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Impact of Spodoptera littoralis (Boisd.)and Agrotis ipsilon (Hufn.) larval frasson oviposition of conspecific insectsA.A.I. Ahmed a , M.Y. Hashem b , Manal M. Adel a , S.M. Mohamedc & S.H. Shimaa Khalil aa Pests & Plant Protection Department, National Research Centre,Dokki, Giza, Egyptb Faculty of Agriculture, Economic Entomology & PesticidesDepartment, Cairo University, Giza, Egyptc Medicinal & Aromatic Plants Department, National ResearchCentre, Dokki, Giza, EgyptVersion of record first published: 17 Dec 2012.

To cite this article: A.A.I. Ahmed , M.Y. Hashem , Manal M. Adel , S.M. Mohamed & S.H. ShimaaKhalil (2013): Impact of Spodoptera littoralis (Boisd.) and Agrotis ipsilon (Hufn.) larval frass onoviposition of conspecific insects, Archives Of Phytopathology And Plant Protection, 46:5, 575-592

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Impact of Spodoptera littoralis (Boisd.) and Agrotis ipsilon (Hufn.)larval frass on oviposition of conspecific insects

A.A.I. Ahmeda, M.Y. Hashemb, Manal M. Adela*, S.M. Mohamedc and S.H. ShimaaKhalila

aPests & Plant Protection Department, National Research Centre, Dokki, Giza, Egypt; bFacultyof Agriculture, Economic Entomology & Pesticides Department, Cairo University, Giza, Egypt;cMedicinal & Aromatic Plants Department, National Research Centre, Dokki, Giza, Egypt

(Received 24 October 2012; final version received 29 October 2012)

Ethanolic extracts of larval frass of the cotton leaf worm and the black cutworm wereprepared and tested to deter the eggs lay of the adults of the same insects. Two dif-ferent types of food were used for larval feeding. Extracts fractions were identifiedusing gas chromatography–mass spectrometry analysis. High concentrations weremore deterrent to oviposition than low. Extracted frass of fed larvae on semi-artificialdiet was more effective than others fed on natural host. Sensitivity of the black cut-worm adult females to the cotton leaf worm frass extract was clearly observed athigh tested concentration of L1–3 and L4 frass extracts which resulted completely ovi-position deterrent. Several fatty acids were identified qualitatively and quantitativelyin frass extracts of different larval instars of both target insects. Type and quantity offatty acid depends mainly on larval food source and larval instar, except palmiticacid which recorded at all larval instars and food sources. Oleic acid and ethyl 9-hexadecaenoic acid were found when semi-artificial diet used as a food source whilemyrisitic acid was observed only in extracted frass of fed larvae on castor oil leaves.

Keywords: larval frass; Spodoptera littoralis; Agrotis ipsilon; oviposition deterrent;gas chromatography–mass spectrometry

Introduction

Two noctuid insect pests, cotton leaf worm Spodoptera littoralis (Boisd.) and black cut-worm Agrotis ipsilon (Hufn.), attack many field crops as well as various vegetablesaffecting production not only in Egypt but also in many other countries (El-Shershaby2010; El-Sayed and Aamir 2011). The intensive use of broad-spectrum insecticidesagainst insects has led to the development of resistance to many registered pesticidesuse for its control (Aydin and Gürkan 2006).

Recently, scientists have become more interested in the utilisation of alternativematerials used as environmentally friendly pesticides to minimise the adverse effects onbeneficial insects, reduce the need for prohibitively expensive chemicals, reduce thedevelopment of resistance and are less polluting the environment (Setiawati et al. 2011).

Oviposition deterrent has been studied in numerous phytophagous insects. Extractsof non-hosts and macerated host plant material, larval and adult frass, as well as

*Corresponding author. Email: mhassanein11@hotmail.com

Archives of Phytopathology and Plant Protection, 2013Vol. 46, No. 5, 575–592, http://dx.doi.org/10.1080/03235408.2012.749691

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non-specific eggs are known to act as oviposition deterrents (Rothschild and Schoonho-ven 1977; Prokopy 1981; Renwick and Radke 1985; Li and Ishikawa 2004).

In several noctuid species, larval frass deters oviposition in conspecific females(Renwick and Radke 1980; Hilker 1985). For example, the Egyptian cotton leaf worm,S. littoralis, is deterred from oviposition by frass from conspecific larvae feeding oncotton leaves (Hilker 1985; Hilker and Klein 1989).

This study aims to evaluate the oviposition deterrent effect of ethanolic extract ofS. littoralis and A. ipsilon larval frass on the adult females of conspecific insects andthe effect of each to other using two different types of food. Different fractions of theextracts should be identifying.

