1 Running Head: Incidence and Morphometrics of amata on Salvadora persica

2 Please address correspondence to: 3 Dr. Shravan M. Haldhar, Scientist 4 Agricultural Entomology 5 ICAR-Central Institute for Arid Horticulture 6 Sri Gananagar Highway, Beechwal Industrial Area P.O. 7 Bikaner-334006, India 8 E-mail: [email protected] 9

10 First report of Colotis amata (: ) on Salvadora

11 persica (Capparales: Salvadoraceae) in Rajasthan, India: incidence

12 and morphometric analysis

13

14 SM Haldhar1*, R Bhargava1, RS Singh1, H Krishna1 & SK Sharma1

15 1Central Institute for Arid Horticulture, ICAR, Bikaner, India

16

17 *Corresponding author; E-mail: [email protected]

18 19

20 Abstract

21

22 An infestation of small salmon Arab, Colotis amata (F.) (Lepidoptera: Pieridae), on pilu

23 (Salvadora persica L.; Capparales: Salvadoraceae) plants was first noticed in 2012 at the

24 Experimental Farm of the Central Institute for Arid Horticulture and other fields of Bikaner

25 District, Rajasthan, India. The maximum incidence (80%) was observed in 18 Dec and the

26 minimum (13%) in 4 Sep. The average numbers of ranged between 6.2 and 22.3 larvae

27 per 3 leaves. Adults of this small were yellow and white in color. Eggs were laid singly

28 on leaves or young shoots and were 0.58-0.72 mm in length, 0.38-0.43 mm in width, and white

29 in color when first laid, later developing red blotches. The length and width of 1st instar larvae

30 were 1.98-2.29 mm and 0.36-0.48 mm, respectively. The lengths of 2nd, 3rd, and 4th instar

31 larvae were 5.04 mm, 9.77 mm, and 14.27 mm, respectively. The 5th instar larvae were 19.30

32 mm long and 3.33 mm wide. Pupae were 14.10 mm long and 5.73 mm wide and laterally

33 compressed. Adults were salmon-pink in color, and females had a body length of 9.71 mm and a

34 wingspan of 33.62 mm. Bodies of males were 7.59 mm long and their wingspan measured 25.67

35 mm. The lengths of male and female antennae were 4.63 mm and 5.46 mm, respectively.

36

37 Key Words: small salmon Arab, pilu, incidence, morphometric characters

38

39

40 41

42 Introduction

43 44 The genus Salvadora belongs to the family Salvadoraceae. It comprises 3 genera (i.e.,

45 Azima, Dobera, and Salvadora) and 10 species distributed mainly in the tropical and subtropical

46 regions of Africa and Asia (Mabberley 2008). On the Indian subcontinent, this family is

47 represented by only 1 genus with 2 species viz. Salvadora persica L. and Salvadora oleoides

48 Decne (Qureshi 1972; Stewart 1972; Perveen & Qaiser 1996). Salvadora persica, or pilu, is a

49 medium-sized tree or shrub with a crooked trunk, seldom more than 1 ft (0.3 m) in diameter. It

50 has a pleasant fragrance as well as a warm and pungent taste. Fruits have a sweet, agreeable,

51 aromatic, and slightly sour flavor. They can be eaten raw, cooked, or dried and stored when

52 dried. Fruits with or without seeds are said to contain 1.7-1.9% sugars when ripe, and delicious

53 drinks can be made from them. Salvadora persica is a popular chewing stick commonly known

54 as “miswak” and is one of the most popular medicinal plants throughout the Indian subcontinent

55 as well as other parts of Asia and the Middle East (Ezmirly et al. 1978; Almas 2002; Al-Otaibi et

56 al. 2003, 2004; Sofrata et al. 2007). When S. persica occurs on river terraces, it is a preferred

57 host of Cistanche tubulosa (Schenk) R. Wight (Lamiales: Orobanchaceae), an obligate

58 phanerogamic root parasitic plant (Sher et al. 2010). Furthermore, defoliating larvae of several

59 beetles and of Colotis ephyia (Klug) (Lepidoptera: Pieridae) often attack the tree, and the mite

60 Eriophyes sp. causes leaf galls (Kumar et al. 2012). An infestation with small salmon Arab has

61 not been reported. Here, we report new information on the incidence of C. amata on pilu,

62 including its morphological characters and taxonomic identification.

