4. RESULTS AND DISCUSSION

Transcription

1 51 4. RESULTS AND DISCUSSION The results achieved in the present study are described and discussed under the following heads: 4.1 Survey and diversity of fruit flies After an extensive survey of commercial cucurbits growing areas of Himachal Pradesh during across eight districts viz. Bilaspur, Chamba, Hamirpur, Kangra, Kullu, Mandi, Solan and Una, the data were compiled to draw inferences as under Fruit flies infestation in cucurbits The fruit flies infestation was encountered (ranging from to per cent) mostly in lower and middle altitude areas of Himachal Pradesh (Table 4.1). The state average fruit flies infestation was per cent, highest being in Kangra at Palampur (80.00%) and lowest in Chamba at Banikhet (44.44%). In district Bilaspur, fruit flies infestation at different locations varied from to per cent with maximum infestation at Ghumarwin (60.00 %) followed by Chandpur (55.55 %). In Chamba, only one location was surveyed i.e. Banikhet and the fruit fly infestation was per cent. The fruit flies infestation ranged between per cent in district Hamirpur, while it varied from per cent at different locations in district Kangra. In district Mandi, Solan, Kullu and Una, the fruit flies infestation varied from to per cent, to per cent, per cent, (at Naggar) and per cent (at Haroli), respectively. The variations of fruit flies infestation in cucurbits at different locations might be due to the variations in the local environmental conditions and relative susceptibility of the crop varieties. For example, highest fruit flies infestation was recorded at Palampur (80.00%) in Kangra district, where Sood et al. (2010) also

2 52 Table 4.1: Species and infestation index of fruit flies at different locations Sr. No. State(s) District(s) Place(s) Species recorded Host collected Per cent infestation* India 1 Himachal Bilaspur Chandpur Bactrocera tau Cucumber Pradesh 2 Ghumarwin B. cucurbitae, B. tau, Cucumber Nihari B. cucurbitae, B. tau, B. scutellaris Chamba Banikhet B. tau Cucumber Hamirpur Bhota B. cucurbitae Cucumber Nadaun B. cucurbitae, B. tau Bitter gourd, Bottle gourd, Cucumber 7 Kangra Indora B. cucurbitae Bitter gourd, Bottle gourd Jawalamukhi B. cucurbitae, B. tau Cucumber Kangra B. cucurbitae, B. tau, B. scutellaris Cucumber Palampur B. cucurbitae, B. tau, B. scutellaris, B. nigrofemoralis, B. latifrons, B. diversa, B.dorsalis, B. zonata, Dacus longicornis, D. sphaeroidalis, Dacus sp., Cyrtostola limbata, Pliomelaena udhampurensis, Dioxyna sororcula Bitter gourd, Bottle gourd, Cucumber, Summer squash, Pumpkin 11 Shahpur B. cucurbitae, B. tau Bitter gourd Paragpur B. dorsalis, B. zonata Kullu Naggar B. tau Cucumber Mandi Barot B. tau, B. scutellaris Pumpkin Mandi B. cucurbitae, B. tau Cucumber Nagwain B. cucurbitae, B. tau Bitter gourd Sundernagar B. cucurbitae, B. tau, Cucumber, Bitter gourd Solan Nauni B. tau, Summer squash Saproon B. cucurbitae Cucumber Una Haroli B. cucurbitae Cucumber Mean Haryana Karnal Karnal B. cucurbitae Cucumber Karnataka Bengaluru Bengaluru B. cucurbitae, B. paraverbascifoliae, B. trilineata, B. correcta, B. - - dorsalis, 23 Delhi Delhi IARI B. cucurbitae Bottle gourd - 24 Bihar Patna Patna B. cucurbitae Cucumber Nalanda Bihar Sharif B. cucurbitae Bottle gourd Samstipur RAU Pusa B. cucurbitae, B. tau, B. dorsalis, B. zonata Uttar Pradesh Ghaziabad Ghaziabad B. cucurbitae Bottle gourd - 28 Maharashtra Solapur Solapur B. cucurbitae Cucumber - Nepal 29 Dhankuta B. cucurbitae, B. tau - - *Visual infestation rating 80.00

3 53 reported per cent fruit damage by fruit flies in cucumber var. Pusa Sanyog (susceptible to fruit flies). High fruit flies activity in Himachal Pradesh might have been facilitated by congenial climatic conditions like high rain fall ( mm annual rainfall) and humidity, with majority rains being received during active cucurbits growing season (May-Sept). This is also supported by faulty insect-pest control practices adopted by the farmers, as they are not using IPM (Integrated Pest Management) approach like field sanitation, MAT (Male Annihilation Technique) and BAT (Bait Application Technique) techniques as observed during the survey. On the other hand, most of the agricultural land have bushy hedges and is surrounded by forest and pastures consisting of many wild cucurbits which could facilitate the fruit flies to rest and pick the resources during and after insecticide application. High fruit flies infestation in cucurbits recorded during present study in the Himachal Pradesh is also in the accordance with Gupta et al. (1992) who had observed per cent fruit flies infestation on different cucurbits in Himachal Pradesh. Outside Himachal Pradesh, six Indian states namely Bihar, Delhi, Haryana, Karnataka, Maharashtra and Uttar Pradesh were surveyed for fruit flies. The infestations were, however recorded at three places outside Himachal Pradesh namely Karnal (77.77%) in Haryana and Patna (77.77%) & Bihar Sharif (70.00%) in Bihar. High fruit infestation in cucurbits due to fruit flies at Karnal (Haryana), might be due to the micro climatic conditions like irrigated farming system (canal irrigation) followed by warm climate during crop season supported by low to moderate rainfall (617 mm annual rainfall) facilitating the rapid fruit fly growth and development. Whereas, at Patna and Bihar Sharif (Bihar) which are located in the east of the Indo-Gangetic plain, the holy river Ganga flow round the year making local climate warm and humid with onset of monsoon (during cucurbits season), in the vicinity of Tropics of Cancer helped rapid expansion of fruit flies and consequently heavy fruit damage. Heavy losses observed in cucurbits by fruit flies in Indo-Gangetic plain are in line with the earlier reports of 30 to 100 per cent fruit infestation in different cucurbits by fruit flies (Dhillon et al. 2005).

4 54 Higher infestation rate (fruit damage) of fruit flies in Himachal Pradesh as well as in other states of India on the crops necessitates large scale adoption of integrated pest management programme with wide-area management programme as an essential component of IPM for fruit fly management with firm cohesion between farmers- government agriculture departments and educational & research institutions like SAUs (State Agricultural Universities) Fruit flies species associated with cucurbits in Himachal Pradesh In the infested cucurbit samples collected from different locations, two species viz. Bactrocera tau and B. cucurbitae were the predominant infesting all the cucurbits at majority locations surveyed. B. cucurbitae and B. tau were observed as the most damaging fruit fly species on different cucurbits at Ghumarwin (Bilaspur), Nadaun (Hamirpur), Jawalamukhi, Kangra, Palampur & Shahpur (Kangra) and Mandi, Nagwain & Sundernagar (Mandi) in Himachal Pradesh (Table 4.1). However, cucurbit samples collected from Chandpur (Bilaspur), Banikhet (Chamba), Naggar (Kullu) and Nauni (Solan) indicated infestation of B. tau only, whereas samples from Bhota (Hamirpur), Indora (Kangra), Saproon (Solan) and Haroli (Una) indicated infestation of B. cucurbitae only. B. scutellaris was the lone species reared from the infested samples of flowers and tender fruits collected from three locations during survey namely, Kangra & Palampur in district Kangra and Barot in district Mandi. During the course of survey, B. tau and B. cucurbitae were recorded as the predominant species infesting cucurbits in Himachal Pradesh. However, B. cucurbitae was earlier considered to be the major fruit fly species infesting cucurbits in the state, has now been observed confined mostly to the low hills of the State. The species has earlier been reported to be the major species infesting cucurbits (Kapoor et al. 1980; Gupta et al. 1992) in Himachal Pradesh. However, Sood and Nath (1999) and Prabhakar et al. (2009a) reported B. tau as a major

5 55 fruit fly species infesting cucurbits in the State. B. tau has also been reported from north-eastern region of India (Borah and Dutta 1996), China (Yang et al. 1994) and Bangladesh (Huque 2006). The reports pertaining to cucurbit infestation by B. tau from across the places indicate a wider geographical distribution of the species. During recent past, B. scutellaris has also been reported as one of the most destructive fruit fly species infesting tender fruits and growing vegetative parts from Himachal Pradesh, damaging not only the fruits but also retarding the plant vigour and growth (Prabhakar et al. 2007, Sunandita and Gupta 2007, Prabhakar et al. 2009a). The reports of different workers on infestation of cucurbits by fruit flies in the state substantially support the present findings, that cucurbits are not damaged by a single fruit fly species but by a complex of species viz. B. cucurbitae, B. tau and B. scutellaris. Cucurbit samples collected from different Indian states outside Himachal Pradesh showed infestation of B. cucurbitae only (Table 4.1). Earlier, Agarwal and Sueyoshi (2005) had reported that B. cucurbitae is widely distributed in India. Stonehouse et al. (2007) also reported activity of B. cucurbitae from five states of India viz. Gujarat, Karnataka, Kerala, Uttar Pradesh and Orissa. Whereas, Agarwal (1984; 1987) reared this species from infested cucurbit samples collected from Bihar, Punjab and Uttar Pradesh Diversity of fruit flies in pheromone traps Pheromone traps were installed at three locations viz. Nihari (Bilaspur), Palampur (Kangra) and Paragpur (Kangra) to assess the diversity of fruit flies associated with cucurbits. Five species of fruit flies from Nihari (Bilaspur) and ten species of fruit flies from Palampur (Kangra) were collected. Whereas, only two species were collected from traps installed at Paragpur (Kangra). Traps installed at two locations outside Himachal Pradesh at Bangaluru (Karnataka) and Samstipur (Bihar) revealed presence of five species at Bengaluru and four species at Samstipur (Table 4.1). Samples of fruit flies from Dhankuta (Nepal) sent by scientist working in Nepal Council of Agricultural Research revealed presence of two species (B. cucurbitae and B. tau) in the region.

6 56 The usefulness of male lures in population monitoring and taxonomic surveys have also been highlighted by White and Hancock (1997), Drew and Hancock (2000), White (2000) and Smith et al. (2003) Identification of fruit flies species Different species of fruit flies attacking cucurbits, damage the crop either individually or collectively, so an attempt was made to identify the associated species using morphological characteristics. The literature pertaining to identification has been given in section Morphological characteristics of fruit fly species identified Subfamily : DACINAE Tribe : DACINI I. Genus Bactrocera Macquart Subgenus Bactrocera Macquart 1. Bactrocera (Bactrocera) correcta (Bezzi) (Plate 4.1) Material examined: Karnataka: Bengaluru district: 3, Bengaluru, 5.i.2010, ME. Description: Face fulvous with transverse, elongate black spots almost meeting in centre. Scutum predominantly black with lateral yellow stripes. Scutellum yellow with a narrow black basal band and two scutellar setae. The wings are mostly clear with a narrow costal band confluent with R 2+3 ending at the apex of this vein and a small fuscous spot around apex of R 4+5. Abdomen oval, tergum I black, tergum II red-brown with a narrow transverse black band that does not reach lateral margins. Abdominal terga III-V red brown with a black T pattern, a pair of oval red-brown shining spots on tergum V. Male with a pectin on tergum III.

