Detailed Larval Biology of Indian Gypsy Moth Lymantria Obfuscata Walker on Quercus Leucotrichophora Roxb

American International Journal of Available online at http://www.iasir.net Research in Formal, Applied & Natural Sciences ISSN (Print): 2328-3777, ISSN (Online): 2328-3785, ISSN (CD-ROM): 2328-3793

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Detailed Larval Biology of Indian Gypsy Moth Lymantria obfuscata Walker on Quercus leucotrichophora Roxb. in Himachal Pradesh (India)

Bhopesh Thakur1, Sumit Chakrabarti2 and Manoj Kumar3 Department of Zoology, University College, Kurukshetra University, Kurukshetra, Haryana, India1 Forest Ecology and Rehabilitation Division, Tropical Forest Research Institute, Madhya Pradesh, India2 Department of Biotechnology, Shoolini University, Solan, Himachal Pradesh, India3

Abstract: Lymantria obfuscata Walker commonly known as Indian Gypsy Moth (IGM), is a serious pest of about 200 broad-leaved tree species throughout India. The pest undergoes complete metamorphosis with six larval instars. The larval stage is the destructive stage as it feeds on the foliage voraciously. The detailed study was done on the larval biology of this pest on the ban oak foliage in Himachal Pradesh. Keywords: Lymantria obfuscata, IGM, larva, feeding, length, width

I. Introduction Quercus leucotrichophora Roxb. (Ban oak), is an evergreen tree found between elevations of 1800 to 2300 m in Himachal Pradesh which grow up to 25 m. The distribution and forest cover of ban oak is highest among five indigenous species of oaks and is also a natural broad-leaf associate of many conifers of Himachal Pradesh [1]. The ban oak is of immense importance for the villagers of Himachal Pradesh, as its foliage is the rich source of nutritious green leaf fodder for livestock during the lean winter months and also provides the best firewood for cooking, heating and charcoal. Out of the 90 species of insects that feed on oak, lepidopteran defoliators cause significant damage to oak and the most important are Lymantria obfuscata, L. concolor, L. mathura, Malacosoma indica, Dasychira sp. and Euproctis sp [2]. Lymantria obfuscata Walker commonly known as Indian Gypsy Moth (IGM), is a serious pest of about 200 broad-leaved tree species, throughout India, namely oak (Quercus spp.), willow (Salix spp.), popular (Populas spp.), walnut (Jugulans spp.), apple (Malus spp.), apricot (Prunus spp.), cherry (Prunus cerasus) and almond (Prunus amygdalis) ([3, 4]). It was reported as the major pest of ban oak [5], and of apple trees at Kotgarh, Shimla (H.P.) [6]. In earlier studies, it was reported as one of the most destructive pests of fruit and forest plantations including apple, walnut willows and poplars in Kashmir ([7, 8, 9, 10, 11]). Incidence of L. obfuscata on apples, poplars, willows and other plantations in India has also been reported ([12, 13]). An outbreak was reported from Sarahan and Narag areas in Sirmour (H.P.), where massive defoliation of oak trees took place [14]. Some preliminary observations on the pest biology of L. obfuscata were made in the past ([5, 15, 16, 17]). These observations on its larval biology were either incomplete or were undertaken in different situations in India. Therefore, present work was undertaken to study the detailed larval biology of L. obfuscata Walker on ban oak in Himachal Pradesh, India. II. Materials and Methods The present study on the biological life cycle of L. obfuscata was conducted in the laboratory of Forest Protection Division, Himalayan Forest Research Institute (HFRI), Shimla (H.P.). Thick population of L. obfuscata was found in the village situated at Sarahan (Himachal Pradesh) and the egg-masses were collected and were placed in the laboratory for over-winter storage. The average temperature and relative humidity, during the course of the investigations (2006-2009) in laboratory were recorded as 19.98±4.535ºC and 58.46±16.627%, respectively [Thermo-Hygro Clock M288CTH, Mextech]. After the hatching of eggs, the 1st instar larvae were placed on fresh tender leaves of Quercus leucotrichophora, in the wire-meshed wooden cages of dimensions, 65 cm × 68 cm × 99 cm. Observations on the larval development such as, the number of instars, colour, moulting and feeding habits were recorded periodically. Morphometric studies were made with the help of Radical stereo zoom microscope [RSM-9] with USB Digital Scale 1.1E software in a digital microscopic workstation.