63

64 65

66 Materials and Methods

67

68 Twenty pilu plants were selected randomly in each of 3 replicates at the Experimental

69 Farm of the Central Institute for Arid Horticulture (CIAH) (N 28° 06' E 73° 21'). Incidence of

70 infestation observed on each plant and numbers of larvae per 3 leaves were recorded from Sep

71 2012 to Feb 2013. The sampling was done by visual observation and manual counting. Average

72 incidence was calculated as the percentage of whole plants infested with C. amata. Average

73 numbers of larvae per 3 leaves were calculated from numbers recorded for 10 randomly selected

74 pilu plants with 3 replications. Twenty insects (10 males and 10 females) were used for

75 observation and measurements. The larvae were reared under laboratory conditions (temperature

76 at 28 ± 2 oC with a relative humidity of 60 ± 5 %) for measurement of different stages. The

77 average linear measurements of various body parts of male and female adults were obtained

78 under a binocular microscope (Radical Instruments, Ambala, Haryana, India) using Jenoptic Pro

79 2.8.0 software. The terminology used to denote different parts of the body was according to

80 Haldhar (2012) and Haldhar & Singh (2014). The butterfly samples collected from the CIAH

81 farm and other fields were preserved in 70% alcohol and deposited at the Biosystematic

82 Section, Division of Entomology, Indian Agricultural Research Institute, New Delhi, for

83 taxonomic identification.

84 Before statistical analysis, data were transformed as necessary to achieve normal

85 distribution. The data on incidence and numbers of insects were analyzed by 1-way ANOVA

86 using SPSS software (O’Connor 2000). When significant differences were detected, means were 87 compared by using Turkey’s honest significant difference (HSD) tests for paired comparisons at

88 a probability level of 5%.

89 Results and Discussion

90 The observed on pilu plants were identified as C. amata. Damaging effects

91 were recorded from Sep to Feb. The percentage of plants infested with C. amata ranged from 13-

92 80%, with the maximum incidence recorded on 18 Dec and the minimum on 4 Sep. The

93 maximum average number of larvae (22.3 per 3 leaves) was recorded on 18 Dec and the

94 minimum (6.2 per 3 leaves) on 4 Sep, followed by 7.6 larvae per 3 leaves on 19 Sep (Table 1).

95 Larvae damaged the new leaves, old leaves, and new or top branches of the pilu plants (Fig. 1).

96 Due to attack by this pest, the growth of pilu plants was suppressed, and new branches and

97 leaves wilted. The data on linear measurements of the small salmon Arab are presented in Table

98 2 and Figure 2. Eggs were laid singly on leaves or young shoots. They were 0.58-0.72 mm in

99 length, 0.38-0.43 mm in width, and white in color when first laid, later developing red blotches.

100 The 1st larval instar was greenish yellow with a black head. The length and width of 1st instar

101 larvae were 1.98-2.29 mm and 0.36-0.48 mm, respectively. The lengths of 2nd, 3rd, and 4th

102 instar larvae were 5.04, 9.77, and 14.27 mm, respectively. The 5th instar larvae were 19.30 mm

103 long, 3.33 mm wide, and light greenish in color with a white stripe on the dorsal surface of the

104 body. Pupae were 14.10 mm long, 5.73 mm wide, and laterally compressed. They had a

105 moderately high dorso-thoracic keel and a pointed but short cephalic projection, slightly up-

106 curved distally. Adults had a salmon-pink color, and females had a body length of 9.71 mm and a

107 wingspan of 33.62 mm. The male body length and wingspan were 7.59 mm and 25.67 mm,

108 respectively. The costa on the forewing was black and thickly overlaid with grayish or pinkish

109 scales. The lengths of male and female antennae were 4.63 mm and 5.46 mm, respectively. 110 Colotis amata infestations were observed on pilu plants in the hot arid region (Thar Desert) of

111 northwestern India. To our knowledge, this is the first report of C. amata on S. persica. In West

112 Africa, this species is a common butterfly of the Sahel (Larsen 2005). The flying capacity of

113 Colotis species is very low and weak. Both sexes are frequently found feeding from flowers and

114 spend much of their time flying around in the vicinity of their larval host plant (S. persica),

115 resting periodically on the ground (Pringle et al. 1994). In Delhi, India, females of C. vestalis

116 were noted ovipositing single eggs on or near old leaves of the host plant, while females of C.

117 amata laid eggs in large clusters on fresh leaves of the same trees. These observations pertained

118 to 2 species of the plant genus Salvadora (Larsen 1988). This butterfly is damaging

119 economically important parts of the plant, such as leaves, flowers, and top branches. Therefore,

120 management practices need to be developed and implemented to minimize the losses caused by

121 this pest.

122 Acknowledgement

123

124 The authors thank V. V. Ramamurthy, National Coordinator, ICAR Network Project on

125 Insect Biosystematics, Division of Entomology, IARI, New Delhi, for the identification of this

126 pest and R. Swaminathan, Associate Professor, Department of Entomology, MPUAT, Udaipur,

127 for critical discussions and suggestions.

128 References

129 Almas K. 2002. The effect of Salvadora persica extract (Miswak) and chlorhexidine gluconate

130 on human dentin: a SEM study. Journal of Contemporary Dental Practice, 3: 27-35. 131 Al-Otaibi M, Al-Harthy M, Söder B, Gustafsson A & Angmar-Mansson B. 2003. Comparative

132 effect of chewing sticks and toothbrushing on plaque removal and gingival health. Oral

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134 Al-Otaibi M, Al-Harthy M, Gustafsson A, Johansson A, Cleasson R & Angmar-Mansson B.