7 57 Adult Male Wing Scutum Scutellum & Abdomen Face Head Legs Spur Plate 4.1: Morphographs of Bactrocera correcta (Bezzi)

8 58 Note: It is similar to B. zonata but is distinguished by the facial spots being united or almost united, to form a black transverse band and scutum predominantly black. Attractant: Methyl eugenol Host: Not recorded Chaetodacus correctus: Bezzi, Bull. Entomol. Res., 7: 107. Syntype,. India (Bihar: Pusa; Tamil Nadu: Coimbatore, Chennai: Guindy, Hagari) (ZSI). Bactrocera zonata: Bezzi, Mem. Indian Mus., 3: 94. [Misidentification]. Strumeta paratuberculatus: Philip, Indian J. Entomol., 10(1): 31. Holotype. Myanmar (Aingyi) (ZSI). Dacus (Strumeta) correctus: Narayanan and Batra, Fruit Flies and Their Control, I.C.A.R., New Delhi, 33. Dacus (Strumeta) dutti: Kapoor, Oriental Insects, 5(4): 480. Holotype. India (Maharashtra: Pune) (NPC). Dacus (Bactrocera) bangaloriensis: Agarwal & Kapoor, J. Entomol. Res., 7(2): 169. Holotype. India (Karnataka: Bangalore) (NPC). Bactrocera (Bactrocera) correcta: Kapoor, Indian Fruit Flies. Oxford & IBH Publ., New Delhi: 73. Bactrocera (Bactrocera) correcta: Drew & Raghu, Raffles Bull. Zool., 50(2): 335. Bactrocera (Bactrocera) correcta: Agarwal & Sueyoshi, Oriental Insects, 39: Bactrocera (Bactrocera) dorsalis (Hendel) (Plate 4.2) Material examined: Himachal Pradesh: Kangra district: 3, 5, Palampur, 9.vii.2009, ex Litchi chinensis; 11, 7, Palampur, 13.vi.2009, ex Mangifera indica; 8,

9 59 Palampur, 13.vi.2009, ME; 9, 5, Palampur, 15.ix.2009, ex Psidium guajava; 9, Paragpur, 19.vii.2010; Karnataka: Bengaluru district: 4, Bengaluru, 29.xii.2009, ME; Bihar: Samastipur district: 9, RAU Pusa, 26.ii.2010, ME. Description: Face fulvous with a pair of medium-sized circular black spots. Scutum predominantly black but may have red-brown areas of varying sizes and shapes. Two broad lateral postsutural vittae, parallel sided and ending behind ia. setae, medial postsutural vittae absent. Scutellum entirely yellow coloured with a narrow basal black band and two apical setae. Wing with a distinct brown costal band ending just beyond the end of vein R 4+5, crossveins r-m and dm-cu not covered by any markings; narrow pale fuscous cubital streak present. Generally, the abdomen with two horizontal black stripes and a longitudinal median stripe extending from the base of the segment III to the apex of the abdomen. These markings may form a T-shaped pattern, but the pattern varies considerably. Legs mostly fulvous with fore tibiae pale fuscous and hind tibiae fuscous. Attractant: Methyl eugenol Host: ex fruit Mango, ex fruit Litchi, ex fruit Guava Musca ferruginea: Fabricius, Entomol. Syst., 4: 342. Preoccupied by Musca ferruginea Scopoli, Entomol. Carn., 340.? Type.? Sex. India Orientali (e. India) (?ZMUC). Dacus ferrugineus: Fabricius, Syst. Antliat., p Dacus dorsalis: Hendel, Suppl. Entomol., 1: 18. Lectotype. Taiwan (Koshun) (BMNH). Bactrocera ferruginea: Bezzi, Mem. Indian Mus., 3: 95. Chaetodacus ferrugineus dorsalis: Bezzi, Bull. Entomol. Res., 7: 104. Dacus (Strumeta) dorsalis: Hardy & Adachi, Bull. Bernice P. Bishop Mus., 14(1): 7.

10 60 Adult Male Wing Scutum Scutellum & Abdomen Face Head Legs Lateral view Plate 4.2: Morphographs of Bactrocera dorsalis (Hendel)

11 61 Dacus (Bactrocera) dorsalis: Hardy, Cat. Diptera Oriental Reg., 3: 49. Bactrocera (Bactrocera) dorsalis: White & Elson-Harris, Fruit Flies of Economic Significance, C.A.B. International Publ., p Bactrocera (Bactrocera) dorsalis: Drew & Raghu, Raffles Bull. Zool., 50(2): 336. Bactrocera (Bactrocera) dorsalis: Agarwal & Sueyoshi, Oriental Insects, 39: Bactrocera (Bactrocera) latifrons (Hendel) (Plate 4.3) Material examined: Himachal Pradesh: Kangra district: 1, 1, Palampur, 12.vii Description: Face with two dark spots. Scutum predominantly black with two lateral postsutural yellow vittae. Scutellum entirely pale yellow with two scutellar setae. Wing with a complete costal band expanded into an apical spot at apex, cubital streak present. Abdomen oval, orange-brown to fuscous, tergum III with a basal transverse dark band and sometimes with a medial stripe down terga III-V. Male with pecten on tergum III. Attractant: Not known Host: On bitter gourd Remarks: New record from Himachal Pradesh Dacus amoyensis: Froggatt, In Official report on fruit fly and other pests in various countries Report on parasitic and injurious insects. N.S.W., Dept. Agric., Sydney, p. 99.? Type. China (Fujian: Amoy) (UMO). (Nomen nudum, attributed to Bigot).

12 62 Adult Female Wing Scutum Scutellum & Abdomen Face Head Legs and Lateral view Plate 4.3: Morphographs of Bactrocera latifrons (Hendel)

13 63 Chaetodacus latifrons: Hendel, Annls. Hist. Nat. Mus. Natl. Hung., 13: 425. Lectotype. Taiwan (Tainan). [Precedent name over Dacus parvulus Hendel, Bull. Zool. Nomencl., 54(1): 68 (1997)] (BMNH). Dacus (Strumeta) latifrons: Hardy & Adachi, Pacif. Sci., 8(2) Dacus (Strumeta) parvulus: Hardy, Pac. Insects Monogr., 31: 49. Dacus (Bactrocera) latifrons: Hardy, Cat. Diptera Oriental Reg., 3: 50. Bactrocera (Bactrocera) latifrons: White & Elson-Harris, Fruit Flies of Economic Significance, C.A.B. International Publ., p Bactrocera (Bactrocera) parvula: Kapoor, Indian Fruit Flies. Oxford & IBH Publ. Co., New Delhi: 76. Bactrocera (Bactrocera) latifrons: Agarwal & Sueyoshi, Oriental Insects, 39: Bactrocera (Bactrocera) nigrofemoralis White & Tsuruta (Plate 4.4) Material examined: Himachal Pradesh: Kangra district: 9, Palampur, 13.vi.2009, CL. Description: Head dark red-brown to fuscous, fulvous laterally and face shining black. Scutum shining black without pale markings. Two narrow lateral yellow postsutural vittae, narrowing posteriorly to end well before infra alar setae. Scutellum yellow except for a moderately broad black basal band with two scutellar setae. Femora mostly shining black, mid femora entirely shining black, hind femora with basal 2/3 fulvous and apical 1/3 shining black, mid tibiae each with an apical black spur. Wings colourless except fuscous cell sc, narrow fuscous costal band confluent with R 2+3 and remaining narrow to end just beyond extremity of R 4+5. A narrow fuscous cubital streak. Abdomen oval, terga I and II black except for a narrow red-brown band along inter-segmental line and a broad transverse fulvous to red

14 64 Adult Male Wing Scutum Scutellum Abdomen Face Legs Lateral view Plate 4.4: Morphographs of Bactrocera nigrofemoralis White & Tsuruta

15 65 brown band across posterior 1/2 of tergum II and narrowing to lateral margins; terga III-V red-brown except for a broad black band across anterior margin of tergum III. A pair of oval black shining spots on tergum. Male with pecten on tergum III. Attractant: Cue lure Host: Not recorded Remarks: New record from Himachal Pradesh as well as north India Bactrocera (Bactrocera) nigrofemoralis: White & Tsuruta, Entomological Sci., 4: 79. Holotype. India (Karnataka: nr. Medikeri, Talakaveri, 1100m) (BMNH). Bactrocera (Bactrocera) nigrofemoralis: Drew & Raghu, Raffles Bull. Zool., 50(2): 339. Bactrocera (Bactrocera) nigrofemoralis: Agarwal & Sueyoshi, Oriental Insects, 39: Bactrocera (Bactrocera) paraverbascifoliae Drew & Raghu (Plate 4.5) Material examined: Karnataka: Bengaluru district: 3, Bengaluru, 30.xii.2009, ME. Description: Face fulvous with medium sized circular to oval black spots. Scutum black with red-brown to dark red-brown below and behind lateral postsutural vittae. Two parallel sided lateral postsutural vittae of medium width. Scutellum yellow except narrow black basal band with two scutellar setae. wings colourless except for dark fuscous cell sc with a narrow fuscous to dark fuscous costal band confluent with R 2+3 and gradually widening to end between extremities of R 4+5 & M and a narrow fuscous cubital streak. Legs fulvous except a small area of dark fuscous

16 66 Adult Male Wing Scutum Scutellum Abdomen Face Legs and Lateral view Plate 4.5: Morphographs of Bactrocera paraverbascifoliae Drew & Raghu

17 67 on base of fore tibiae, fuscous on base of mid tibiae and hind tibiae dark fuscous basally to fuscous apically. Mid tibiae each with an apical black spur. Abdomen oval, tergum I fuscous anterocentrally, tergum II red-brown except for a narrow transverse black anteriorly which ends laterally just before the dark fuscous lateral margins, terga III- V orange-brown except for a distinct black 'T' pattern. A pair of oval red- brown shining spots on tergum V. Male with pecten on tergum III. Attractant: Methyl eugenol Host: Not recorded Remarks: Not present in Himachal Pradesh Bactrocera (Bactrocera) paraverbascifoliae: Drew & Raghu, Raffles Bull. Zool., 50(2): 341. Holotype. India (Kerala: New Amarambalam Forest) (BMNH). Bactrocera (Bactrocera) paraverbascifoliae: Agarwal & Sueyoshi, Oriental Insects, 39: Bactrocera (Bactrocera) zonata (Saunders) (Plate 4.6) Material examined: Himachal Pradesh: Kangra district: 6 Palampur, 13.vi.2009, ME; 8, Pragpur, 19.vii.2010, ME; Bilaspur district: 4, Nihari, 15.v.2010, ME; Bihar: Samastipur district: 5, RAU Pusa, 26.ii.2010, ME. Description: Face fulvous with two medium sized oval black spots. Scutum red-brown with a pale fuscous pattern posteriorly with lateral yellow or orange postsutural stripes, scutellum entirely pale yellow coloured except for a narrow dark red-brown basal band and with two scutellar setae. Wing lacks a complete costal band (reduced to an isolated apical spot), cubital streak absent but with a very small pale

18 68 Adult Male Wing Scutum Scutellum Abdomen Face Head Lateral view with Legs Plate 4.6: Morphographs of Bactrocera zonata (Saunders)

19 69 fuscous tint in the cell cup. Legs fulvous with apices of femora red-brown and hind tibiae pale fuscous to fuscous, mid tibiae each with an apical black spur. On abdomen there is usually a pair of dark marks on tergum III and no medial dark line except on tergum V. Male abdomen with pecten. Attractant: Methyl eugenol Host: Not recorded Note: B. zonata is a general red-brown species having a wing pattern which is similar to that of B. correcta i.e. an incomplete costal band with a fuscous spot around apex of R 4+5. B. correcta is different in having a black scutum and black "T" pattern on abdominal terga III-V. Dasyneura zonatus: Saunders Trans. Entomol. Soc. London, 3: 61.?Type.?Sex. India (Bengal) (?UMO). Bactrocera maculigera: Doleschall, Natuurk. Tijdschr. Ned.-Indië, 17: 122.?Type. Indonesia (Moluccas: Ambon) (ZMHU). Rivellia persicae: Bigot, Indian Mus. Notes, 1:192. Syntype,. India (Jharkhand: Chota Nagpur: Ranchi) (?ZSI). Dacus ferrugineus var. mangiferae: Cotes, Indian Mus. Notes, 3(1):17. Lectotype. India (Bihar: Tirhut region) (?ZSI). Chaetodacus zonatus: Bezzi, Bull. Entomol. Res., 7: 105. Dacus (Strumeta) zonatus: Hardy, Pac. Insects Monogr., 31: 54. Dacus (Bactrocera) zonatus: Hardy, Cat. Diptera Oriental Reg., 3: 53. Bactrocera (Bactrocera) zonata: White & Elson-Harris, Fruit Flies of Economic Significance, C.A.B. International Publ., p Bactrocera (Bactrocera) zonata: Drew & Raghu, Raffles Bull. Zool., 50(2): 347. Bactrocera (Bactrocera) zonata: Agarwal & Sueyoshi, Oriental Insects, 39: 379.

20 70 Subgenus Hemigymnodacus Hardy 7. Bactrocera (Hemigymnodacus) diversa (Coquillett) (Plate 4.7) Material examined: Himachal Pradesh: Kangra district: 1, 2, Palampur (Sagur), 17.vi.2010, ME. Description: Face of male entirely pale yellow without black spots while face of female with a transverse black line above mouth. Scutum black with broad parallel sided lateral yellow vittae and a narrow medial postsutural vittae. Scutellum yellow with a narrow black basal band and 2 or 4 scutellar setae. Wing with a narrow dark costal band confluent with R 2+3 and widening across apex of wing, dark cubital streak present. Abdominal terga III-V red-brown with a black T pattern. Male without pectin. Attractant: Methyl eugenol Host: Not recorded Dacus diversus : Coquillett, Proc. Entomol. Soc. Wash., 6: 139. Syntype,. Sri Lanka (Colombo), India (Karnataka: Bangalore) (USNM). Bactrocera diversa: Bezzi, Mem. Indian Mus., 3: 94. Chaetodacus diversus: Bezzi, Bull. Entomol. Res., 7: 108. Dacus quadrifidus: Hendel, Entomol. Mitt., 17(5): 343. Holotype. Sri Lanka (DEI). Dacus (Gymnodacus) diversus: Hardy, Proc. Entomol. Soc. Wash., 56(1): 18. Dacus (Melanodacus) citronellae: Kapoor & Katiyar, Bull. Entomol., 10(2): 123. Holotype. India (Bihar: Pusa) (NPC). Dacus (Hemigymnodacus) diversus: Hardy, Pac. Insects Monogr., 31: 19.