A. Preparation of permanent microscopic slides During the period of investigation, larvae of different instars were preserved in 70% alcohol in specimen tubes. 70% alcohol was decanted from the sample and distilled water was added. Thereafter, the sample was kept in

AIJRFANS 15- 224; © 2015, AIJRFANS All Rights Reserved Page 31 Bhopesh Thakur et al., American International Journal of Research in Formal, Applied & Natural Sciences, 10(1), March-May 2015, pp. 31-34 hot water bath for 5 minutes. Distilled water was decanted from the sample and 10% KOH solution was added and again kept in hot water bath for 15-20 minutes, till the inner tissues of the sample were completely dissolved and appeared clearer. KOH solution was decanted from the sample and distilled water was added and kept in hot water bath for 10-15 minutes. It was repeated till the excess of KOH was removed from the sample. After that dehydration was done in ascending grades of 30%, 50% and 70% alcohol and kept in hot water bath for 10-15 minutes. Finally, a puncture was made in the last abdominal segment and by pressing the larval body gently, the inner dissolved gut tissues came out making the specimen clearer. 70% alcohol was decanted from the sample and 90% alcohol was added and kept in hot water bath for 5 minutes. 90% alcohol was decanted from the sample and absolute alcohol was added and kept in hot water bath for 2-3 minutes. The sample was then dried with the help of blotting paper and Bersale’s mounting media was added to the sample and fixed with cover slip.

III. Results and Discussion In the laboratory rearing of L. obfuscata, it was observed that from a single egg-mass around 300-400 neonate larvae emerged in spring just when the new leaves emerged. And rearing of single larvae individually showed that L. obfuscata has six larval instars separated by five moulting stages. The caterpillars were nocturnal and fed gregariously, from the periphery towards the mid rib of the leaves. Young caterpillars spread to new locations by spinning silken thread. Each larval instar had a cylindrical, elongated body that was differentiated into head, thorax and abdomen. The colour changed from greyish-black in the first instar larva to reddish-brown in the last instar larva. The splitting of the exuvae started near the first thoracic segment, and the head capsule got itself split to the opposite direction. The head was hypognathus, black to reddish-brown in colour with small microscopic setae. It was composed of two parietal plates differentiated from each other by a distinct Y-shaped pericardial suture. In the head, the median epicranial sulcus was well-developed and the frons was represented by a pair of narrow oblique plates termed the adfrontals. Both clypeus and labrum were evident and the typical number of ocelli was six which were situated just behind, and a little above the bases of the segmented antennae. The mouthparts were found to be of mandibulate type and each mandible had three teeth, which were powerful and helpful in mastication of food. Larvae bore three thoracic segments which had a pair of legs each which end in a single curved