135 2004. Subgingival plaque microbiota in Saudi Arabians after use of miswak chewing

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137 Ezmirly ST, Cheng JC & Wilson SR. 1978. Saudi Arabian medicinal plants: Salvadora persica.

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139 Haldhar SM. 2012. Report of Homoeocerus variabilis (Hemiptera: Coreidae) on khejri (Prosopis

140 cineraria) in Rajasthan, India: incidence and morphometric analysis. Florida

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142 Haldhar SM & Singh RS. 2014. Report of Dictyla cheriani Drake (Hemiptera: Tingidae)

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149 Larsen TB. 2005. Butterflies of West Africa. Apollo Booksellers, Vester Skerninge, Denmark.

150 Mabberley DJ. 2008. Mabberley's Plant Book: A Portable Dictionary of Plants, their

151 Classifications, and Uses. 3rd ed. Cambridge University Press, Cambridge, United

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154 parallel analysis and Velicer’s MAP test. Behavior Research Methods, Instruments, and

155 Computers, 32: 396-402.

156 Perveen A & Qaiser M. 1996. Pollen flora of Pakistan – VI. Salvadoraceae. Pakistan Journal of

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158 Pringle EL, Henning GA & Ball JB [eds]. 1994. Pennington’s Butterflies of Southern Africa, 2nd

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160 Sher H, Al-Yemeni MN, Masrahi YS & Shah AH. 2010. Ethnomedicinal and ethnoecological

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163 Sofrata A, Lingström P, Baljoon M & Gustafsson A. 2007. The effect of miswak extract on

164 plaque pH: an in vivo study. Caries Research, 41: 451-454.

165 166 Table 1. Mean percent of Salvadora persica plants infested by the small salmon Arab, Colotis

167 amata, and mean number of larvae per 3 leaves of plant in 2012-2013 at the experimental

168 farm of the ICAR-Central Institute for Arid Horticulture, Bikaner District, Rajasthan,

169 India.

Sampling date Mean incidence Mean no. of C. amata larvae

(%)a,b per 3 leavesb

4 Sep 2012 13.33 (21.14)a 6.20a

19 Sep 2012 16.67 (23.85)a 7.63a

4 Oct 2012 30.00 (32.99)b 10.37b

19 Oct 2012 36.67 (37.21)bc 11.93bc

3 Nov 2012 56.67 (48.83)de 13.33cd

18 Nov 2012 60.00 (50.83)de 16.30ef

3 Dec 2012 70.00 (56.98)efg 18.30gh

18 Dec 2012 80.00 (63.90)g 22.30i

2 Jan 2013 66.67 (54.76)ef 19.00hi

17 Jan 2013 63.33 (52.75)ef 16.77fg

01 Feb 2013 46.67 (43.06)cd 12.80cd

16 Feb 2013 36.67 (37.21)bc 10.80bc

170 a Parentheses contain the angular transformation value.

171 b Values followed by different letters are significantly different (Tukey’s HSD test, P < 0.05).

172 173 Table 2. Mean (± SEM) linear morphometric measurements of different life stages of the small 174 salmon Arab, Colotis amata. Life stage Length (mm) Width (mm)

Egg 0.62 ± 0.009 0.41 ± 0.007

1st instar larva 2.15 ± 0.023 0.42 ± 0.012

2nd instar larva 5.04 ± 0.027 1.15 ± 0.022

3rd instar larva 9.77 ± 0.022 1.96 ± 0.011

4th instar larva 14.27 ± 0.052 2.27 ± 0.021

5th instar larva 19.30 ± 0.100 3.33 ± 0.012

Pupa 14.10 ± 0.046 5.73 ± 0.020

Adult female with wing expansion 9.71 ± 0.007 33.62 ± 0.031

Adult male with wing expansion 7.59 ± 0.021 25.67 ± 0.035

Female antenna 5.46 ± 0.016 not applicable

Male antenna 4.63 ± 0.017 not applicable

175 a Mean of ten specimens.

176

177 178 Figure Captions

179

180 Fig. 1. Aggregation of Colotis amata on a pilu plant (Salvadora persica) during the winter

181 season at the ICAR-Central Institute for Arid Horticulture, Bikaner, India.

182

183 Fig. 2. Different developmental stages of Colotis amata. A, eggs; B, 1st instar larvae; C, 5th

184 instar larva; D, pupae; E, adult male; F, adult female.

185

186

187 188

189 Fig. 1. Aggregation of Colotis amata on a pilu plant (Salvadora persica), during the winter

190 season at the ICAR-Central Institute for Arid Horticulture, Bikaner, India.

191

192 193 194

195 Fig. 2. Different developmental stages of Colotis amata. A, eggs; B, 1st instar larvae; C, 5th

196 instar larva; D, pupae; E, adult male; F, adult female.

197