21 71 Adult Male Wing Scutum Scutellum Abdomen (Female) Face Head Lateral view with Legs Plate 4.7: Morphographs of Bactrocera diversa (Coquillett)

22 72 Bactrocera (Hemigymnodacus) diversa: White & Elson-Harris, Fruit Flies of Economic Significance, C.A.B. International Publ., p Bactrocera (Hemigymnodacus) diversa: Agarwal & Sueyoshi, Oriental Insects, 39: 381. Subgenus Javadacus Hardy 8. Bactrocera (Javadacus) trilineata (Hardy) (Plate 4.8) Material examined: Karnataka: Bengaluru district: 3, Bengaluru, 2.i.2010, ME. Description: Face entirely fulvous without black marking. Scutum black with broad parallel sided lateral yellow vittae and a narrow medial postsutural vittae, anterior supra alar seate absent. Scutellum yellow with a narrow black basal band and 2 scutellar setae. Wings with complete costal band not overlapping R 4+5 and expanding slightly at wing apex. Legs fulvous with apices of femora red-brown. Abdominal terga I and II black except for a narrow red-brown band along intersegmental line and a broad transverse fulvous to red brown band across posterior 1/2 of tergum II and narrowing to lateral margins. Terga III-IV entirely black except fulvous at posterosubmedially part of terga IV, terga V entirely fuscous to light brown. Attractant: Methyl eugenol Host: Not recorded Dacus (Afrodacus) trilineatus: Hardy, J. Kans. Entomol. Soc., 28(1): 12. Holotype. India (Karnataka: Bangalore: Sarakki village) (BMNH).

23 73 Adult Male Wing Scutum Scutellum Abdomen Head Legs Lateral view Plate 4.8: Morphographs of Bactrocera trilineata (Hardy)

24 74 Bactrocera (Afrodacus) trilineata: Kapoor, Indian Fruit Flies. Oxford & IBH Publ. Co., New Delhi: 73. Bactrocera (Javadacus) trilineata: White & Elson-Harris, Fruit Flies of Economic Significance, C.A.B. International Publ., p Bactrocera (Javadacus) diversa: Agarwal & Sueyoshi, Oriental Insects, 39: 382. Subgenus Zeugodacus Hendel 9. Bactrocera (Zeugodacus) cucurbitae (Coquillett) (Plate 4.9) Material examined: Himachal Pradesh: Bilaspur district: 2, 1, Ghumarwin, 7.viii.2009, ex Cucumis sativus; 8, Nihari, 15.v.2010, CL; Hamirpur district : 2, 2, Bhota, 29.v.2009, ex Cucumis sativus; 1, 2, Nadaun, 25.v.2009, ex Momordica charantia; Kangra district: 4, 2, Indora, 9.viii.2009, ex Lagenaria siceraria, ex Momordica charantia; 2, 2, Jawalamukhi, 19.viii.209, ex Cucumis sativus; 5, 1, Kangra, 26.vi.2009, ex Cucumis sativus; 4, Palampur, 26.vi.2009, CL; 5, 3, Shahpur, 3.ix.2009, ex Momordica charantia; Mandi district: 2, 1, Mandi, 2.vi.2009, ex Cucumis sativus; 2, 2, Nagwain, 10.viii.2009, ex Momordica charantia; 2, 1, Sundernagar, 2.vi.2009, ex Cucumis sativus; Solan district: 3, 2, Saproon, 3.vii.2009, ex Cucumis sativus; Una district: 1, 2, Haroli, 2.ix.2009, ex Cucumis sativus; Haryana: Karnal district: 4, 2, Karnal, 16.x.2009, ex Cucumis sativus; Karnataka: Bengaluru district: Bengaluru, 27.xii.2009, CL; Delhi: Delhi: IARI, 19.vii.2009, ex Lagenaria siceraria; Bihar: Patna district: 4, 5, Patna, 15.vii.2009, ex Cucumis sativus; Nalanda district: 3, 2, Bihar Sharif, 15.vii.2009, ex Lagenaria siceraria; Samstipur district: 6, RAU Pusa, 25.ii.2010, CL; Uttar Pradesh: Ghaziabad district: 4, 2, Ghaziabad, 19.vii.2009, ex Lagenaria siceraria; Maharashtra: Solapur district: 5, 2, Solapur, 9.vii.2009, ex Cucumis sativus; Nepal: 3, Dhankuta, 5.vii.2010, CL.

25 75 Adult Female Wing Scutum Scutellum Abdomen Face Head Legs and Lateral view Plate 4.9: Morphographs of Bactrocera cucurbitae (Coquillett)

26 76 Description: Face fulvous with a pair of medium sized elongate oval black spots. Scutum entirely red-brown or fuscous to dark fuscous markings with both lateral and medial yellow stripes. Scutellum yellow with a narrow dark fuscous basal band, one pair scutellar setae and rarely two pairs. Wing with complete costal band, which is expanded into a spot at apex, crossvein dm-cu and r-m covered by infuscate area, broad fuscous cubital streak present. Legs with femora fulvous except for apical dark patterns which are red-brown on fore femora and fuscous to dark fuscous on mid and hind femora, fore tibiae fuscous, mid tibiae fulvous with fuscous basally, hind tibiae dark fuscous, all tarsi fulvous. On abdomen transverse band across tergum III, medial longitudinal stripe on terga III-V. Male with a row of setae (the pecten) on tergum III. Attractant: Cue lure Host: Cucurbits Dacus cucurbitae: Coquillett, Entomol. News, 10: 129. Lectotype. Hawaii (Honolulu) (USNM). Dasyneura caudata: Walker, List Dipt. Ins. Coll. Brit. Mus., 4: (Misidentification). Bactrocera cucurbitae: Bezzi, Mem. Indian Mus., 3: 96. Chaetodacus cucurbitae: Bezzi, Bull. Entomol. Res., 7: 109. Dacus (Strumeta) cucurbitae: Swezey, Bull. B. P. Bishop Mus., 10: 199. Dacus (Zeugodacus) cucurbitae: Drew, Queensl. Dep. Indus., Div. Plant Indus. Bull., 652, p. 23. Zeugodacus cucurbitae: Munro, Entomol. Mem. S. Afr. Dep. Agr., 61: 18. Bactrocera (Zeugodacus) cucurbitae: Drew, Mem. Queensl. Mus., 26: 212. Bactrocera (Zeugodacus) cucurbitae: Drew & Raghu, Raffles Bull. Zool., 50(2): Bactrocera (Zeugodacus) cucurbitae: Agarwal & Sueyoshi, Oriental Insects, 39: 385.

27 Bactrocera (Zeugodacus) scutellaris (Bezzi) (Plate 4.10) Material examined: Himachal Pradesh: Bilaspur district: 5, Nihari, 15.v.2010, CL; Kangra district: 4, 2, Kangra, 26.vi.2009, ex Cucumis sativus; 8, Palampur 13.vi.2009, CL; Mandi district: 2, 2, Barot, 12.viii.2009, ex Cucurbita maxima. Description: Face fulvous with a pair of transverse oval black spots pointed towards centre. Scutum shining black with narrow lateral and median postsutural yellow vittae. Scutellum yellow with a distinct black apical spot and two pairs of scutellar setae. Wings with a narrow dark fuscous complete costal band which is extremely narrow beyond apex of vein R 2+3 and expanding into a distinct apical spot around apex of R 4+5, cubital streak dark and broad. Abdominal terga III-V mostly dark fuscous to black. Male with pecten on tergum III. Attractant: Cue lure Host: Cucurbits Dacus ornatypes: Froggatt, In Official report on fruit fly and other pests in various countries Report on parasitic and injurious insects. N.S.W., Dept. Agric., Sydney, p. 99.?Type. India (UMO). (Nomen nudum, attributed to Bigot). Bactrocera scutellaris: Bezzi, Mem. Indian Mus., 3: 98. Syntype,. India (Meghalaya: Shillong; W. Bengal: Siliguri, Kurseong; Uttaranchal: Kumaon: Bhowali) (ZSI). Chaetodacus scutellaris: Bezzi, Bull. Entomol. Res., 7: 113. Dacus (Paradacus) pusaensis: Kapoor & Katiyar, The Entomologist, 103: 252. Holotype. India (Bihar: Pusa) (NPC).

28 78 Adult Male Wing Scutum Scutellum Abdomen Face Head Legs and Lateral view Plate 4.10: Morphographs of Bactrocera scutellaris (Coquillett)

29 79 Dacus (Zeugodacus) scutellaris: Hardy, Pac. Insects Monogr., 31: 68. Bactrocera (Zeugodacus) scutellaris: White and Elson-Harris, Fruit Flies of Economic Significance, C.A.B. International Publ., p Bactrocera (Paradacus) pusaensis: Kapoor, Indian Fruit Flies. Oxford & IBH Publ. Co., New Delhi: 79. Bactrocera (Zeugodacus) scutellaris: Agarwal & Sueyoshi, Oriental Insects, 39: 386. Bactrocera (Zeugodacus) scutellaris: Prabhakar et. al., Pest Manage. Econ. Zool, 15(2): Bactrocera (Zeugodacus) tau (Walker) (Plate 4.11) Material examined: Himachal Pradesh: Bilaspur district: 2, 4, Chandpur, 29.viii.2009, ex Cucumis sativus; 3, 3, Ghumarwin, 7.viii.2009, ex Cucumis sativus; 5, Nihari, 15.v.2010, CL; Chamba district: 2, 3, Banikhet, 5.viii.2009, ex Cucumis sativus; 1, 2, Hamirpur district: 1, 2, Nadaun, 25.v.2009, ex Lagenaria siceraria, ex Momordica charantia, ex Cucumis sativus; Kangra district: 4, 5, Jawalamukhi, 19.viii.209, ex Cucumis sativus, ex Momordica charantia; 3,2, Kangra, 26.vi.2009, ex Cucumis sativus; 5, 9, Palampur, 26.vi.2009, ex Cucumis sativus; 12.viii.2009, ex Momordica charantia; 2, 2, Shahpur, 3.ix.2009, ex Momordica charantia; Kullu district: 1, 1, Naggar, 22.viii.2009, ex Cucumis sativus; Mandi: district: 2, 2, Barot, 12.viii.2009, ex Cucurbita maxima; 2, 2, Nagwain, 10.viii.2009, ex Momordica charantia; 2, 3, Sundernagar, 2.vi.2009, ex Cucumis sativus; Solan district: 1, 1, Nauni, 2.vii.2009, ex Cucurbita pepo; Bihar: Samastipur: 5, RAU Pusa, 26.ii.2010, CL; Nepal: 4, Dhankuta, 5.vii.2010, CL.

30 80 Adult Male Wing Scutum Scutellum Abdomen Face Head Legs and Lateral view Plate 4.11: Morphographs of Bactrocera tau (Walker)

31 81 Description: Face fulvous with medium sized black oval spots. Scutum black with large redbrown areas located centrally and anterocentrally, lateral and median postsutural yellow vittae present. Scutellum entirely yellow with four setae. Wings with a narrow dark fuscous complete costal band overlapping vein R 2+3 and expanding into a distinct apical spot at wing apex, cubital streak dark and broad. Abdominal terga III-V fulvous with a black T pattern. Male with pecten on tergum III. Attractant: Cue lure Host: Cucurbits Dasyneura tau: Walker, List Dipt. Ins. Coll. Brit. Mus., 4: Syntype. China (Fujian: Foochow) (BMNH). Dacus pictus: Froggatt, In Official report on fruit fly and other pests in various countries Report on parasitic and injurious insects. N.S.W., Dept. Agric., Sydney, p. 99.?Type. Sri Lanka (UMO). (Nomen nudum, attributed to Bigot). Bactrocera caudata: Bezzi, Mem. Indian Mus., 3: 97. India, Nepal (Kathmandu), Myanmar. (Misidentification). Chaetodacus hageni: Bezzi, Bull. Entomol. Res., 7: 109. Zeugodacus bezzianus: Hering, Arb. Morphol. Taxon. Entomol., 8(1): 26. Holotype. China (Sichuan: Mou-Pin) (BMNH). Dacus (Zeugodacus) tau: Hardy, Bull. Brit. Mus. (Nat. Hist.) Entomol., 8(5): 233. Bactrocera (Zeugodacus) tau: White and Elson-Harris, Fruit Flies of Economic Significance, C.A.B. International Publ., p Bactrocera (Zeugodacus) tau: Agarwal & Sueyoshi, Oriental Insects, 39: Bactrocera (Zeugodacus) tau: Prabhakar et. al., J. Insect Sci., 22(3):

32 82 II Genus Dacus Fabricius Subgenus Callantra Walker 12. Dacus (Callantra) longicornis Wiedemann (Plate 4.12) Material examined: Himachal Pradesh: Kangra district: 7, Palampur, 2.vi.2009, CL. Description: Face fulvous with a pair of oval black spots. Scutum dark red-brown without distinct black patterns, lateral and medial postsutural vittae absent. Scutellum yellow with broad red-brown basal band and two scutellar setae. Wing with cells bc and c fuscous, a broad dark fuscous complete costal band overlapping vein R 4+5 for its entire length. Legs with fore femora dark red-brown to fuscous, mid femora dark red-brown to fuscous except basal ¼ fulvous, hind femora dark fuscous, fore and mid tibiae dark red brown to fuscous, hind tibiae dark fuscous. Abdomen elongate-oval, strongly petiolate, abdominal terga III-V dark fuscous to black, large orange brown spots posterocentrally on terga IV and V with oval shining spots on tergum V. Male with pecten on tergum III. Attractant: Cue lure Host: Not recorded Remarks: New record from Himachal Pradesh as well as north western Himalayas Dacus longicornis: Wiedemann, Aussereuropäische Zweiflügelige Insekten., 2: 524. Lectotype. Indonesia (Java) (ZMUC). Callantra smieroides: Walker, J. Proc. Linn. Soc. London, Zool., 4: 154. Lectotype. Indonesia (Sulawesi: Makassar) (BMNH).