claw. There were ten abdominal segments and 3rd, 4th, 5th, 6th and 10th abdominal segments bore a pair of prolegs each. A typical abdominal leg was a fleshy and was provided with a series of hooks which helped in locomotion. A pair of eversible glands was present on the dorsum of the 6th and 7th abdominal segments. Small hairs were scarcely distributed dorsally over the whole body of the larva and an anal comb was present prior to anus. The armature of the larval body consisted of simple hairs, tubercles and verrucae, bearing tufts of setae. The double row of tubercles on the dorsal surface of matured larvae were more prominent as the first five pairs were bluish and last six pairs brick red in colour. A. First instar larva The freshly hatched first instar larva was very small, active, and greyish in colour, with a small black head. The body of the larva was found to be covered with tufts of hairs, having a pair of small dots on dorsal side of each segment. The mean body length and width of first instar larva measured were 4.65±0.382 mm and 0.65±0.072 mm, respectively. The mean head length and width recorded were 0.78±0.042 mm and 0.75±0.038 mm, respectively, whereas, the mean mandible length and width were 0.18±0.015 mm and 0.16±0.013 mm, respectively. The mean inter-ocular distance was measured as 0.60±0.011 mm. This stage lasted for 11-14 days, with a mean of 12.75±1.50 days. Reference [17] reported that the first instar larvae of L. obfuscata measured 3.26 mm × 0.36 mm and their body was covered with tufts of hairs with a faint light strips, having a pair of small dots on each segment on the back, and this stage lasted for 8.26 days (range 7-10 days). B. Second instar larva The colour of the second instar larva was brown, and the hairs covering the body of the larva were very conspicuous. Its head was dark brown in colour. A double row of small tubercles was found along the back of the larva. The mean body length and width of second instar larva measured were 9.23±1.253 mm and 1.10±0.049 mm, respectively. The mean head length and width larva were recorded as 1.20±0.305 mm and 1.19±0.205 mm, respectively. The mean mandible length and width were 0.24±0.053 mm and 0.23±0.030 mm, respectively, and the mean inter-ocular distance was 0.93±0.183 mm. This stage lasted for 9-13 days, with a mean of 10.75±1.71 days. According to reference [17] the second instar larvae measured 9.5 mm × 2.03 mm and they have a brownish tint, with hairs covering the body. The head of the larva was dark brown in colour. He concluded that the stage was of shortest duration and lasted for 5.46 days (range 4-7 days). C. Third instar larva The third instar larva was dark grey in colour. The dark brown head of this stage was marked by yellow striations. The mean body length and width of third instar larva measured were 13.81±1.787 mm and 1.87±0.227 mm, respectively. Mean head length and width were recorded as 1.71±0.360 mm and 1.78±0.367 mm, respectively. The mean mandible length and width were 0.41±0.084 mm and 0.34±0.070 mm, respectively, and the mean inter-ocular distance was 1.44±0.365 mm. This stage lasted for 12-15 days, with a mean of