Material and methods

Insects rearing and extract preparation

Two polyphagous noctuid insects, S. littoralis (Boisd.) and A. ipsilon (Hufn.), were usedin this study. Number of eggs was collected from the stock culture of Pests and PlantProtection Department, National Research Center. Hatched larvae were fed on castor oilleaves (Ricinus communis L.). Black cutworm larvae at 4th, 5th and 6th instars werereared individually in plastic vials to avoid cannibalism. Emerged adults were kept inglass jars supplied with 10% sucrose solution for feeding. Black gauze and Tafla leavesNerium oleander L. were used for eggs lay of A. ipsilon and S. littoralis, respectively.

Frass of larval instars (L1–3, L4, L.5 and L6) of experimental insects fed on castoroil leaves and/or semi-artificial diet (Schmidt et al. 1997) was collected separately andleft to dry at room temperature (25 ± 2 °C).

Twenty grams of dry frass was soaked in 25ml of the solvent (ethanol 95%) for2 days before filtration and solvent evaporation at room temperature. Frass soaking andextract filtration were repeated two times using fresh solvent.

Experiments

Three different concentrations (5.0, 2.5 and 1.25%) of each crude extract were preparedin water using some drops of Tween 20 for good mixing. The oviposition deterrenteffect of each concentration on every one of target insects was evaluated using fiveglass jars (1 L volume of each) which contains three pairs of moths, suitable treatedoviposition site and a piece of cotton soaked in 10% sucrose solution. Five jars suppliedwith untreated oviposition sites were used as a control for each experimental insect.

Number of laid eggs were counted after 2 days at all treatments and for the control.Insects rearing and all experiments were maintained under laboratory conditions of

27 °C ± 2 °C, 65 ± 5 RH and normal daylight.The oviposition repellency of each extract concentration was calculated according to

(Setiawati et al. 2011) using the following formula:

OR ð%Þ ¼ MC�MT

MC� 100 ð%Þ

whereOR=Oviposition RepellencyMC=Mean number of eggs in ControlMT=Mean number of eggs in Treatment

576 A.A.I. Ahmed et al.

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Statistical analysis was carried out using (One-Way ANOVA test) through“SPSS-Computer Program”.

Gas chromatography–mass spectrometry (GC/MS) analysis of frass extracts

The polar fractions of extracts of larval frass were identified according to the followingsteps that described by Li and Ishikawa (2004). One gram of dry frass was extracted inethanol 95% by overnight soaking technique before filtration and solvent evaporation atroom temperature. Amount of hexane was added to the dry extract for overnight andthe extract left for dryness. Using of hexane for extracting was repeated three timesusing fresh solvent each time then evaporated to near dryness. The residue was dis-solved again in hexane to be ready for injection in the GC/MS at condition: CapillaryColumn HP6890 Series (Agilent), TR-5MS (5% Phenyl Polysil–Phenylene–Siloxane),30m� 0.25mm ID� 0.25� μm film; oven temprerature 50–180 rate (5 °C/min); Injec-tor temperature: 250 °C; Detector temperature: 250 °C; Carrier Gas: helium at 1ml/min;Detector: MSD and Mass Spectrometer HP 5973 (Agilent). Sample injection volume:2 μl.

Results

Effect of ethanolic extract of S. littoralis and A. ipsilon larval frass on oviposition ofadult females of the same insects

Frass extract of S. littoralis larvae fed on castor oil leaves and /or semi-artificial dietobtained different levels of oviposition deterrent to adult females of the conspecificinsect and A. ipsilon. High concentration (5%) of L6 frass extract of S. littoralis larvaefed on semi-artificial diet was the most effective concentration against adult females ofthe same insect recording the minimum number of laid eggs (140.4 eggs/female).

Females of S. littoralis laid in average 1073.6 eggs at the lowest effective extract(L1–3 frass extract) which obtained insignificant difference with control (1091.4 eggs/female) (Table 1). On the other hand extract of frass of fed larvae on natural host wasmore effective on eggs lay of A.ipsilon adult females. Sensitivity of the black cutwormadult females to the cotton leaf worm frass extract was clearly observed at high testedconcentration of L1–3 and L4 frass extracts which resulted completely oviposition deter-rent. The mean number of laid eggs at other treatments was ranged between 33.2 eggs/female at high concentration of L6 frass extract and 544.0 eggs/female at low concentra-tion of L5 frass extract, while it was 792.8 eggs/female at control (+) and 848.2 eggs/female at control (�), (Table 2). Frass extract of larvae fed on semi-artificial diet wassignificantly more effective against conspecific insect at all concentrations of L1–3 frassextract and concentration 1.25% of L4 and L6 frass extracts in contrast with thatobserved at treatments of another target insect which obtained almost more sensitivityto extract of fed larvae on natural host than semi-synthetic diet. (Tables 1 and 2)