33 83 Adult Male Wing Scutum Scutellum and Abdomen Last abdominal segment Face Head Legs and Lateral view Plate 4.12: Morphographs of Dacus longicornis Wiedemann

34 84 Callantra smicroides: Bezzi, Mem. Indian Mus., 3: 84. (Emend. C. smieroides Walker). Mellesis destillatoria: Bezzi, Bull. Entomol. Res., 7: 118. Holotype. Myanmar (Kachin: Bhamo) (MCSNM). Mellesis bioculata: Bezzi, Philipp. J. Sci., 15(5): 437. Lectotype. Philippines (Luzon: Mt. Makiling) (BAKER, presently in MCSNM). Dacus (Callantra) smieroides: Malloch, Proc. Linn. Soc. N. S. W., 64: 411. Callantra eumenoides: Perkins, Proc. R. Soc. Queensl., 48(9): 54. Callantra eumenoides: Hardy, Pac. Insects Monogr., 31: 11. Callantra longicornis: Hardy, Cat. Diptera Oriental Reg., 3: 45. Dacus (Callantra) variegatus: Liang et al., J. Aust. Entomol. Soc., 32: 139. Dacus (Callantra) eumenoides: Kapoor, Indian Fruit Flies. Oxford & IBH Publ., New Delhi: 83. Dacus (Callantra) longicornis: Drew et al., Inverterbr. Taxon., 12: 604. Dacus (Callantra) longicornis: Agarwal & Sueyoshi, Oriental Insects, 39: Dacus (Callantra) sphaeroidalis (Bezzi) (Plate 4.13) Material examined: Himachal Pradesh: Kangra district: 2, 1, Palampur, 17.vi.2010, 2.vii.2010, CL. Description: Face red brown with two large elongated black spots. Scutum red brown without dark markings. Scutellum yellow with a narrow black basal band and 2 scutellar setae. Wings with a pale fuscous tint across membrane except for fuscous cell

35 85 Adult Male Wing Scutum Scutellum Abdomen Face Head Legs and Lateral view Plate 4.13: Morphographs of Dacus sphaeroidalis (Bezzi)

36 86 sc, broad fuscous costal band confluent with R 4+5 and expanding apically in to a large round dark spot which overlaps M; pale fuscous anal streak present. Legs with fore and mid femora red-brown, hind femora fulvous except red-brown around apical 1/3, fore and mid tibiae red-brown, hind tibiae red-brown basally. Abdomen elongate-oval, petiolate but not as strongly as in D. longicornis. Abdominal terga III-V red-brown except for a narrow transverse black band across anterior margin of tergum III, a pair of oval fuscous to dark fuscous shining spots on tergum V. Male with pecten on tergum III. Attractant: Cue lure Host: Not recorded Mellesis sphaeroidalis: Bezzi, Bull. Entomol. Res., 7: 115. Holotype. India (Uttaranchal: Dehra Dun) (BMNH). Callantra sphaeroidalis: Hardy, Pac. Insects Monogr., 31: 11. Callantra discophora: Agarwal, Biol. Bull. India, 9(2): 135. [Misidentification]. Dacus (Callantra) sphaeroidalis: Liang et al., J. Aust. Entomol. Soc., 32: 139. Dacus (Callantra) sphaeroidalis: Agarwal & Sueyoshi, Oriental Insects, 39: Dacus (Callantra) sp. (Plate 4.14) Material examined: Himachal Pradesh: Kangra district: 1, Palampur, 2.vii.2010, CL. Description: Face pale brown with a transverse black line above mouth. Scutum red brown with a narrow black line at centre. Scutellum yellow without a narrow black basal

37 87 Adult Male Wing Scutum Scutellum Abdomen Face Head Legs and Lateral view Plate 4.14: Morphographs of Dacus (Callantra) sp.

38 88 band and two scutellar setae. Wings with a pale fuscous tint across membrane except for fuscous cell sc, broad fuscous costal band confluent with R 4+5 and expanding apically into a large round dark spot which overlaps M, pale fuscous anal streak present. Legs with fore femora red-brown, mid and hind femora fulvous except red-brown around apical 1/3; fore, mid and hind tibiae red-brown. Abdomen elongate-oval and petiolate. Abdominal terga I-II pale brown with black or dark brown at middle, terga III-V red-brown except for a narrow transverse black band across anterior margin of tergum III. Attractant: Cue lure Host: Not recorded Remarks: New record from Himachal Pradesh Tribe: GASTROZONINI III Genus Cyrtostola Hancock & Drew 15. Cyrtostola limbata (Hendel) (Plate 4.15) Material examined: Himachal Pradesh: Kangra district: 1, Palampur, 10.viii Description: Scutum with four longitudinal black vittae placed submedially and sublaterally. Scutellum with a large apical black spot with four scutellar setae. Wing with fulvous basal area, including most of cell sc (which is brown basally) and narrow transverse and marginal brown bands, across r-m crossvein reaching costa well distal of cell sc, that across dm-cu crossvein converging with and weakly joined to the subapical band over apex of vein M. Abdomen with transverse black bands anteriorly on terga III-V.

39 89 Adult Female Wing Scutum & Scutellum Abdomen Face Head Legs and Lateral view Plate 4.15: Morphographs of Cyrtostola limbata (Hendel)

40 90 Attractant: Unknown Host: Not recorded Remarks: New record from Himachal Pradesh Taeniostola limbata Hendel, 1915, Ann. Hist. Nat. Mus. Natl. Hung., 13: 435. Holotype. Taiwan (Taihorinsho) (DEI). Cyrtostola limbata: Hancock & Drew, J. Nat. Hist., 33(5): 699. Cyrtostola limbata: Agarwal & Sueyoshi, Oriental Insects, 39: 394. Subfamily TEPHRITINAE Tribe PLIOMELAENINI IV Genus Pliomelaena Bezzi 16 Pliomelaena udhampurensis Agarwal & Kapoor (Plate 4.16) Material examined: Himachal Pradesh: Kangra district: 1, Palampur, 18.viii Description: Small sized, blackish-brown species. Head with width more than height. Thorax black with yellow dust and small yellow pubescence. scutellum wider than long, rounded at apical end, black, with yellow dust, lateral margins of scutellum yellow. Four scutellar bristles, all equal in size. Wings hyaline at base, marking dark brown, costal spine two, both cross veins straight, wing marking light in axillary lobe. Legs yellow, fore tibiae with long yellowish-white bristles. Abdomen black-brown with few small yellow pubescence on abdomen. Attractant: Unknown Host: On cucurbits

41 91 Adult Male Wing Scutum Scutellum Abdomen Face Legs Lateral view Plate 4.16: Morphographs of Pliomelaena udhampurensis Agarwal & Kapoor

42 92 Remarks: New record from Himachal Pradesh Pliomelaena udhampurensis: Agarwal & Kapoor, J. Entomol. Res., 12(2): 119. Holotype. India (Jammu and Kashmir: Udhampur) (NPC). Pliomelaena udhampurensis: Agarwal & Sueyoshi, Oriental Insects, 39: 418. Tribe TEPHRITINI V Genus Dioxyna Frey 17. Dioxyna sororcula (Wiedemann) (Plate 4.17) Material examined: Himachal Pradesh: Kangra district: 1, Palampur, 6.vii Description: A small species with head longer than height. Body black in ground colour and covered with dense yellow-grey dust and scale-like setae. Two pairs of frontal and orbital setae; proboscis slender, elongate and geniculate. Two scutellar setae. Wing markings as 3 large hyaline spots filling cell r 1, 4-5 hyaline round spots in cell bm. Attractant: Unknown Host: On cucurbits Trypeta sororcula: Wiedemann, Aussereuropäische Zweiflügelige Insekten., 2: 509.?Type. Canary Is. (Teneriffe) (NMW, Holotype probably lost). Leptomyza variipennis: Wulp, Természetr. Füz., 20: 143. Holotype. Sri Lanka (Kandy) (?TMB). Ensina bisetosa: Enderlein, Zool. Jahrb. Abt. Syst. Geogr. Biol. Tiere, 31: 455. Lectotype. Taiwan (Takao) (PAN).

43 93 Adult Male Wing Scutum Head Face Legs & Lateral view Plate 4.17: Morphographs of Dioxyna sororcula (Wiedemann)

44 94 Oxyna sororcula: Bezzi, Mem. Indian Mus., 3: 159. Ensina sororcula: Hendel, Ann. Hist. Nat. Mus. Natl. Hung., 13: 465. Paroxyna sororcula f. madeirensis: Lindner, Konowia, 7: 30. Syntype. Madeira (nr. Funchal) (SMN). Paroxyna seguyi: Zia, Sinensia, 10(1-6): 12. Lectotype. China (Guangxi: Yangso [Yangshuo]) (IZAS). Dioxyna sororcula: Frey, Commentat. Biol. Soc. Sci. Fenn. (1944), 8(10): 62. Stylia sororcula: Hardy & Adachi, Bull. Bernice P. Bishop. Mus., 14(1): 21. Dioxyna sororcula: Hardy & Drew, Inverterbr. Taxon., 10: 241. Dioxyna sororcula: Agarwal & Sueyoshi, Oriental Insects, 39: Simple keys to known species of Bactrocera and Dacus of Himachal Pradesh 1. Abdomen oval or elongate (Genus Bactrocera) - Abdomen petiolate and elongate (Genus Dacus) 2 (1). Lateral and medial postsutural yellow vittae present Lateral postsutural yellow vittae present, medial postsutural yellow vittae absent (2). Scutum black Scutum mostly red brown (3). Scutellum yellow without an apical black spot Bactrocera (Hemigymnodacus) diversa (Coquillett) - Scutellum yellow with an apical black spot Bactrocera (Zeugodacus) scutellaris (Bezzi)

45 95 5 (3). Wings with cubital streak and costal band with a distinct large spot in wing apex... Bactrocera (Zeugodacus) tau (Walker) - Wings with infuscation on dm-cu crossveins in addition to cubital streak and costal band with a distinct large spot in wing apex Bactrocera (Zeugodacus) cucurbitae (Coquillett) 6 (2). Scutum base colour red brown Scutum base colour black (6). Wings with costal band but either discontinuous or with an extremely narrow section distal to apex R 2+3 before expanding into a spot in wing apex... Bactrocera (Bactrocera) zonata (Saunders) 8(6) Wings with costal band but either discontinuous or with an extremely narrow section distal to apex R 2+3 before expanding into a spot in wing apex... Bactrocera (Bactrocera) correcta (Bezzi)* - Wings with continuous costal band confluent with R (8). All femora with dark black marking Bactrocera (Bactrocera) nigrofemoralis White & Tsuruta - All femora entirely fulvous (9) Costal band confluent with R 2+3 not expanding into a distinct spot in wing apex... Bactrocera (Bactrocera) dorsalis (Hendel) - Costal band confluent with R 2+3 expanding into a small spot in wing apex... Bactrocera (Bactrocera) latifrons (Hendel) 11 (1). Scutum red brown with postsutural medial yellow vittae, lateral postsutural yellow vittae absent..... Dacus (Callantra) discophorus (Hering)* - Scutum red brown without postsutural lateral and medial yellow vittae... 12

46 96 12(11). Costal band narrow, confluent with vein R 2+3 except at apex... Dacus (Didacus) ciliatus Loew* - Costal band broad, usually confluent or overlapping vein R (12). Costal band broad, usually confluent or overlapping vein R 4+5 not expanding into a large spot in wing apex.... Dacus (Callantra) longicornis Wiedemann - Costal band broad, usually confluent or overlapping vein R 4+5 expanding into a large spot in wing apex, reaching and crossing vein M... Dacus (Callantra) sphaeroidalis (Bezzi) * Not recorded in the present study from Himachal Pradesh 4.3 Molecular characterization of fruit fly species prevalent in India Homoplasmy in morphology, economic importance, adaptation to varied climatic conditions, a wide host range and little work on the genetic relationship among the members of tephritid fruit flies make them an excellent candidate for the study of species diversity and evolutionary processes. Among different DNA markers, mitochondrial cytochrome oxidase I (mtcoi) gene is reasonably well conserved and evolving approximately 10 times faster than single-copy nuclear DNA (Brown et al Brown 1985). Nevertheless, mtcoi sequences are at the base of the barcoding identification system (Hebert et al. 2003; Hajibabaei et al. 2006) that, besides being a valuable tool for species identification and discovery, has been proposed as a powerful methodology in biosecurity and invasive species identification (Armstrong and Ball 2005). Therefore in the present study, mitochondrial cytochrome oxidase (mtcoi) gene sequences were exploited for characterization of fruit fly and their isolates collected from different regions of India.

