|

2003vitellogenin

[Nilaparvata

vitellogenin [Laodelphaxstriatella]

Unique

Peptide

Spectrum

Fold change

85.6

3

50

3.04

63.1

3

109

0.76

73.3

4

289

2.84

53.8

3

177

0.78

72.2

7

68

3.25

53.5

5

275

0.78

27.8

4

132

2.16

16.6

1

79

0.75

46.2

14

65

1.3

45.0

15

222

0.73

99.9

1

12

3.33

59.5

1

245

0.76

29.1

4

9

2.37

12.7

2

12

0.7

AGJ26477.1|

gi|478647670|gb|

vitellogenin [Laodelphax striatella]

AGJ26478.1|

gi|723001888|dbj|

vitellogenin [Nilaparvata lugens]

BAP87098.1|

Unigene2074_MI

2777

X2

vitellogenin [Pteromalus puparum]

4069

Unigene21177_M

3

IX2

[Nilaparvata lugens]

875

minus

strand

minus strandvitellogenin

Unigene22608_M

125

IX2

protein [Maconellicoccus hirsutus]

691

putative small heat shock

22


Page 23 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


CL4798.Contig 2_MIX2

CL824.Contig1 _MIX2



Unigene2054_ MIX2

Unigene2839_ MIX2

283 636 eukaryotic translation initiation factor 4E binding protein [Nilaparvata lugens]

multiple inositol polyphosphate phosphatase 1 precursor, putative [Pediculus humanus Corporis]

AGAP004734-PA gambiae str. PEST]

[Anopheles

1 526 LEN=1213; translated

hypotheticalprotein Phum_PHUM300620 humanusCorporis]

50.0

4

17

1.97

34.7

2

13

0.86

44.0

14

28

2.73

35.9

12

104

0.8

75.6

23

126

1.92

30.4

10

99

0.8

45.5

7

28

1.95

61.9

8

51

0.78

43.6

9

13

1.96

9.8

3

7

0.81

43.5

11

88

2.84

44.2

10

75

0.71

[Pediculus

hypothetical protein DICPUDRAFT_84489 [Dictyostelium purpureum]



c: The number of amino acids in the unique peptide identified by this protein, as a percentage of the total protein amino acid. d: Identified unique peptide number of protein. 23



e: Identified unique spectrum number of protein.

24


Page 24 of 37

Page 25 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 4. Differentially expressed proteins related to reproduction, reproductive process, biological regulation, and metabolic processes following JGM and, separately, CBM treatments. Genes encoding the two proteins in bold font were investigated in dietary dsRNA experiments. Protein_ID