13.75±1.26 days. Reference [17] showed that the third instar larvae measured 19.31 mm × 4.65 mm and were dark grey in colour. The dark brown head of this stage had yellow marking. This stage lasted for 7.6 days (range 5-9 days). D. Fourth instar larva The fourth instar larva was reddish-brown in colour with the double row of tubercles more prominent, and the ventral surface, prolegs and legs were greyish-brown in colour. The body was covered with hairs. A light strip between the tubercles and spiracles on each side of the larva was present. The mean body length and width of fourth instar larva measured were 18.90±0.974 mm and 2.61±0.109 mm, respectively. Mean head length and width were recorded as 3.10±0.360 mm and 3.05±0.268 mm, respectively. The mean mandible length and width were 0.86±0.190 mm and 0.68±0.068 mm, respectively, and the mean inter-ocular distance was 2.52±0.177 mm. This stage lasted for 15-19 days, with a mean of 16.50±1.91 days. Reference [17] observed during the study, that fourth instar larvae measured 26.40 mm × 6.64 mm and the hairy body was brownish in colour with double row of tubercles prominent. A light strip between the tubercles and spiracles on each side of the larva was present. This stage lasted for 10.00 days (range 8-12 days). E. Fifth instar larva The fifth instar larva was bigger than that of fourth instar larva. It had short dorsal and long lateral tufts of hairs. The mean body length and width of fifth instar larva measured were 21.22±1.208 mm and 3.21±0.349 mm, respectively. Mean head length and width were recorded as 3.09±0.440 mm and 3.26±0.285 mm, respectively. The mean mandible length and width were 0.91±0.128 mm and 0.55±0.059 mm, respectively, and the mean inter-ocular distance was 2.72±0.405 mm. This stage lasted for 13-18 days, with a mean of 15.25±2.22 days. According to reference [17] the fifth instar larvae measured 40.17 mm × 11.62 mm and the general appearance and feeding behavior of fifth instar was almost same as that of fourth instar larva. It had short dorsal and long lateral tufts of hair. According to him, the fifth instar lasted for 16.06 days (range 14-19 days). F. Sixth instar larva The sixth instar larvae were the largest of all the previous instars. The mean body length and width of sixth instar larva measured were 36.30±6.764 mm and 5.38±0.869 mm. Mean head length and width were recorded as 4.12±0.424 mm and 4.28±0.049 mm. The mean mandible length and width were 1.06±0.141 mm and 0.70±0.106 mm, respectively, and the mean inter-ocular distance was 3.68±0.120 mm. This stage lasted for 10- 12 days, with a mean of 11.25±0.96 days. The observations made in the present study, were in complete harmony with [18], according to which the tribe Lymantriini has shorter, more even tufts of hair, arising in clumps from the verrucae and conspicuous yellowish- red dorsal glands on sixth and seventh abdominal segments. Earlier, [19] described the full grown larvae of L. obfuscata, [16] the other stages and [20] the head capsule, but only briefly. According to [21], the caterpillars were nocturnal, brown with black specks on the dorsum, head was prominent, brownish-yellow having two dark brown specks on the median suture. Body was covered with tufts of brown hairs. Besides three thoracic, there were four pairs of abdominal pseudolegs and the full grown larva measured 3.3 cm × 0.5 cm. In the present study, the larval period of L. obfuscata was recorded as 76-84 days, with average of 80.25±3.30 days (from mid March to May). It has been observed that the larva moults five times during the larval period. The larvae of L. obfuscata closely resembled those of L. dispar. According to Reference [17], the respective stages 1st, 2nd, 3rd, 4th and 5th lasted for 8.26, 5.46, 7.6, 10.00 and 16.06 days, respectively. Similarly, in a previous study the 1st, 2nd, 3rd, 4th, 5th and 6th instars of L. obfuscata lasted for 7-8, 9-11, 9-11, 10, 8 and 7 days, respectively [3]. During the day the larvae rested either on tree trunks or under stones. When full grown, they seek sheltered spots either in a hollow on the trunk of a tree or in the crevices in stones on the ground. G. Larval feeding The larvae of different instars were observed regarding their feeding habits. The newly hatched larvae rested for sometime on the top of their egg-mass. Thereafter, larvae moved to the leaf surface, where they started feeding on the outer soft parenchyma tissue of the leaf and formed minute holes in the leaf surface. The second instar larvae also preferred softer and fresh leaves and formed patches and holes in the epidermis. Feeding along the margins of the leaves was also observed. In the past it was observed that first instar larvae made small perforations on upper surface of leaves, and late instars devoured the leaves completely leaving the mid-rib of leaves only [17]. The third instar larva fed upon the softer leaf tissues along with the softer veins of the leaves. More larvae were found feeding along leaf margins as compared to previous stages. The fourth and fifth instars were found to cause maximum damage to the foliage by feeding upon the harder tissues of the ban oak leaves. Sixth instar larvae had slow rate of feeding and after sometime they stopped feeding and transformed into the next pupal stage. All the larvae fed gregariously, and moved to the other part of the foliage in search of food, showing their voracious feeding nature. Later instars of gypsy moth move down the trees at dawn and climb back at the dusk. This daily migration appeared to be influenced by environmental conditions, including abiotic factors and sunlight was probably responsible for stimulating large caterpillars to migrate up and down the trees [22]. It was noticed that when larvae were crowded or partially starved, pupal weights were reduced but the period of larval development was extended by 0-3 (crowding) and 8 (starvation) days [23].