47 Mitochondrial cytochrome oxidase (mtcoi) gene analysis of Bactrocera cucurbitae PCR analyses of mtcoi region of 33 B. cucurbitae individuals collected from 20 locations with universal primer pair (UEA7 and UEA10) amplified a ~650 bp amplicons, characteristics of this region (Plate 4.18). Sequencing of PCR product using custom services revealed that various sequences of test isolates consisted of 611 bp in all isolates Sequences submission and blast analysis The obtained sequences were first blast search against the B. cucurbitae sequences available online in the NCBI GenBank using BLASTN programme and confirm their identity as B. cucurbitae. Comparative analysis and sequences of the test and reference isolates from GenBank revealed that identity of all isolates was in confirmation with that of the morphological characteristics, thus establishing their taxonomic status. All the sequences were submitted to GenBank database (NCBI) vide accession numbers HQ to HQ Multiple alignments of test isolates of B. cucurbitae All the sequences of 33 isolates were compared by multiple alignments using ClustalW programme in Molecular Evolutionary Genetics Analysis (MEGA) software version 4.1. Multiple alignments of 33 sequences of B. cucurbitae revealed 15 variable positions in various sequences and 4 indels. Variable sites were characterized by 11 singletons and 4 parsimonious informative, giving an overall 2.45 per cent sequence variation of total length (611 bp) Pair-wise genetic distance between B. cucurbitae isolates The data pertaining to genetic distance between sequences (below diagonal) and standard error (above diagonal) is presented in Table 4.2. The pair wise genetic distance between the isolates ranged from to 0.009, thereby indicating very low genetic variation between the B. cucurbitae isolates used in

48 98 Table 4.2: Pair wise genetic distance based on mtcoi gene sequences of Bactrocera cucurbitae using the K2P method in MEGA4.1. Sr. No. B. cucurbitae isolates Pair wise genetic distance B. cucurbitae isolates HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ B. tau GU L. migratoria All results are based on the pairwise analysis of 35 sequences. Below diagonal and above diagonal values are number of base substitution per site and standard error estimate(s) respectively, and were obtained by a bootstrap procedure (500 replicates).

49 99 Plate 4.18: mtcoi gene PCR product of Bactrocera cucurbitae isolates amplified by using gene specific markers.(lane 1 and 35, DNA ladder 100bp; lane 2 to 34 showing mtcoi gene amplification of Bactrocera cucurbitae isolate P101, P102, P103, P104, P106, P106A, P106B, P106C, P107, P108, P109, P110, P111, P111A, P111B, P111C, P111D, P112, P113, P114, P115, P117, P119A, P119B, P119C, P119D, P119E, P119F, P120, P121, P122, P123 and P123A)

50 100 this study. Whereas, genetic distance with outgroup members ranged from to (B. cucurbitae/ B. tau) and to (B. cucurbitae/ L. migratoria) base substitution per site. Hu et al. (2008) reported (base substitution per site) pairwise genetic distance between different B. cucurbitae isolates collected from China and south east Asia. The observed variations in pair wise genetic distance among B. cucurbitae suggested that B. cucurbitae populations present in Indian subcontinent have little more variation than those present in China and south east Asia Estimation of population genetic structure of B. cucurbitae from Indian subcontinent For population genetic structure determination, haplotype numbers, haplotypes distribution, haplotype frequency, polymorphic sites and nucleotide diversity were assessed using ARLEQUIN 3.1. To estimate population genetic structure of B. cucurbitae in Indian subcontinent mtcoi gene sequences of all the 33 isolates were divided into 5 groups on the basis geographical origin (Table 4.3). The first group contained B. Table 4.3: Population groups of B. cucurbitae isolates based on their geographical origin Sr. No. B. cucurbitae population groups Geographical origin of isolates Number of individuals genotyped (n) Sequences accession number 1 North west India Himachal Pradesh 11 HQ378195, HQ378196, HQ378197, HQ378198, HQ378206, HQ378212, HQ378213, HQ378214, HQ378215, HQ378216, HQ East India Bihar 6 HQ378199, HQ378200, HQ378201, HQ378202, HQ378203, HQ South India Maharashtra and Karnataka 6 HQ378207, HQ378208, HQ378209, HQ378210, HQ378211, HQ North India Delhi, Haryana, Uttar Pradesh 8 HQ378204, HQ378205, HQ378217, HQ378218, HQ378219, HQ378220, HQ378221, HQ Nepal Nepal 2 HQ378226, HQ378227

51 101 cucurbitae isolates of Himachal Pradesh named as North West India, the second group contained isolates from Bihar and referred as East India, the third group constituted of isolates from Maharashtra and Karnataka designated as South India, the fourth group named North India had isolates from Haryana, Delhi and Uttar Pradesh and fifth group contained isolates from Nepal kept in Nepal population. Among 11 isolates of B. cucurbitae from North West India population, sequence analysis exhibited 7 variable positions characterized by 3 substitutions (2 transitions and 1 transversion) and 4 indels (Table 4.4). In East India population of B. cucurbitae, no variable site was observed whereas, 16 variable sites (4 transitions, 2 transversions, 6 substitutions and 10 indels) were observed in South India population of B. cucurbitae. However, in North India population, only 5 variable sites with 4 transitions and 1 transversion (5 substitutions) with no indels were observed. B. cucurbitae population from Nepal contained only two sequences with 1 transition, 1 substitution and 1 indels. Within and between the population groups, haplotypes diversity was determined on the basis of sequence variations. Table 4.4: Molecular diversity indices of B. cucurbitae Statistics North-West India East India South India North India Nepal Sample Size No. of transitions No. of transversions No. of substitutions No. of indels No. of transitions sites No. of transversions sites No. of substitutions sites No. of indel sites

52 102 Overall 14 sequence variants (haplotypes) were identified in the five B. cucurbitae populations (Table 4.5) and most of the haplotypes differ by two or three mutations only with the exception of Bengaluru isolate of South India population that showed 12 mutations (Fig 4.1 and Plate 4.19). Haplotype H1 is the predominant haplotype among different groups except South India with an overall frequency of 0.52 (52% of the individuals of B. cucurbitae belong to haplotype H1 ) followed by haplotype H6 having overall frequency of 0.09 (9%) and shared two groups namely South India and North India. Other haplotypes are localized in their geographical group (Fig 4.2). Literature pertaining to B. cucurbitae population genetic structure is scanty and during literature searches only one published study was encountered. Recently, Hu et al. (2008) published population genetic structure of B. cucurbitae from China and south-east Asia using 64 individuals from eight geographically distinct populations excluding Indian population. They reported 8 haplotypes (12.50%) from 64 individuals. Whereas, 14 haplotypes (42.42%) were observed in 33 individuals distributed over populations in the present study. This suggests that the haplotypes diversity is more among B. cucurbitae population present in Indian subcontinent than that of China and south-east Asia. The present findings are also supported by the results of Virgilio et al. (2010), who studied phylogeography and genetic structure of 25 populations of B. cucurbitae collected from different countries by using 13 microsatellite loci and suggested that B. cucurbitae populations are more diverse in central Asia (India, Pakistan and Bangladesh) than any other populations of the world. They also suggested that the central Asia is the most possible centre of origin of B. cucurbitae. The present finding on haplotype diversity in B. cucurbitae population in Indian subcontinent also propounds the possible origin of B. cucurbitae from central Asia and more precisely in India. However, sole predominance of H1 haplotypes prevalent in Indian subcontinent supported that the fruit flies might have travelled long distances through single or multiple sources. Two systems of dispersal could be possible for the B. cucurbitae in Indian subcontinent, i) this fly might be

53 103 Table 4.5: Distribution and frequency of different mitochondrial haplotypes in populations Population groups Mitochondrial haplotypes n* H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 North-West India East India 6 6 South India North India Nepal Overall Frequency *n indicates the number of individuals genotyped

54 104 Fig 4.1: Minimum spanning tree (MST) of mitochondrial haplotypes of B. cucurbitae generated by population genetic analysis software Arlequin 3.1

55 105 Plate 4.19: Minimum spanning network of the 14 mitochondrial haplotypes, observed in a set of 33 individuals from all 5 Bactrocera cucurbitae populations. Sampling region of each haplotype is colour coded as: North west India, green; East India, yellow; South India, light blue, North India, blue and Nepal, brown. One, two and three step haplotypes are shown.

56 106 Fig 4.2: Distribution map of different mitochondrial haplotypes of Bactrocera cucurbitae populations in India

57 107 flying or traveling with the temperature gradients from north to south with the start of winter season in the northern regions, where winter temperature fall below 0 O C and at the same time, temperature is favourable round the year in south India and vice-versa with the northward rise in temperature from south-middle-north India. This statement is supported by the collection of B. cucurbitae from Bengaluru (South India) during Dec-Jan, when no activity of this fly was observed in Himachal Pradesh (North West India). ii) This fly could easily be transported along with their host fruit to any place in India, as no regulation has been enacted against transportation and trade of host fruit and planting materials for fruit fly in India i. e. no domestic quarantine regulations have been enforced in India against fruit fly. Besides above two systems of dispersal, large scale tourist movement as well as personal commutation by vast majority of population also facilitates the dispersal of fruit flies with in India. However in India, the first system of dispersal may not be working solely, as the highest dispersal capability of the fly is reported to be as high as 2 km per 2 weeks (Peck et al. 2005). Their observations pertain to the terrain topography consisting of hills and plains, without big rivers and high mountains, whereas in Indian subcontinent, the north-western and north-eastern Himalayan terrain consists of big rivers and high mountains, whereas, east and west Indo-Gangetic plains and middle & south Indian terrain harbour big rivers. Therefore the second system of the fly dispersal could be of major significance in the Indian subcontinent Phylogenetic analysis of B. cucurbitae isolates from Indian subcontinent based on mtcoi gene The optimal phylogenetic tree is presented in Fig. 4.3 had branch length of base substitutions per site. The bootstrap value for each branch is given in the tree. It is clear from the Fig. 4.3 that all the B. cucurbitae isolates were clustered in a single clade with no significant variations. Comparing B. cucurbitae mtcoi sequences with outgroups revealed that interspecific pair wise distances

58 108 HQ B. cucurbitae Nalanda India HQ B. cucurbitae Indora India HQ B. cucurbitae Indora India HQ B. cucurbitae RAU Pusa India HQ B. cucurbitae Nadaun India HQ B. cucurbitae Karnal India HQ B. cucurbitae Patna India HQ B. cucurbitae Ghumarwin India HQ B. cucurbitae Jawalamukhi India HQ B. cucurbitae Dhankuta Nepal 10 HQ B. cucurbitae Patna India HQ B. cucurbitae Solapur India HQ B. cucurbitae Haroli India HQ B. cucurbitae Mandi India HQ B. cucurbitae Nagwain India HQ B. cucurbitae Karnal India 7 HQ B. cucurbitae Nihari India HQ B. cucurbitae Karnal India HQ B. cucurbitae Patna India HQ B. cucurbitae Patna India 10 HQ B. cucurbitae Karnal India HQ B. cucurbitae Bhota India HQ B. cucurbitae Dhankuta Nepal HQ B. cucurbitae Karnal India 0 HQ B. cucurbitae Solapur India HQ B. cucurbitae Solapur India 23 HQ B. cucurbitae Ghaziabad India 43 HQ B. cucurbitae Solapur India 2 HQ B. cucurbitae Solapur India 35 HQ B. cucurbitae Karnal India 100 HQ B. cucurbitae Sundernagar India HQ B. cucurbitae Bengaluru India HQ B. cucurbitae Delhi India HQ B.tau GU Locusta migratoria Fig 4.3: UPGMA tree based on mtcoi gene sequences showing the relationships between thirty three B. cucurbitae isolates of India and rooted at Locusta migratoria. Number above the branches are bootstrap values calculated by UPGMA (500 replicates). Phylogenetic analyses were conducted in software MEGA4.1.