When the larvae fed on castor oil leaves, frass extract of 4th instar larvae ofA. ipsilon obtained the highest activity as oviposition deterrent to adult females of con-specific insect in comparison with other treatments which resulting the minimum numberof laid eggs (15.0 eggs/female) at high concentration increased to 189.4 eggs/female atthe lowest tested concentration (1.25%) of the same extract. The extract activity wasdecreased sharply at oldest larval instars (L6) especially at low concentration resulting332.2 eggs/female in average, in contrast, extract of frass of old larvae was more

Archives of Phytopathology and Plant Protection 577

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Table

1.Effectof

ethano

licextractof

S.littoralis

larval

frasson

oviposition

ofconspecificinsect

usingtwodifferenttypesof

larval

food.

Ethanolic

extract

oflarval

frass

Meanno

.of

laid

eggs/fem

ale

Con

c.(%

)

Frass

ofS.

litoralislarvae

fedon

Con

t.(+)

Con

t.(�

)Fv.

Castoroilleaves

Sem

i-artificial

diet

L1–3

5.0

503.6±30

.6Ba

303.2±28

.4Aa

1064

.8±79

.5C

1091

.4±81

.7C

57.939

⁄⁄

2.5

789.8±28

.3Bb

431.4±12

.7Aa

1064

.8±79

.5C

1091

.4±81

.7C

37.119

⁄⁄

1.25

1073

.6±40

.4Bc

849.4±33

.9Ab

1064

.8±79

.5B

1091

.4±81

.7B

4.34

7⁄

Con

t.(+)

1064

.8±79

.5c

1064

.8±79

.5c

Con

t.(�

)10

91.4±81

.7c

1091

.4±81

.7c

Fv.

26.151

⁄⁄58

.146

⁄⁄

L4

5.0

275.4±9.6A

a27

1.4±10

.7Aa

1064

.8±79

.5B

1091

.4±81

.7B

91.777

⁄⁄

2.5

345.2±14

.9Aa

573.8±12

.1Bb

1064

.8±79

.5C

1091

.4±81

.7C

56.969

⁄⁄

1.25

936.6±13

.5Bb

742.4±8.8A

c10

64.8±79

.5BC

1091

.4±81

.7C

10.726

⁄⁄

Con

t.(+)

1064

.8±79

.5bc

1064

.8±79

.5d

Con

t.(�

)10

91.4±81

.7c

1091

.4±81

.7d

Fv.

82.366

⁄⁄62

.309

⁄⁄

L5

5.0

245.0±19

.9Aa

496.2±9.2B

a10

64.8±79

.5C

1091

.4±81

.7C

73.300

⁄⁄

2.5

559.2±26

.2Ab

645.8±21

.9Ab

1064

.8±79

.5B

1091

.4±81

.7B

29.610

⁄⁄

1.25

694.2±30

.4Ab

855.0±27

.8Bc

1064

.8±79

.5C

1091

.4±81

.7C

12.893

⁄⁄

Con

t.(+)

1064

.8±79

.5c

1064

.8±79

.5d

Con

t.(�

)10

91.4±81

.7c

1091

.4±81

.7d

Fv.

56.689

⁄⁄31

.954

⁄⁄

L6

5.0

219.6±34

.8Aa

140.4±10

.9Aa

1064

.8±79

.5B

1091

.4±81

.7B

102.54

1⁄⁄

2.5

367.2±22

.2Ab

250.6±14

.3Aab

1064

.8±79

.5B

1091

.4±81

.7B

80.624

⁄⁄

1.25

925.0±16

.6Bc

378.6±14

Ab

1064

.8±79

.5BC

1091

.4±81

.7C

45.644

⁄⁄

Con

t.(+)

1064

.8±79

.5d

1064

.8±79

.5c

Con

t.(�

)10

91.4±81

.7d

1091

.4±81

.7c

Fv.

75.185

⁄⁄10

7.80

9⁄⁄

Notes:⁄⁄Highlysign

ificant

�Means

follo

wed

bydifferentletters

aresign

ificantly

differentfrom

each

otherat

p<0.05

(Duncantest).

L1–3=frassof

1st–3rdLarvalinstars,L6=frassof

6thLarvalinstar,L4=frassof

4thLarvalinstar,L1–6=frassof

1st–6thLarvalinstarsandL5=frassof

5thLarvalinstar.

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Table

2.Effectof

ethano

licextractof

S.littoralis

larval

frasson

oviposition

ofA.ipsilonadultfemales

usingtwodifferenttypesof

larval

food.