Description

Protein Unique Unique Fold Coverage Peptide Spectrum change

Unigene17436_MIX2

Ribosomal protein S5

25.0

1

4

0.64

Unigene12975_MIX2

LOW QUALITY PROTEIN: e3 ubiquitin-protein ligase UHRF1-like

12.9

1

3

0.69

37.6

5

26

0.79

gi|751425399|gb|AJF93905.1| 60S ribosomal protein L9 Unigene20656_MIX2

L-3-hydroxyacyl-Coenzyme A dehydrogenase

15.9

2

35

0.81

CL4798.Contig2_MIX2

Eukaryotic translation initiation factor 4E 34.7 binding protein

2

13

0.86

Unigene24032_MIX2

Mitochondrial-processing peptidase subunit beta

62.5

19

92

1.26

Unigene867_MIX2

l-xylulose reductase-like

30.9

7

15

1.34

CL1556.Contig2_MIX2

Matrix metalloproteinase-16-like isoform 13.9 1

6

10

1.39

CL1550.Contig1_MIX2

Tropomyosin 1 isoform A

56.5

5

17

1.4

Unigene24107_MIX2

Short-chain dehydrogenase

28.1

7

15

1.41

CL3264.Contig1_MIX2

Cuticular protein analogous to peritrophins 3-C5 isoform 1 precursor

39.5

9

18

1.42

Unigene2964_MIX2

Triosephosphate isomerase-like

73.7

13

79

1.44

Unigene3015_MIX2

Dihydropteridine reductase-like

46.8

7

17

1.48

Unigene647_MIX2

Hydroxysteroid dehydrogenase-like protein 2-like

40.3

14

54

1.48

Unigene20988_MIX2

Muscle protein 20-like protein

99.9

17

109

1.53

Unigene1429_MIX2

Non-specific lipid-transfer protein

37.3

16

40

1.55

Unigene8961_MIX2

Lysosomal alpha-mannosidase-like

58.8

4

9

1.56

CL3806.Contig1_MIX2

3-ketoacyl-CoA thiolase, mitochondrial-like

69.8

17

62

1.57

28.5

1

4

1.58

gi|478346283|gb|AGI96989.1| Troponin H, partial 25



Page 26 of 37

Unigene17611_MIX2

Similar to short-chaindehydrogenase

35.1

6

11

1.58

Unigene20963_MIX2

RNA and export factor binding protein

47.0

7

15

1.58

Unigene21606_MIX2

Peroxiredoxin 5

99.9

13

90

1.6

Unigene24685_MIX2

Myophilin

59.6

8

30

1.61

CL2100.Contig1_MIX2

Methionine sulfoxide reductase B3 isoform 1

23.6

3

8

1.62

CL4256.Contig1_MIX2

Peroxiredoxin-6-like

35.1

8

13

1.62

Unigene6334_MIX2

Alanine aminotransferase 2-like

44.6

2

4

1.68

CL4237.Contig1_MIX2

6-phosphogluconolactonase-like

60.8

10

27

1.69

CL1826.Contig1_MIX2

I-type lysozyme

23.2

2

4

1.72

CL3879.Contig1_MIX2

TAR DNA-binding protein 43-like

16.2

4

6

1.73

Unigene2087_MIX2

Fibroblast growth factor receptor 1-A-like 50.8

5

13

1.73

Unigene22548_MIX2

Cuticular protein analogous to peritrophins 3-D1

24.0

5

6

1.83

Unigene23980_MIX2

Phosphomannomutase

36.5

8

15

2.36



c: The number of amino acids in the unique peptide identified by this protein, as a percentage of the total protein amino acid. d: Identified unique peptide number of protein. e: Identified unique spectrum number of protein.

26


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Figure legends: Fig 1: JGM and CBM influence L. striatellus reproductive parameters. Panel A: Fecundity as numbers of eggs laid. Panel B: Oviposition period (OPD). Panel C: Preoviposition period (POVP).Panel D: Female longevity. Histogram bars report the indicated parameters and error bars show 1 SEM. Bars annotated with different letters are significantly different at P< 0.05. Fig 2:

Effect of JGM and CBM on female SBPH fresh weight, body length and

oviduct length. Panel A: Fresh, wet weight (mg). Panel B: Body length (mm). Panel C: valvula length (mm). Histogram bars report the indicated parameters and error bars show 1 SEM. Bars annotated with different letters are significantly different at P< 0.05. Fig 3: GO biological process analysis of differentially expressed proteins in JGM-treated and CBM-treated SBPHs. Panel A: JGM-treatment. Panel B: CBM treatment. The data are presented as circle graphs and the arcs within each graph indicate the proportions of total proteins in each category. The numbers outside each arc show numerical approximation as proportions. Fig 4: The influence of gene silencing on relative accumulations of mRNAs encoding each enzyme. Panel A: LCHAD silencing, B: HSD silencing. The histogram bars indicate relative accumulations of mRNA and the error bars indicate 1 SEM. Bars annotated with different letters are significantly different at P< 0.05.