59 109 ranged from (between B. cucurbitae and B. tau) to (between B. cucurbitae and L. migratoria). In the phylogenetic tree shown in Fig. 4.3, B. tau occupied a position closer to B. cucurbitae with high confidence values. This finding suggested that the B. cucurbitae populations in Indian subcontinent is homogeneous and also deny the presence of any cryptic species complex as suggested in other tephritid fruit flies like B. tau (Jamnongluk et al. 2003) and B. dorsalis (Baimai et al. 1995; 1999; 2000a) Phylogenetic analysis of B. cucurbitae populations across the world available in GenBank (NCBI) based on mtcoi gene All the sequences of 33 isolates of B. cucurbitae from Indian subcontinent and 23 global mtcoi sequences of B. cucurbitae available in NCBI GenBank were compared by multiple sequence alignments using ClustalW programme in MEGA software version 4.1. The identity and accession number of GenBank sequences is given in Table 3.5. Pair wise genetic distance between 56 isolates of B. cucurbitae based upon substitutions per site was obtained by bootstraps procedure (500 replicates) using K2P method in MEGA 4.1 to elucidate the relationship among them. The pair wise genetic distance between the isolates varied from to (1%). The phylogenetic tree is presented in Fig The evolutionary distances were compared using K2P and were in units of base substitution per site. The optimal tree had branch length of base substitutions per site. The same tree topology was obtained when the phylogenetic analysis was carried out with NJ method. The bootstrap values calculated by UPGMA and NJ method with 500 replicates are given above and below the branches in the phylogenetic tree. It is clear from tree presented in Fig. 4.4 that all the global isolates including Indian were clustered in a single clade with no significant variation among isolates irrespective of their origin or geographical distributions.

60 110 HQ B. cucurbitae Nadaun India AB B. cucurbitae Japan EU B. cucurbitae China HQ B. cucurbitae Indora India HQ B. cucurbitae Haroli India HQ B. cucurbitae Ghumarwin India FJ B. cucurbitae Malaysia HQ B. cucurbitae Indora India HQ B. cucurbitae Solapur India HQ B. cucurbitae Nagwain India HQ B. cucurbitae Patna India AY B. cucurbitae USA AB B. cucurbitae Sri Lanka AY B. cucurbitae USA HQ B. cucurbitae Patna India HQ B. cucurbitae Karnal India EU B. cucurbitae China EU B. cucurbitae China HQ B. cucurbitae Nalanda India AY B. cucurbitae UAS HQ B. cucurbitae Karnal India EU B. cucurbitae China AY B. cucurbitae USA HQ B. cucurbitae Patna India AY B. cucurbitae USA HQ B. cucurbitae Dhankuta Nepal HQ B. cucurbitae Patna India AY B. cucurbitae Japan EU B. cucurbitae China AY B. cucurbitae USA HQ B. cucurbitae Jawalamukhi India HQ B. cucurbitae Karnal India HQ B. cucurbitae RAU Pusa India AF B. cucurbitae Thailand HQ B. cucurbitae Nihari India HQ B. cucurbitae Mandi India HQ B. cucurbitae Karnal India HQ B. cucurbitae Bhota India EU B. cucurbitae China AY B. cucurbitae China HQ B. cucurbitae Karnal India EU B. cucurbitae China HQ B. cucurbitae Solapur India HQ B. cucurbitae Ghaziabad India HQ B. cucurbitae Solapur India AB B. cucurbitae Thailand HQ B. cucurbitae Dhankuta Nepal EU B. cucurbitae China AY B. cucurbitae USA HQ B. cucurbitae Karnal India HQ B. cucurbitae Solapur India EU B. cucurbitae China HQ B. cucurbitae Sundernagar India HQ B. cucurbitae Bangluru India HQ B. cucurbitae Delhi India HQ B. cucurbitae Solapur India HQ B. tau HQ B. scutellaris FJ Musca domestica GU Locusta migratoria Fig 4.4: Phylogenetic tree based on mtcoi gene sequences showing the relationships between fifty six B. cucurbitae isolates of India and other countries, rooted at Locusta migratoria. Numbers above and below the branches are bootstrap values calculated by UPGMA (500 replicates) and neighbour-joining method (500 replicates), respectively. Phylogenetic analyses were conducted in software MEGA4.1.

61 111 Comparing B. cucurbitae mtcoi sequences with outgroups, interspecific K2P distances ranged from , , and with B. tau, B. scutellaris, Musca domestica and L. migratoria, respectively. This analysis further support the homogeneity in Indian B. cucurbitae population as observed during comparison of only Indian isolates (Section ). The present finding is in concordance with that of Hu et al. (2008), who observed that B. cucurbitae populations present in China and south-east Asia did not possess significant variation. The results of the present study also support the fact that B. cucurbitae populations are homogeneous irrespective of their geographical distribution because of very low intraspecific pairwise genetic distance. However, high intraspecific distances were previously reported within the species Bactrocera oleae (Ochando and Reyes 2000; Nardi et al. 2005), Bactrocera depressa (Mun et al. 2003) and Bactrocera dorsalis (Shi et al. 2005; 2010). Based on studies of insect mtdna, Brower (1994b) suggested that the molecular clock could be calibrated to 2.3% pairwise sequence divergence per million years. Using this value, B. cucurbitae could have originated some 0.4 million years ago, exhibiting its recent evolution in nature. Using the same estimate of Brower (1994b), Jamnongluk et al. (2003) suggested that the B. tau complex (complex of eight cryptic species) and B. dorsalis complex could have arisen some 5 and 15 million years ago, respectively. This B. tau species is similar in morphology and host range like B. cucurbitae. High genetic uniformity in the present studies also suggested that the B. cucurbitae origin is recent as compared to B. tau and B. dorsalis and the saturation of nucleotide sequences might not have occured to form the cryptic species or origin of complex as in case of B. tau (Jamnongluk et al. 2003). The similarity of B. cucurbitae population of India with that of Japan, where it has been eradicated with SIT (sterile insect technique) programme (Koyama et al. 2004), make this fly a suitable candidate for exploiting its management by SIT

62 112 in India too. This also suggests that any large scale B. cucurbitae management programme may work in all populations including India as the genetic makeup of the populations is same. However, the success of large scale (international/ intercontinental) SIT programme depends on the mating compatibility of the released laboratory strain with the wild flies. To support the above phenomenon, mating tests among three melon fly, B. cucurbitae populations from Mauritius, Seychelles and Hawaii (genetic sexing strain) were conducted by Sookar et al. (2010), they observed that the sexual activity among the three melon fly populations was similar and no significant non-random, assortative mating was observed. Therefore, they concluded that melon flies from Mauritius, Seychelles and the Hawaii are compatible, at least under semi-natural conditions. The compatibility of Hawaii population with other geographically isolated population is in agreement with the high genetic similarity observed in the present study between India and Hawaii (USA) melon fly population. This also suggested that the B. cucurbitae invasion in different countries is recent in nature; most possible source might be India (origin place) and presence of B. cucurbitae in Asian countries as well as Hawaii Island of USA showed no historical separation as they are coming from geographically distinct places but forming single clade. However, any international or Indian government programme may use SIT technique with the help of sterile strain of B. cucurbitae used in Japan for the eradication of this dreaded pest for sustainable vegetable production and better livelihood of the farming community. This programme would also save the environment from the indiscriminate use of pesticide as well as the farming community and consumers from different health problems Mitochondrial cytochrome oxidase I (mtcoi) gene analysis of B. tau prevalent in Himachal Pradesh PCR analyses of mtcoi gene of 16 B. tau isolates were amplified using UEA7 and UEA10 primers. Gel electrophoresis photograph of mtcoi gene of 16 isolates of B. tau is presented in Plate Sequencing of PCR product using custom services revealed that all the test isolates consisted of optimum 611bp sequences length.

63 113 Plate 4.20: mtcoi gene PCR product of Bactrocera tau isolates amplified by using gene specific markers. (Lane 1 and 18, 100bp ladder; lane 2 to 17 showing mtcoi gene amplification of Bactrocera tau isolate P1, P2, P4, P5, P7, P8, P9, P10, P11, P12, P13, P14, P15, P16, P18 and P20)

64 Sequences submission and blast analysis The obtained sequences were first subjected to blast search against the sequences of B. tau available online in the NCBI GenBank using BLASTN programme and their identity confirmed as B. tau. Comparative sequence analysis of the test and reference isolates confirmed the taxonomic status of the test isolates determined on morphological characteristics. All the sequences were submitted to GenBank database (NCBI) under accession number HQ HQ Multiple alignment of mtcoi gene of B. tau isolates All the sequences of 16 isolates were compared by multiple alignment using ClustalW programme in MEGA 4.1. Multiple alignment of 16 sequences of B. tau revealed 16 variable positions with no indels. Variables sites were characterized by 12 singletons and 4 parsimonious informative Pair wise genetic distances between B. tau isolates The pairwise genetic distance between sequence pairs is presented in below diagonal and standard error above diagonal in Table 4.6. The pair wise genetic distance between the isolates varied from to 0.012, thereby indicating very low genetic divergence without any significant variation among B. tau isolates. However, contrary to our observations Jamnongluk et al. (2003) noticed higher divergence (0.006 to 0.280) in sequences of the mtcoi gene of eight species of the B. tau complex from Thailand. The present findings revealed that B. tau population from Thailand possess comparatively high genetic divergence in comparison to the Indian population, however, this could be further established by including more number of isolates from various regions of the country.

65 HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ GU HQ Table 4. 6: Pair wise genetic distance based on mtcoi gene sequences between B. tau isolates of India using the K2P method in MEGA4.1 B. tau isolates Pair wise genetic distance B. tau isolates HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ GU L. migratoria HQ B. cucurbitae All results are based on the pairwise analysis of 16 B. tau mtcoi gene sequences. Below diagonal and above diagonal values are number of base substitution per site and standard error estimate(s) respectively, and were obtained by a bootstrap procedure (500 replicates).

66 Phylogenetic analysis of B. tau isolates based on mtcoi gene Phylogenetic tree presented in Fig. 4.5 had branch length of base substitutions per site. It is clear from the Fig. 4.5 that all the isolates were clustered in a single clade with no significant variation among B. tau isolates. Comparing B. tau mtcoi sequences with outgroups, interspecific genetic distances ranged from (between B. tau and B. cucurbitae) to (between B. tau and L. migratoria). The present results showed that no historical separation had taken place among B. tau isolates of Himachal Pradesh (India) infesting cucurbits, although variations were observed in the B. tau populations of Thailand (Jamnongluk et al. 2003) Comparative analysis of Indian isolates and other Asian isolates of B. tau available in NCBI GenBank Sequences of 16 test isolates from India (H.P.) were compared with sequences of B. tau available online in GenBank (NCBI) by multiple sequence alignment using ClustalW programme in MEGA software version 4.1. The identity and accession number of GenBank sequences is given in Table 3.6. The pair wise genetic distance among various isolates of different origin ranged between to base substitutions per site (Table 4.7). The genetic distance among B. tau isolates from India, China, Japan, Malaysia and one isolates from Thailand was very low ranged from to base substitution per site and varied non-significantly (Bootstrap support < 50%). However, the distance (genetic) of Indian isolates of B. tau was very high with other species of B. tau complex (B, C, D, E, F, G, H, I) of Thailand ranged from to base substitution per site (Table 4.7). The phylogenic analysis performed with MEGA 4.1 software using UPGMA method clustered all the B. tau isolates in one clade, whereas, Thailand isolates showed wide variation (Fig. 4.6). This further revealed a narrow genetic makeup of B. tau populations prevalent in north-west India, China, Japan and Malaysia along with species A from Thailand. The optimal tree had branch length of base substitutions per site.

67 FJ AY AY AF AY AF AF AF AF AF AF EU HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ HQ FJ GU Table 4.7: Pair wise genetic distance based on mtcoi gene sequences between B. tau isolates of India and other countries using the K2P method in MEGA4.1 B. tau isolates Pair wise genetic distance B. tau isolates FJ Malaysia AY China AY Japan AF Thailand A AY Thailand G AF Thailand I AF Thailand F AF Thailand E AF Thailand D AF Thailand C AF Thailand B EU China HQ India HQ India HQ India HQ India HQ India HQ India HQ India HQ India HQ India HQ India HQ India HQ India HQ India HQ India HQ India HQ India FJ M. domestica GU L. migratoria All results are based on the pairwise analysis of 28 mtcoi gene sequences. Below diagonal and above diagonal values are number of base substitution per site and standard error estimate(s) respectively, and were obtained by a bootstrap procedure (500 replicates).

68 118 HQ B. tau Banikhet India HQ B. tau Nagwain India HQ B. tau Jawalamukhi India HQ B. tau Palampur India 35 HQ B. tau Nadaun India HQ B. tau Barot India 30 HQ B. tau Shahpur India HQ B. tau Jawalamukhi India 24 HQ B. tau Chandpur India HQ B. tau Nadaun India 15 HQ B. tau Ghumarwin India 49 HQ B. tau Nihari India 35 HQ B. tau Palampur India 54 HQ B. tau Mandi India 99 HQ B. tau Nadaun India HQ B. tau Nauni India HQ B. cucurbitae India GU Locusta migratoria Fig 4.5: Phylogenetic tree based on mtcoi gene sequences showing the relationships between sixteen B. tau isolates of Himachal Pradesh and rooted at Locusta migratoria. Numbers above branches are bootstrap values calculated by UPGMA (500 replicates). Phylogenetic analyses were conducted in software MEGA4.1.