Ethanolic

extract

oflarval

frass

Meanno

.of

laid

eggs

Con

c.(%

)

Frass

ofS.

litoralislarvae

fedon

Con

t.(+)

Con

t.(�

)Fv.

Castoroilleaves

Sem

i-artificial

diet

L1–3

5.0

0Aa

66.4±5.9A

a79

2.8±64

.4B

848.2±27

.5B

168.24

9⁄⁄

2.5

16.2±2.7A

a12

1.8±8.4A

a79

2.8±64

.4B

848.2±27

.5B

152.96

3⁄⁄

1.25

42.0±2.9A

a49

6.2±22

.6Bb

792.8±64

.4C

848.2±27

.5C

100.43

8⁄⁄

Con

t.(+)

792.8±64

.4b

792.8±64

.4c

Con

t.(�

)84

8.2±27

.5b

848.2±27

.5c

Fv.

196.16

8⁄⁄

120.40

0⁄⁄

L4

5.0

0Aa

99.4±15

.9Aa

792.8±64

.4B

848.2±27

.5B

155.10

7⁄⁄

2.5

41.0±4.9A

a23

4.4±14

.16B

b79

2.8±64

.4C

848.2±27

.5C

126.18

5⁄⁄

1.25

151.0±16

.8Ab

405.6±36

.5Bc

792.8±64

.4C

848.2±27

.5C

66.884

⁄⁄

Con

t.(+)

792.8±64

.4c

792.8±64

.4d

Con

t.(�

)84

8.2±27

.5c

848.2±27

.5d

Fv.

167.94

4⁄⁄

82.852

⁄⁄

L5

5.0

179.0±13

.5Aa

136.2±10

.6Aa

792.8±64

.4B

848.2±27

.5B

113.23

9⁄⁄

2.5

317.0±23

.1Ab

234.0±12

.2Aa

792.8±64

.4B

848.2±27

.5B

72.031

⁄⁄

1.25

544.0±33

.9Ac

503.2±23

.5Ab

792.8±64

.4B

848.2±27

.5B

18.212

⁄⁄

Con

t.(+)

792.8±64

.4d

792.8±64

.4c

Con

t.(�

)84

8.2±27

.5d

848.2±27

.5c

Fv.

62.508

⁄⁄89

.594

⁄⁄

5.0

38.2±4.1A

a33

.2±3.2A

a79

2.8±64

.4B

848.2±27

.5B

166.83

6⁄⁄

L6

2.5

54.4±3.7A

a34

6.0±21

.9Bb

792.8±64

.4C

848.2±27

.5C

105.21

9⁄⁄

1.25

216.2±17

.5Ab

504.0±18

.8Bc

792.8±64

.4C

848.2±27

.5C

61.062

⁄⁄

Con

t.(+)

792.8±64

.4c

792.8±64

.4d

Con

t.(�

)84

8.2±35

.6c

848.2±35

.6d

Fv.

134.76

1⁄⁄

97.417

⁄⁄

Notes:⁄⁄Highlysign

ificant�M

eans

follo

wed

bydifferentletters

aresign

ificantly

differentfrom

each

otherat

p<0.05

(Duncantest).

L1–3=frassof

1st–3rdLarvalinstars,L4=frassof

4thLarvalinstar,L5=frassof

5thLarvalinstar

andL6=frassof

6thLarvalinstar.

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Table

3.Effectof

ethano

licextractof

A.ipsilonlarval

frasson

oviposition

ofconspecificinsect

usingtwodifferenttypesof

larval

food

.

Ethanolic

extract

oflarval

frass

Meanno

.of

laid

eggs/fem

ale

Con

c.(%

)

Frass

ofA.ipsilonlarvae

fedon

Con

t.(+)

Con

t.(�

)Fv.

Castoroilleaves

Sem

i-artificial

diet

L1–3

5.0

162.6±7A

a64

.8±4.7A

a79

2.8±64

.4B

848.2±27

.5B

135.49

9⁄⁄

2.5

227.0±14

.2Aab

136.8±13

Aa

792.8±64

.4B

848.2±27

.5B

104.42

6⁄⁄

1.25

280.2±20

.1Ab

329.6±16

.9Ab

792.8±64

.4B

848.2±27

.5B

63.988

⁄⁄

Con

t.(+)

792.8±64

.4c

792.8±64

.4c

Con

t.(�

)84

8.2±27

.5c

848.2±27

.5c

Fv.