H. Moulting behaviour Before moulting in each instar larvae, the feeding was arrested and the movement slowed down. Shedding of the old skin and head capsule followed different patterns as the head capsule was found intact or broken into two halves occasionally. Shedding of the skin commenced at the first thoracic segment and receded to the rear abdominal end where it was found to be removed off the larval body.

Acknowledgements The corresponding author is highly thankful to University Grants Commission, New Delhi, India for providing financial assistance in the form of Research Fellowship (Junior Research Fellowship), during the present study. Acknowledgments are also owed to Himalayan Forest Research Institute, Shimla, Himachal Pradesh, India for providing the necessary laboratory facilities during the course.

References [1] S. S. Negi and H. B. Naithani, Oaks of India, Nepal and Bhutan, International Book Distributors, Dehra Dun, 1995. [2] R. N. Mathur and B. Singh, A list of insect pests of forest plants in India and the adjacent countries, Part 1-9. List of insect pest of plants genera C-Z. Part IV-X-Indian Forest Bulletin, 1958-1961, vol. 171. [3] P. R. Dharmadhikari, G. Ramaseshiah and P. D. Achan, “Survey of Lymantria obfuscata and its natural enemies in India,” Entomophaga, vol. 30, 1985, pp. 399-408. [4] N. D. Rishi and K. A. Shah, “Survey of bio-ecological studies on the natural enemies of Indian gypsy moth Lymantria obfuscata Walker (Lepidoptera: Lymantriidae),” Journal of Entomological Research, vol. 9, 1985, pp. 82-93. [5] C. F. C. Beeson, Ecology and control of the forest insects of India and the neighboring countries, Forest Research Institute, Dehra Dun, 1941. [6] K. A. Rahman, “Occurrence of the gypsy moth Lymantria obfuscata Walker in Shimla Hills,” Indian Journal of Entomology, vol. 3, 1941, p. 338. [7] R. A. Malik, A. A. Punjabi and A. A. Bhat, “Survey and study of insect and non-insect pests in Kashmir,” Horticulture, vol. 3, 1972, pp. 29-44. [8] G. A. Dar, A. G. Sheikh and B. L. Ganjoo, “Relative efficacy of some insecticides in suppressing Lymantria obfuscata Walker on apple trees in Kashmir,” Pesticides, vol. 11, 1977, pp. 27-29. [9] A. G. Sheikh, “The effect of repeated defoliators caused by Lymantria obfuscata Walker on apple in Kashmir,” Indian Journal of Plant Protection, vol. 3, 1975, pp. 170-172. [10] M. A. Masoodi, A. R. Trali and A. M. Bhat, “Suppression of Lymantria obfuscata Walker by sex pheromone trapping of males,” Indian Journal of Entomology, vol. 52, 1990, pp. 414-417. [11] P. A. Kumar, N. Dorjay, M. S. Mir and A. S. Bilal, “Major insect pest associated with forest plantations in cold arid region, Ladakh of Jammu and Kashmir,” Journal of Entomological Research, vol. 31, 2007. [12] H. S. Pruthi and H. N. Batra, Important fruit pests of North-West India, The Indian Council of Agricultural Research, New Delhi, 1960. [13] P. S. Singh and S. S.Singh, Insect pests and diseases of poplar, Forest Research Institute Publications, 1986. [14] R. Singh, S. Kumar, S. Chakrabarti and A. Kumar, “Resurgence of Indian gypsy moth, Lymantria obfuscata Walker (Lepidoptera: Lymantriidae) on ban oak (Quercus leucotrichophora) forests in Rajgarh Forest Division, Himachal Pradesh,” Indian Journal of Forestry, vol. 30, 2007, pp. 83-85. [15] K. A. Rahman and A. N. Kalra, “Apple hairy caterpillars in the Shimla Hills,” Indian Farming, vol. 5, 1944, pp. 312-314. [16] M. L. Roonwal, “Life history and control of the Kashmir willow defoliator Lymantria obfuscata (Lepidoptera: Lymantriidae),” Journal of Indian Academy of Wood Science, vol. 8, 1977, pp. 97-104. [17] M. A. Masoodi, “Biological studies on Lymantria obfuscata Walker (Lepidoptera: Lymantriidae) in Kashmir,” Indian Forester, vol. 177, 1991, pp. 644-651. [18] D. C. Ferguson, The Moths of America: North of Mexico, Noctuoidea, Lymantriidae, E. W. Classey, London, vol. 22. [19] J. C. M. Gardner, “Immature stages of Indian Lepidoptera (1) Lymantriidae,” Indian Forest Record, vol. 3, 1938, pp. 187-212. [20] S. Adhikari, “Studies on the head capsule of the mature larva of Lymantria obfuscata Walker (Lymantriidae: Lepidoptera),” Nepal Journal of Agriculture, vol. 14, 1979, pp. 93-102. [21] T. D. Verma, J. R. Thakur and G. S. Dogra, “Outbreak of Indian Gypsy moth Lymantria obfuscata Wlk., on oak in Himachal Pradesh,” Indian Forester, vol. 105, 1979, pp. 594-597. [22] R. M. Weseloh, “Behavioural responses of gypsy moth (Lepidoptera: Lymantriidae) larvae to abiotic environmental factors,” Environmental Entomology, vol. 18, 1989, pp. 361-367. [23] D. R. Lance, J. S. Elkinton and C. P. Schwalbe, “Components of density-related stress as potential determinate of population quality in the gypsy moth Lymantria dispar (Lepidoptera: Lymantriidae),” Environmental Entomology, vol. 15, 1986, pp. 914-918.