Fig 5: The influence of LCHAD silencing on SBPH reproductive parameters. Panel A: 27



Fecundity as numbers of eggs laid. Panel B: Oviposition period (OPD). Panel C: Preoviposition period (POVP). Panel D: Female longevity. Histogram bars report the indicated parameters and error bars show 1 SEM. Bars annotated with different letters are significantly different at P< 0.05. Fig 6: The influence of HSD silencing on SBPH reproductive parameters. Panel A: Fecundity as numbers of eggs laid. Panel B: Oviposition period (OPD). Panel C: Preoviposition period (POVP). Panel D: Female longevity. Histogram bars report the indicated parameters and error bars show 1 SEM. Bars annotated with different letters are significantly different at P< 0.05. Fig 7: LCHAD silencing led to disrupted SBPH ovarian development. Panel A: Control treatments led to normal development. Panel B: GFP control treatment similarly led to normal development. Panel C: JGM treatment led to disrupted development. Panel D: dsCHAD silencing similarly led to disrupted development. Fig 8: HSD silencing led to disrupted SBPH ovarian development. Panel A-C: the control experiments led to normal ovaries. Panel D: HSD silencing led to developmental abnormalities.

28


Page 28 of 37

Page 29 of 37

Fig. 1

a

A

30 Oviposition period (D)

Number of eggs laid

350 300 250

b

200 150

c

100 50 Control

6

C

JGM

a

25

b

20

b

15 10 5 Control

CBM

30

a b

Female longevity (D)

5 4

B

0

0

Preoviposition period (D)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


b

3 2 1

JGM

a

D

25

CBM

ab

b

20 15 10 5 0

0 Control

JGM

Control

CBM

Treatment

JGM

Treatment

29


CBM


2.0

A

a b c

1.5 1.0 .5 0.0

Control JGM

CBM

3.5

B

b

3.0

a c

2.5 2.0 1.5 1.0 .5

1.4

V a lv u la le n g th (m m )

2.5

F e m a le b o d y le n g th (m m )

Fig. 2

F e m a le w e ig h t (m g )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 37

1.2 1.0

C

a b c

.8 .6 .4 .2 0.0

0.0

Control JGM

CBM

Treatment

30


Control JGM CBM

Page 31 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig.3 PANEL A

PANEL B

31



Fig.4 1.2

A

a

a

Relative HSD expression

Relative LCHAD expression

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 37

1.0 .8

b

.6

b

.4 .2 0.0

ol G F P G M AD J nt r s CH L Co ol+d s l+d nt r tr o Co n Co

2.5

B

a

a

2.0 1.5

b

1.0

c .5 0.0

ol FP nt r ds G o + C M CB

D M CB dsHS + M CB

Treatment

Treatment

32


Page 33 of 37

Fig.5

a

a


A

140 120

b

100

b

80 60 40 20 0

20 18 16 14 12 10 8 6 4 2 0

7

C

a

b

b

4

b

b

3 2 1 0

ol FP JGM HA D nt r ds G o + C s LC l o l+d tr o n r Co nt Co

D

a

25

b

20

b

b

15 10 5 0

ol FP JGM HA D nt r ds G o + C s LC l o l+d tr o n r Co nt Co

a

5

30

a

a

B

6

ol FP JGM HA D ntr dsG o + C sLC l +d l t ro o n Co nt r Co


Number of eggs laid

160

Oviposition period (D)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ol FP JGM HA D sG nt r C Co ol+d ds L r + l t o n Co nt r Co

Treatment

Treatment

33



Fig.6

a

200


A

a

b

150 100

c

50 0

25

ol G F P s nt r Co M +d B C

C

a b

c 10 5

a

a

3 2 1 0

ol FP nt r ds G + M CB

D

a

D M C B ds H S + M CB

a

25 20

b

b

15 10 5 0


a a

4

30

a

15

ol FP sG nt r Co M +d CB

B

Co

20

0

5

D M CB dsHS + M CB


Number of eggs laid

250

Oviposition period (D)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 37

ol FP nt r ds G o + C M CB

Treatment


Treatment

34


Page 35 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig.7

35



Fig.8

36


Page 36 of 37

Page 37 of 37

Table of Contents Graphic

160 Number of eggs laid

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A

a

a

140 120

b

100

b

80 60 40 20 0

l tro sGFP JGM CHA D n Co ol+d ds L r + l t o n Co nt r o C

Treatment

37


Two Fungicides Alter Reproduction of the Small Brown Planthopper

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