98.132

⁄⁄12

4.35

3⁄⁄

L4

5.0

15.0±2

Aa

17.2±2.9A

a79

2.8±64

.4B

848.2±27

.5B

175.78

1⁄⁄

2.5

24.2±2.3A

a70

.0±5A

ab79

2.8±64

.4B

848.2±27

.5B

162.20

6⁄⁄

1.25

189.4±9.1A

b15

6.2±9.1A

b79

2.8±64

.4B

848.2±27

.5B

110.81

8⁄⁄

Con

t.(+)

792.8±64

.4c

792.8±64

.4c

Con

t.(�

)84

9.2±27

.5c

849.2±27

.5c

Fv.

171.40

2⁄⁄

166.06

5⁄⁄

L5

5.0

48.0±3.8A

a16

.2±2.1A

a79

2.8±64

.4B

848.2±27

.5B

168.78

4⁄⁄

2.5

69.6±4.6A

a22

.8±2.5

Aa

792.8±64

.4B

848.2±27

.5B

162.71

7⁄⁄

1.25

282.2±20

.1Bb

28.6±1.6

Aa

792.8±64

.4C

848.2±27

.5C

119.43

7⁄⁄

Con

t.(+)

792.8±64

.4c

792.8±64

.4b

Con

t.(�

)84

8.2±27

.5c

848.2±27

.5b

Fv.

140.67

3⁄⁄

194.52

0⁄⁄

5.0

77.2±7.2B

a13

.4±3.1A

a79

2.8±64

.4C

848.2±27

.5C

162.20

1⁄⁄

L6

2.5

170.2±5.9B

a38

.4±3.4A

a79

2.8±64

.4C

848.2±27

.5C

140.80

0⁄⁄

1.25

332.2±14

.7Bb

75.2±4.8A

a79

2.8±64

.4C

848.2±27

.5C

107.511⁄

⁄

Con

t.(+)

1064

.8±79

.5d

1064

.8±79

.5c

Con

t.(�

)10

91.4±81

.7d

1091

.4±81

.7c

Fv.

75.185

⁄⁄10

7.80

9⁄⁄

Notes:⁄⁄Highlysign

ificant�M

eans

follo

wed

bydifferentletters

aresign

ificantly

differentfrom

each

otherat

p<0.05

(Duncantest).

L1–3=frassof

1st–3rdLarvalinstars,L4=frassof

4thLarvalinstar,L5=frassof

5thLarvalinstar

andL6=frassof

6thLarvalinstar.

580 A.A.I. Ahmed et al.

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Table

4.Effectof

ethano

licextractof

A.ipsilonlarval

frasson

oviposition

ofS.

littoralis

adultfemales

usingtwodifferenttypesof

larval

food.

Ethanolic

extract

oflarval

frass

Con

c.(%

)

Meanno

.of

laid

eggs/fem

ale

Frass

ofA.ipsilonlarvae

fedon

Con

t.(+)

Con

t.(�

)Fv.

Castoroilleaves

Sem

i-artificial

diet

L1–3

5.0

140.0±13

.6Aa

79.2±6.3A

a10

64.8±79

.5B

1091

.4±81

.7B

132.91

9⁄⁄

2.5

345.4±8.7B

b18

0.4±9.2A

a10

64.8±79

.5C

1091

.4±81

.7C

96.581

⁄⁄

1.25

671.4±19

.3Ac

864.0±19

.6Bb

1064

.8±79

.5C

1091

.4±81

.7C

15.426

⁄⁄

Con

t.(+)

1064

.8±79

.5d

1064

.8±79

.5c

Con

t.(�

)10

91.4±81

.7d

1091

.4±81

.7c

Fv.

91.404

⁄⁄12

3.36

6⁄⁄

L4

5.0

360.6±14

.4Ba

146.0±17

.3Aa

1064

.8±79

.5C

1091

.4±81

.7C

96.603

⁄⁄

2.5

655.2±19

.6Bb

271.6±10

.9Aa

1064

.8±79

.5C

1091

.4±81

.7C

62.034

⁄⁄

1.25

906±26

.7Ac

941.8±14

.3Abb

1064

.8±79

.5B

1091

.4±81

.7B

3.26

3⁄

Con

t.(+)

1064

.8±79

.5d

1064

.8±79

.5bc

Con

t.(�

)10

91.4±81

.7d

1091

.4±81

.7c

Fv.

45.093

⁄⁄10

6.18

4⁄⁄

L5

5.0

552.4±13

.7Aa

528.2±17

.1Aa

1064

.8±79

.5B

1091

.4±81

.7B

39.898

⁄⁄

2.5

743.8±21

.5Ab

779.4±12

.2Ab

1064

.8±79

.5B

1091

.4±81

.7B

13.739

⁄⁄

1.25

890.2±29

.9Ac

1052

±53

Abc

1064

.8±79

.5AB

1091

.4±81

.7B

2.57

0ns

Con

t.(+)

1064

.8±79

.5d

1064

.8±79

.5c

Con

t.(�

)10

91.4±81

.7d

1091

.4±81

.7c

Fv.