69 AY B. tau Japan HQ B. tau Nadaun India HQ B. tau Shahpur India HQ B. tau Barot India HQ B. tau Chandpur India EU B. tau China HQ B. tau Jawalamukhi India HQ B. tau Palampur India HQ B.tau Jawalamukhi India HQ B. tau Baniket India HQ B. tau Nadaun India AY B. tau China HQ B.tau Nagwain India HQ B.tau Ghumarwin India HQ B. tau Nihari India HQ B. tau Mandi India FJ B. tau Malaysia HQ B.tau Palampur India HQ B. tau Nadaun India AF B. tau A Thailand HQ B. tau Nauni India AF B. tau D Thailand AY B.tau G Thailand AF B. tau E Thailand AF B. tau F Thailand AF B. tau B Thailand AF B. tau I Thailand AF B. tau C Thailand FJ Musca domestica GU Locusta migratoria Fig 4.6: Phylogenetic tree based on mtcoi gene sequences showing the relationships between twenty eight B. tau isolates of India and other countries and rooted at Locusta migratoria. Numbers above branches are bootstrap values calculated by UPGMA (500 replicates). Phylogenetic analyses were conducted in software MEGA4.1.

70 120 Jamnongluk et al. (2003) compared sequences of the mitochondrial cytochrome oxidase I gene of eight species of the Bactrocera tau complex from Thailand. The sequence divergence between species in the B. tau complex ranged from to However in the present study, the sequence divergence was slightly less than that observed by Jamnongluk et al. (2003). This variation could be ascribed due to the length of sequences included in the analysis and the boot strap support analysis. Jamnongluk et al. (2003) included 639bp sequences and used 1000 replication for bootstrap test whereas, in the present analysis, 611 bp sequences and 500 replications (Bootstrap) were used. All B. tau isolates collected from Himachal Pradesh (India) were clustered with B. tau Thailand species A, this propounds that the cucurbits infestation in Himachal Pradesh is by B. tau Thailand species A of the B. tau species complex as Thailand species A has been considered as the generalist cucurbits pest (Jamnongluk et al. 2003). The presence of other species of B. tau species complex in H.P. as well as in India should not be ignored as a total of eight species have been reported in B. tau species complex. This however needs further detailed investigations Molecular phylogeny of Bactrocera and Dacus species based on mtcoi gene PCR amplification of mtcoi gene of Bactrocera (other than B. cucurbitae and B. tau) and Dacus species is presented in Plate Sequencing of PCR product using custom services revealed that sequences of all test isolates consisted of 611bp Sequence submission and blast analysis The obtained sequences were first blast searched against the sequences of Bactrocera and Dacus species available in online NCBI GenBank using BLASTN programme. After blast search of mtcoi gene of different Bactrocera and Dacus species isolates, it was found that mtcoi gene of three species namely B. nigrofemoralis, Dacus longicornis, Dacus sphaeroidalis sequenced in

71 121 Plate 4.21: mtcoi gene PCR product of fruit fly species isolates amplified by using gene specific markers. (Lane 1 and 14, 100bp DNA ladder; lane 2 to 13 showing mtcoi gene amplification of fruit fly species, lane 2-3, Bactrocera scutellaris; lane 4, Bactrocera zonata; lane 5-9, Bactrocera dorsalis; lane 10, Bactrocera nigrofemoralis; lane 11, Dacus longicornis and lane 12-13, Dacus sphaeroidalis)

72 122 present study were new and the mtcoi sequences of these species were not present in NCBI GenBank. All the mtcoi gene sequences of Bactrocera and Dacus species were submitted to NCBI GenBank vide accession number HQ (B. nigrofemoralis), HQ (Dacus longicornis), HQ HQ (Dacus sphaeroidalis), HQ HQ (B. scutellaris), HQ (B. zonata) and HQ HQ (B. dorsalis). mtcoi gene sequences of B. nigrofemoralis, Dacus longicornis and Dacus sphaeroidalis were new to NCBI GenBank and constitute first record in the global GenBank database (GenBank, NCBI, USA) Multiple alignment and comparison of different Bactrocera and Dacus spp. isolates All the sequences of 61 isolates of different Bactrocera and Dacus spp. were compared by multiple alignment using ClustalW programme in MEGA Genetic distance within and between species Although pair-wise genetic distance was calculated to compare any two isolates but it gave little idea about genetic distance between and within different Bactrocera and Dacus species groups. Keeping this in view mean genetic distance between and within species groups was calculated and presented in Table 4.8, revealed very low genetic distance within each species group varying from to The maximum genetic distance was within B. dorsalis (0.014) followed by B. tau (0.004), B. cucurbitae (0.002), B. scutellaris (0.00) and Dacus sphaeroidalis (0.00). The genetic distances within a species were not calculated for the species B. zonata, B. nigrofemoralis, D. longicornis, Musca (out group) and Locusta (out group) because of single sequence.

73 123 Table 4.8. Estimates of evolutionary divergence over sequence pairs between species using the K2P method in MEGA4.1 Species Number of base substitutions per site B. scutellaris B. zonata B. dorsalis B. nigrofemoralis Species Dacus longicornis Dacus sphaeroidalis B. cucurbitae B. tau Musca Locusta B. scutellaris B. zonata nc* B. dorsalis B. nigrofemoralis nc Dacus longicornis nc Dacus sphaeroidalis B. cucurbitae B. tau Musca nc Locusta nc *nc not calculated All results are based on the pairwise analysis of 63 sequences. Below diagonal and above diagonal values are number of base substitution per site and standard error estimate(s) between the species respectively, and were obtained by a bootstrap procedure (500 replicates). Diagonal values are within species sequence divergence

74 124 Mean genetic distance (below diagonal) between Bactrocera and Dacus species is given in Table 4.8. The mean genetic distance between species group varied from to with minimum distance between B. dorsalis/ B. nigrofemoralis (0.013) followed by B. cucurbitae/ B. tau (0.047), B. zonata/ B. scutellaris (0.093), B. zonata/ B. dorsalis (0.97). The mean genetic distance between out group and all other isolates was very high (0.226 to 2.342) indicating their distinct genetic makup. Very low mean genetic distance between B. cucurbitae and B. tau confirmed their classical taxonomic relationships as these two species have been placed in Zeugodacus group. Lower genetic distance among B. dorsalis, B. nigrofemoralis and B. zonata is signifying their relatedness as a member of Bactrocera group Phylogenetic relationship between different Bactrocera and Dacus species using mtcoi gene sequences In order to infer relationships among eight species of Bactrocera and Dacus using mitochondrial DNA sequences. The optimal phylogenetic tree with a branch length of base substitutions per site was generated using UPGMA method and is given in Fig. 4.7 revealed that all the isolates were divided into their respective species group and clustered together indicating that all the species can be differentiated on the basis of mtcoi gene. Further isolates of B. cucurbitae, B. tau and B. scutellaris were clustered in a larger clade as per classical group Zeugodacus (Fig 4.7). Branch topology of phylogenetic tree in the present study revealed that the evolution of group Zeugodacus recovered as monophyletic and emerging from Bactrocera group of species with high bootstrap support (74 percent). However, B. zonata, B. dorsalis and B. nigrofemoralis members of the subgenus Bactrocera have been clustered together outside Zeugodacus. In the present study, the genus Bactrocera also recovered as monophyletic in origin with 81 per cent bootstrap support. Two species of genus Dacus namely Dacus longicornis and Dacus sphaeroidalis have been clustered together, forming Callantra group. Whereas, out group members i.e. Musca domestica from the order Diptera and Locusta migratoria from the order Orthoptera of class Insecta were placed outside in the phylogenetic tree (Fig 4.7).

75 HQ B. cucurbitae Patna India HQ B. cucurbitae Karnal India HQ B. cucurbitae Patna India HQ B. cucurbitae Jawalamukhi India HQ B. cucurbitae Karnal India HQ B. cucurbitae Dhankuta Nepal HQ B. cucurbitae Nihari India HQ B. cucurbitae Ghumarwin India HQ B. cucurbitae Karnal India HQ B. cucurbitae Patna India 7 HQ B. cucurbitae Indora India HQ B. cucurbitae Karnal India HQ B. cucurbitae Nadaun India HQ B. cucurbitae Nagwain India HQ B. cucurbitae Mandi India HQ B. cucurbitae Nalanda India 0 HQ B. cucurbitae Solapur India HQ B. cucurbitae Indora India HQ B. cucurbitae RAU Pusa India HQ B. cucurbitae Patna India HQ B. cucurbitae Haroli India HQ B. cucurbitae Bhota India 1 HQ B. cucurbitae Ghaziabad India HQ B. cucurbitae Solapur India 47 HQ B. cucurbitae Solapur India HQ B. cucurbitae Dhankuta Nepal 2 HQ B. cucurbitae Karnal India HQ B. cucurbitae Solapur India HQ B. cucurbitae Karnal India HQ B. cucurbitae Sundernagar India HQ B. cucurbitae Bengaluru India 100 HQ B. cucurbitae Delhi India 31 HQ B. cucurbitae Solapur India HQ B. tau Nauni India HQ B. tau Nadaun India 100 HQ B. tau Mandi India HQ B. tau Palampur India HQ B. tau Ghumarwin India HQ B. tau Nihari India 33 HQ B. tau Nagwain India HQ B. tau Jawalamukhi India HQ B. tau Nadaun India 7 HQ B. tau Baniket India HQ B. tau Chandpur India 9 HQ B. tau Jawalamukhi India HQ B. tau Shahpur India 16 HQ B. tau Nadaun India HQ B. tau Palampur India HQ B. tau Barot India HQ B. scutellaris Palampur India 100 HQ B. scutellaris Palampur India HQ B. zonata Palampur India 41 HQ B. dorsalis Palampur India 100 HQ B. dorsalis Palampur India 100 HQ B. dorsalis Palampur India 79 HQ B. dorsalis Palampur India HQ B. dorsalis Palampur India 67 HQ B. nigrofemoralis Palampur India HQ Dacus longicornis Palampur India HQ Dacus sphaeroidalis Palampur India 100 HQ Dacus sphaeroidalis Palampur India FJ Musca domestica GU Locusta migratoria Zeugodacus group Bactrocera group Callantra group Fig 4.7: Phylogenetic tree based on mtcoi gene sequences showing the relationships between eight species of Bactrocera and Dacus spp. of India rooted at Locusta migratoria. Numbers near the branches are bootstrap values calculated by UPGMA (500 replicates). Phylogenetic analyses were conducted in software MEGA 4.1.

76 126 Some researchers have proposed a phylogenetic analysis of the Bactrocera subgenera groupings based on morphological characters (Drew 1989b; Drew and Hancock 2000; White 2000). According to Drew (1989b), the subgenera of Bactrocera are divided into 4 groups, the Bactrocera group, Queenslandacus group, Zeugodacus group, and Melanodacus group. In the present study, the subgenus Bactrocera is placed in the Bactrocera group and the subgenus Zeugodacus in the Zeugodacus group. This is in agreement with the classification by Drew (1989b). Analysis suggested that the subgenus Bactrocera and Zeugodacus were monophyletic in the present study. These results are in accordance with the findings of Drew and Hancock (2000) and Muraji and Nakahara (2001), who suggested that the Bactrocera (Zeugodacus) group is monophyletic, whereas, Smith et al. (2003) recovered subgenera of Zeugodacus group as paraphyletic. In the present study, subgenus Bactrocera was recovered as monophyletic in origin. This is supported by the study of Drew and Hancock (2000), who suggested that the Bactrocera group of subgenera, represented here by B. (Bactrocera) is monophyletic. However, Smith et al. (2003) as well as White (2000) recovered subgenera of Bactrocera group as paraphyletic. Bactrocera and Dacus species sequenced in this study were found as sister groups and rooted to Musca domestica (outgroup). This result is supported by Munro (1984) and White (2000) who suggested that these two genus as sister groups based on the morphological characteristics. Results of the present study suggest that the sequence analysis of mtcoi gene is very useful to elucidate phylogeny of Bactrocera and Dacus taxa. However, more taxa must be analyzed to infer genetic diversity among them and more genomic data must be generated that exhibit sufficient genetic variation to resolve some of the internal nodes ambiguity.

77 Isolation of gut bacteria of fruit fly Gut bacteria were isolated from nine populations of fruit fly B. tau on two culture media viz. PYEA and BHIA (enriched culture media). It was found that bacteria were associated with all the nine populations of B. tau (Table 4.9). A total of 63 different bacterial colonies were observed on two culture media. Out of which, 28 were isolated on PYEA and 35 on BHIA (Table 4.9). On the basis of colony morphology, 16 and 14 isolates obtained on PYEA and BHIA, respectively, were chosen for screening purpose to select the five most promising gut bacteria as fruit fly attractant (Table 4.9) and for their further characterization. Bacterial association with Tephritidae in general and Bactrocera in particular is well known and has been confirmed by many workers. Gupta et al. (1982a) isolated bacteria from different organs of B. cucurbitae, but they could record only one type of bacteria from the fruit flies. Sood and Nath (2002) isolated 11 types of bacteria from B. tau and B. cucurbitae and established the true association of fruit fly type bacteria with Bactrocera spp. which is in conformity with the present findings. The presence of bacteria in alimentary track of B. tryoni in Australia has been well documented (Drew and Lloyd 1987). Unlike, Gupta et al. (1982b), who reported only one species (Pseudomonas pseudomalaii) in B. cucurbitae, Drew and Lloyd (1987) reported six types of bacteria in B. tryoni. Whereas, three types of bacteria were reported in B. tau by Prabhakar et al. (2009b). The variation in number and types of bacteria associated with Bactrocera by different workers from different species might be due to different geographical location and species variation.