23.747

⁄⁄24

.032

⁄⁄

L6

5.0

360.8±10

.8Aa

388.6±10

.9Aa

1064

.8±79

.5B

1091

.4±81

.7B

69.979

⁄⁄

2.5

757.6±19

.4Ab

649.8±11.5

Ab

1064

.8±79

.5B

1091

.4±81

.7B

20.080

⁄⁄

1.25

936.4±13

.7Ac

1004

.6±33

.6Ac

1064

.8±79

.5A

1091

.4±81

.7A

1.80

7ns

Con

t.(+)

1064

.8±79

.5cd

1064

.8±79

.5c

Con

t.(�

)10

91.4±81

.7d

1091

.4±81

.7c

Fv.

45.438

⁄⁄44

.964

⁄⁄

Notes:n.s.no

n-significant,⁄ significant

and

⁄⁄high

lysign

ificant�M

eans

follo

wed

bydifferentletters

aresign

ificantly

differentfrom

each

otherat

p<0.05

(Duncantest).

L1–3=frassof

1st–3rdLarvalinstars,L4=frassof

4thLarvalinstar,L5=frassof

5thLarvalinstar

andL6=frassof

6thLarvalinstar.

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Table5.

Fatty

acid

compo

nentspercentage

offrassextracted

from

differentlarval

instar

ofS.

littoralis

and

A.ipsilon

fed

oncastor

oilleaves

and

semi-artificial

diet.

Chemicals

%of

frassextractsfatty

acids

L1–3

L4

L5

L6

S.littoralis

A.ipsilon

S.littoralis

A.ipsilon

S.littoralis

A.ipsilon

S.littoralis

A.ipsilon

AB

AB

AB

AB

AB

AB

AB

AB

Palmiticacid

ethy

leester

35.4

10.50

40.33

13.21

50.09

12.8

37.61

13.04

43.6

2.85

17.01

15.63

13.97

6.68

22.69

34.28

Myristic

acid

0.99

1.12

1.31

2.63

1.79

1.31

�1.13

Linolenic

acid

�20

.33

5.24

��

5.71

�6.96

23.64

41.95

�26

.25

7.79

linoleic

�5.02

3.31

�5.28

�4.51

4.49

12.35

3.89

Oleic

acid

11.09

10.33

5.70

11.09

16.98

Pentadecano

icacid

2.22

1.48

5.44

5.82

7.10

.13-hexadecatrieno

icacid

methy

lester

��

��

��

�5.60

Stearic

acid

��

�7.11

3.05

��

3.43

3.60

4.82

�3.67

2.76

Ethyl

9-hexadecaenoicacid

�1.07

3.54

�2.02

2.76

��

7.00

��

6.41

Totalfatty

acids

36.39

36.92

52.22

16.75

51.4

38.3

41.72

34.89

45.39

20.02

49.52

41.81

74.4

16.17

59.56

68.22

Notes:A=fatty

acids%

extractedfrom

larvae

feed

oncastor

oilleaves

andB=fatty

acids%

extractedfrom

larvae

fedon

semi-artifi

cial

diet.

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effective when the larvae fed on semi-artificial diet, that clearly observed at treatmentwith L5 which resulted 16.2, 22.8 and 28.6 eggs/female at concentrations 5.0, 2.5 and1.25%, respectively, while the oviposition effect of extracted frass of young larvae (L1–3)was very week obtaining the maximum number of laid eggs in the experiment (Table 3).Extracted frass of A. ipsilon larvae obtained significant different between mean numberof laid eggs at all tested concentrations of each treatment also between each concentra-tion and the control in the case of castor oil leaves as a food source while using of semi-artificial diet resulting insignificant difference between the two concentrations 5.0 and2.5% at both of treatments L1–3 and L4. Extract of frass of old larvae L5 and L6 obtainedsignificant difference between all tested concentrations and between each of them andthe control. Extract of frass of the youngest tested larvae (L1–3) was the most effectiveextract on the oviposition of S. littoralis adult females especially when the larvae fed onsemi-artificial diet resulting the minimum number of laid eggs (79.2 eggs/female) at hightested concentration (5.0%) (Table 4).

Fractionation of frass extracts and oviposition repellency of treated insect

Several fatty acids were identified qualitatively and quantitatively in frass extracts ofdifferent larval instars of both target insects. Type and quantity of fatty acid dependsmainly on larval food source and larval instar, except palmitic acid which recorded atall larval instars and food sources. Oleic acid and ethyle 9-hexadecaenoic acid werefound when semi-artificial diet used as a food source while myrisitic acid was observedonly in extracted frass of fed larvae on castor oil leaves. Percentage of total fatty acidsin frass extracts of Agrotis larvae was ranged between 16.75% at young larvae (L1–3)and 68.22% at old larvae (L6) when the semi-artificial diet used for feeding, whileextracted frass of larvae feeding on natural host contained relatively more amount offatty acids percentages which recorded 41.72% at low level and 59.56% at high level.On the other hand, extracted frass of old Spodoptera larvae (L6) obtained the lowestamount of fatty acids (16.17%) while it was 36.92% at young larvae (L1–3) when thelarvae fed on semi-artificial diet in contrast with that found by using natural host for

Figure 1. Effective repellency percentage of the female adult of S. littoralis affected by sourceof feeding and frass extraction concentrations of conspecific insects.

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feeding recording 74.4 and 36.39% fatty acids in frass extracts of L6 and L1–3,respectively (Table 5).

Fluctuated oviposition repellency percentages were observed at different treatmentsaccording to larval instars of tested insects, extract concentration and source of larvalfood. The highest tested concentration (5.0%) was generally the most effectiveconcentration in contrast with the lowest concentration (1.25%). Frass extract of S. litto-ralis larvae fed on semi-artificial diet resulted higher oviposition repellency when testedagainst the adult females of conspecific insect while the opposite was occurred againstA. ipsilon adult females (Figures 1 and 2). The oviposition repellency of A. ipsilonlarval frass was more than 90% at young larvae only fed on semi-artificial diet againstS. littoralis adult females while the same effect was observed at all larval instars whentested against the conspecific insect which also relatively affected by frass extract oflarvae fed on castor oil leaves (Figures 3 and 4).

Figure 2. Effective repellency percentage of the female adult of A. ipsilon affected by source offeeding and frass extraction concentrations of S. littoralis.

Figure 3. Percentage of effective repellency of A. ipsilon affected by source of feeding and frassextraction concentrations of conspecific insect.

584 A.A.I. Ahmed et al.

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GC/MS analysis of larval frass

The chemical composition of tested extracts was identified using GC/MS analysis tech-nique and the data are represented in Table 5 and Figures 5–8. The frass extract ofyoung S. littoralis larvae (L1–3) fed on castor oil leaves or semi-artificial diet revealedthat, presence of fatty acids as esters in percentages 36.39 and 36.92%, respectively, ofthe total extract while a group of aliphatic and aromatic hydrocarbons was identifiedand represented the rest of the total extract. The analysis recorded that, palmitic acidethyl ester was the major percentage of fatty acid representing 35.4% of total extractfollowed by myristic acid (0.99%) of frass extract of larvae fed on castor oil leaves(Figure 5(a)), while when the larvae fed on semi-artificial diet the frass analysesobtained that, four fatty acids ethyl esters with different percentages were represented.Unsaturated major one was linolenic acid constituted 20.33% followed by saturated onepalmitic which represent 10.50% and the minimum recorded levels were 5.02% atlinoleic acid and 1.07% at ethyl 9-hexadecaenoic acid (Figure 5(b)).

Analysis of frass extract of old larvae (6th instar) of the same insect obtained thehighest percentage of fatty acids (74.4%) when castor oil leaves were used for feeding,linolenic and myristic acid were represented the highest and lowest percentages of thetotal fatty acids (41.94 and 1.31%, respectively), while the lower amount of total fattyacids was identified in frass extract of fed larvae on semi-artificial diet (Figure 6(a) and(b), respectively).

On the other hand, analysis of the tested extracts of frass of A. ipsilon larvae fed onnatural food or semi-artificial diet revealed the presence of aliphatic and aromatichydrocarbons in addition to volatile fatty acids ethyl esters. The relative percentages oftotal fatty acids in frass extract of the L1–3 and L6 instars larvae were 52.22, 59.65%and 16.75, 68.22% at castor oil leaves and semi-artificial feeding, respectively. Palmiticacid presented the major percentage in L1–3 frass extract recording 40.33% when castoroil leaves were used and 13.21% by using semi-artificial diet for larval feeding. Asanalysis obtained, linolenic acid was the most abundant one in frass extract of L6 fedon castor oil leaves (26.25%),while at extracted frass of 6th instar larvae fed on

Figure 4. Percentage of effective repellency of S. littoralis affected by source of feeding andfrass extraction concentrations of A. ipsilon.

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Figure5.

GC/M

Sanalysisof

hexane

extractsof

frassof

S.littoralis

L1–3instarslarvae

fedon

(a)castor

oilleaves

and(b)semi-artificial

diet.

586 A.A.I. Ahmed et al.

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Figure6.

GC/M

Sanalysisof

hexane

extractsof

frassof

S.littoralis

L6instar

larvae

fedon

(a)castor

oilleaveand(b)semi-artificial

diet.

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Figure7.

GC/M

Sanalysisof

hexane

extractsof

frassof

A.ipsilonL1–3instarslarvae

fedon

(a)castor

oilleaves

and(b)semi-artificial

diet.

588 A.A.I. Ahmed et al.

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Figure8.

GC/M

Sanalysisof

hexane

extractsof

frassof

A.ipsilonL6instar

larvae

fedon

(a)castor

oilleaves

and(b)semi-artificial

diet.

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semi-artificial diet, the palmitic acid was the predominate fatty acid constituted 34.28%(Figures 7(a, b) and 8(a, b)).

In general, it was observed that, fatty acids of extracted frass of different larvalinstars obtained varied quantitatively and qualitatively in spite of all instars fed on thesame source of food (natural host or semi-artificial diet) as shown in (Table 5).

Discussion

The oviposition deterrent effect of ethanolic extract of frass of S. littoralis or A. ipsilonlarvae fed on castor oil leaves was very clear at high tested concentrations against adultfemales of the same species. In a similar study by Hilker and Klein (1989), the authorsused different solvents e.g. acetone, water, ethanol, chloroform and pentane to extract thefrass of S. littoralis larvae fed on natural host. Fluctuated oviposition deterrent effect toadult females of the same insect was observed by obtaining relatively more effect by ace-tone than ethanol. Extracted frass of Helicoverpa armigera larvae fed on Gossypium hirsu-tum leaves by hexane resulted in a positive oviposition deterrent to adult females, suchresponse that was discussed by Xu et al. (2006), may be due to the non-specific fatty acidsproduced by the larvae and independence of food type, which play a great role in oviposi-tion deterrent. Effect of ethanolic extract of larval frass of S. littoralis was more deterrentto oviposition in case of A. ipsilon adult females than in S. littoralis adult females.

Hexane extract of frass of H. armigera larvae fed on G. hirsutum leaves obtainedpositive oviposition deterrent to adult females, such response that was discussed by Xuet al. (2006), may be due to the non-specific fatty acids produced by the larvae andindependence of food type which play a great role in oviposition deterrent. Our resultsalso indicated that, the oviposition of A. ipsilon adult females was more affected thanS. littoralis adult females when oviposition sites treated with ethanolic extract ofS. littoralis larval frass.

On the other hand, Anderson et al. (1995) studied the effect of methanolic extractof S. littoralis or Agrotis segetum frass of larvae fed on several types of synthetic foodon the oviposition of the adult females of both insects, they concluded that, ovipositionbehaviour was influenced by larval experience which need to be transferred to the adultstage, where the information storage takes place during the larval development in thecentral nervous system (CNS). Such observation was accepted by the finding of Jermyet al. (1968), they reported that the induced larval feeding preference, which persistover several larval moults have been considered to be due to a storage of informationin the CNS. That conclusion is in contrast with our observation in all evaluation inthese experiments by using emerged adults from reared larvae on natural host whichobtained oviposition deterrent on treated sites with extracted frass collected from larvaefed on semi-artificial diet. The obtained results are in agreed with that reported byAnderson and Löfqvist (1996), the filed females of A. segetum responded to thedeterrents similar to females from the laboratory culture. Oviposition activity of etha-nolic extract of larval frass that obtained in this study was accepted by the finding ofMitchell and Heath (1985); they stated that, the oviposition deterrent in larval frass ofSpodoptera exigua L. and Spodoptera eridania (Cramer) could be extracted with bothwater and organic solvents like ethanol and dichloromethane.

Fractionation and identification of larval frass of both tested insects S. littoralis and A.ipsilon revealed that the presence of several aliphatic and aromatic hydrocarbons in addi-tion to volatile fatty acids ethyl esters with different concentrations. Two main fatty acids,tetraecanoic acid ethyl ester and palmatic acid ethyl ester, were identified in the extract.

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These fatty acids play an important role in oviposition deterrence as reported by Xu et al.(2006) and that agree with the results in this study, which obtained fluctuated ovipositiondeterrent of larval frass of different instars according to amounts of fatty acids.

Variations of the effect of frass extract of different larval instars depend mainly onthe type of fatty acids and some other components. Ratio of each others plays also agreat role in this effect as was described by Weaver et al. (1989), Klein et al. (1990),Anderson et al. (1993), Parr et al. (1998), Li and Ishikawa (2004), Xu et al. (2006),Guo and Li (2009).

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