78 128 Table 4.9 : Isolation of gut bacteria from different populations of B. tau B. tau isolates used for bacterial isolation Culture media used Peptone Yeast Extract Agar (PYEA) Bacterial Bacterial isolates isolate number selected for attractancy screening Bacterial colonies isolated P1 4 4 P1A, P1B, P1C, P1D Brain Heart Infusion Agar (BHIA) Bacterial isolates selected for attractancy screening Bacterial colonies isolated Bacterial isolate number 4 2 B1A, B1B P2 3 2 P2A, P2B 4 1 B2A P3 3 1 P3A 3 2 B3A, B3B P4 2 1 P4A 5 2 B4A, B4B P5 4 2 P5A, P5B 4 1 B5A P9 2 1 P9A 3 1 B9A P P10A, P10B 4 2 B10A, B10B P P15A 5 2 B15A, B15B P P18A, P18B 3 1 B18A Total

79 Screening of fruit fly gut bacteria as fruit fly attractant A perusal of data presented in Table 4.10 revealed that majority of bacteria isolated from the gut of B. tau attracted more number of B. tau adult (female and male) than control (un-inoculated PYE broth). The five most attractive bacterial isolates were selected on the basis of adult fruit flies visits/ 30 min. to different bacterial cultures (treatments) which were initially coded as P1B, P3A, P10A, B4A and B10B. These bacterial isolates were characterized, evaluated as attractant to fruit fly and subjected to GCMS analysis for identification of volatile bacterial metabolites. 4.5 Identification of gut bacterial isolates of fruit fly The five gut bacterial isolates of fruit fly were identified on the basis of cultural, morphological, biochemical and 16S rdna characteristics. The results of different cultural morphological, biochemical tests and 16S rdna sequences for the identification of these bacterial isolates are presented in Table Morphological characterization All bacterial isolates were rod shaped and Gram- negative in nature (Table 4.11). Isolates P1B, P3A, B4A and B10B were found motile, whereas isolate P10A was non-motile. On the basis of colony characteristics, bacterial isolate P10A was found to produce yellow pigment, while P1B, P3A, B4A and B10B did not produce any pigment. All the isolates P1B, P3A, P10A, B4A and B10B were found to form sediment in PYEB medium Biochemical characterization Isolate P1B gave a positive reaction for MR, oxidase and catalase where as, a negative reaction for citrate, VP, TSI, D-glucose and gas production in glucose medium. Isolate P3A was oxidase, catalase and D-glucose positive and citrate, TSI and gas production in glucose medium negative while the reaction was doubtful for citrate and VP (Table 4.11).

80 130 Table 4.10: Attractancy of bacterial isolates against fruit fly, B. tau (Walker) Sr. No. Bacterial Isolates (72 hrs old, 2 ml broth culture) Fruit flies visited /30 min Female* Male* Total* 1 P1A P1B** P1C P1D P2A P2B P3A** P4A P5A P5B P9A P10A** P10B P15A P18A P18B B1A B1B B2A B3A B3B B4A** B4B B5A B9A B10A B10B** B15A B15B B18A Control (Un-inoculated PYEA broth) CD *Mean of six replications ** Selected for characterization and analysis

81 131 Table 4.11: Morphological, biochemical and molecular characteristics of promising gut bacteria of B. tau Characteristics Bacterial isolates P1B P3A P10A B4A B10B Morphological Shape Rod Rod Rod Rod Rod Gram's reaction Pigment production - - Y - - growth in broth Sediment Sediment Sediment Sediment Sediment medium Motality Biochemical Citrate test - d - - d Methyl red V P test - d TSI Catalase Oxidase D- Glucose d Gas Production in glucose medium Molecular 16S rdna sequence blast similarity Bacteria identified as 91 % with Delftia acidovorans Delftia acidovorans 97 % with Pseudomonas putida Pseudomonas putida 95 % with Flavobacterium sp. Flavobacterium sp. 98 % with Defluvibacter sp. Defluvibacter sp. 99 % with Ochrobactrum sp. Ochrobactrum sp. Y yellow pigment, - negative reaction, + positive reaction, d doubtful result

82 132 A positive reaction for oxidase, catalase and D-glucose was obtained for isolate P10B and it was negative for citrate, MR, VP, TSI and gas production in glucose medium tests (Table 4.11). Isolate B4A also gave same result as isolate P10B except that D-glucose was negative. Isolate B10B was positive for oxidase and catalase, negative for MR, VP, TSI, and gas production in glucose medium and doubtful for citrate and D- glucose (Table 4.11) Sequencing of 16S rrna bacterial gene and sequence analysis An attempt was made to characterize the promising bacteria isolated from the fruit fly gut using 16S rrna gene sequences to identify and to decipher their phylogenetic affiliation. The 16S rrna gene serve as molecular chronometer, since it is the most conserved part during evolution (Clarridge 2004). Therefore, 16S rrna gene sequencing is accepted worldwide for authenticated identification and for phylogenetic analysis of the bacterium. The PCR amplified products of 16S rrna gene using specific primers of five gut bacterial isolates from B. tau are presented in Plate Sequence data of 16S rrna gene obtained after custom sequencing of the PCR products using specific primers revealed the presence of 975bp in all the test isolate i.e. P1B, P3A, P10A, B4A and B10B. Nucleotide sequence analysis of test isolates using online BLAST nucleotide similarity search program ( revealed that test bacterial isolates showed maximum homology (similarity) with Delftia acidovorans (91%), Pseudomonas putida (97%), Flavobacterium sp. (95%), Defluvibacter sp. (98%) and Ochrobactrum sp. (99%). Thus on the basis of cultural, morphological, biochemical and 16S rdna gene characteristics, the bacterial isolates P1B, P3A, P10A, B4A and B10B were identified as Delftia acidovorans, Pseudomonas putida, Flavobacterium sp., Defluvibacter sp. and Ochrobactrum sp., respectively (Plate 4.23).

83 133 Plate 4.22: 16S rrna gene PCR product of gut bacterial isolates of B. tau (Walker) amplified by using gene specific markers (Lane M1: double digested DNA ladder and Lane M2: and DNA ladder 100bp)

84 134 Delftia acidovorans (P1B) Pseudomonas putida (P3A) Flavobacterium sp. (P10A) Defluvibacter sp. (B4A) Ochrobactrum sp. (B10B) Plate 4.23: Promising gut bacteria of Bactrocera tau

85 135 The 16S rdna gene nucleotide sequences of these isolates were submitted to GenBank nucleotide database under accession number HQ to HQ Multiple sequence alignment and pair wise genetic distance All the sequences of 5 bacterial isolates were compared with other 42 bacterial sequences available online in GenBank (NCBI) by multiple sequence alignment tool using ClustalW programme. The identity and accession number of the sequences used for analysis is given in Table 3.8. The per cent pair wise genetic distance (Table 4.12) of the five promising isolates with other selected sequences ranged from 0.00 to 0.70 nucleotide per site. The pair wise genetic distance was minimum (0.01) between the test Delftia acidovorans P1B and other Delftia sequences. However, genetic distance of this genus was very high with other bacterial genera such as Pseudomonas ( ), Ochrobactrum & Brucellaceae ( ), Defluvibacter & Phyllobacteriaceae ( ) and Flavobacterium & Myroides ( ) used in the analysis (Table 4.12). The pair wise genetic distance among test bacterial isolate P3A (Pseudomonas putida, HQ446524) and other isolates of Pseudomonas spp. isolates varied from 0.00 to 0.20, however, least genetic distance (0.00) among different Pseudomonas spp. was observed with Pseudomonas putida (AY and DQ387441) followed by Pseudomonas spp. (HM152635, EU372964, FJ472861, FJ472858, AM and AM930519, 0.01) and maximum genetic distance (0.20) was observed with Pseudomonas geniculata (HM805109). Gut bacterial isolate P10A identified as Flavobacterium sp. (HQ446525) showed minimum genetic distance (0.01) with Flavobacterium sp. (FJ965845) followed by 0.03 with Myroides odoratus (AB517709), Myroides sp. (GU350455),

86 136 Myroides odoratus (M58777) and Flavobacterium odoratum (D14019). Whereas, maximum genetic distance (0.16) was observed with Uncultured Flavobacterium sp. (AM910365), though overall genetic distance among various spp. used in the analysis was very low (Table 4.12). Genetic distance between B4A isolate identified as Defluvibacter sp. (HQ446526) a member of bacterial family Phyllobacteriaceae, and other Phyllobacteriaceae bacteria ranged from 0.00 to Defluvibacter sp. (HQ446526) showed minimum genetic distance (0.00) with Defluvibacter sp. (FJ542910), Defluvibacter lusatiensis (EU870446) & Phyllobacteriaceae bacterium (GQ and AM884147) followed by 0.01 genetic distance with Defluvibacter lusatiensis (FJ982919) & Phyllobacteriaceae bacterium (AM884148) and maximum genetic distance (0.06) with Phyllobacteriaceae bacterium (AM884144). The test organism, Ochrobactrum sp. isolate B10B (HQ446527) of the present study was found genetically similar to Ochrobactrum guangzhouense (EF125185) with genetic distance of The genetic distance of this species with other member of the genus Ochrobactrum was Whereas, genetic distance was 0.03 with closely related genus Pseudochrobactrum (Table 4.12). Dendrogram constructed by phylogenetic analysis presented in Fig 4.8 shows that the all bacterial isolates viz., P1B, P3A, B4A and B10B clustered with Delftia, Pseudomonas, Defluvibacter and Ochrobactrum respectively, all Proteobacteria except P10A (Flavobacterium sp., HQ446525). Isolate P10A was clustered with Flavobacterium, a typical Bacteroidetes. Based on their affinity with known sequences in databank, the isolate P1B belongs to class β-proteobacteria, P3A to class γ-proteobacteria and B4A & B10B to class α-proteobacteria. Many workers suggested that nucleic acid sequence approaches, particularly 16S rrna genes, have proved an important tool to settle the taxonomic position of the microbial community of insects (Paster et al. 1996; Brauman et al. 2001; Toth et al. 2001). Because of an immense library of

87 HQ Delftia acidovorans HQ Pseudomonas putida HQ Flavobacterium sp. HQ Defluvibacter sp. HQ Ochrobactrum sp. HQ Delftia acidovorans FR Delftia sp. AF Delftia acidovorans AF Delftia acidovorans FJ Delftia sp. AM Uncultured Delftia acidovorans EF Delftia sp. GQ Delftia acidovorans AB Myroides odoratus GU Myroides sp. M58777 Myroides odoratus D14019 Flavobacterium odoratum GQ Myroides sp. AJ Myroides odoratimimus AM Uncultured Flavobacterium FJ Flavobacterium sp. EF Ochrobactrum guangzhouense FJ Pseudochrobactrum sp. EF Brucellaceae bacterium DQ Ochrobactrum sp. AM Ochrobactrum sp. AM Ochrobactrum oryzae EU Ochrobactrum sp. AJ Ochrobactrum shiyianus HM Pseudochrobactrum sp. GQ Phyllobacteriaceae bacterium AM Phyllobacteriaceae bacterium FJ Uncultured Defluvibacter sp. EU Defluvibacter lusatiensis AM Phyllobacteriaceae bacterium FJ Defluvibacter lusatiensis AM Phyllobacteriaceae bacterium HM Uncultured Pseudomonas sp. AM Uncultured Pseudomonas sp. EU Pseudomonas sp. FJ Pseudomonas putida FJ Pseudomonas putida AM Pseudomonas sp. AM Pseudomonas putida DQ Pseudomonas putida AY Pseudomonas putida HM Pseudomonas geniculata 137 Table 4.12: Pair wise genetic distance based on 16S rdna sequences of gut bacteria of Bactrocera tau and other bacterial sequences Pair wise genetic distance Organisms Organisms HQ Delftia acidovorans HQ Pseudomonas putida HQ Flavobacterium sp HQ Defluvibacter sp HQ Ochrobactrum sp HQ Delftia acidovorans FR Delftia sp AF Delftia acidovorans AF Delftia acidovorans FJ Delftia sp AM Uncultured Delftia acidovorans EF Delftia sp GQ Delftia acidovorans AB Myroides odoratus GU Myroides sp M58777 Myroides odoratus D14019 Flavobacterium odoratum GQ Myroides sp AJ Myroides odoratimimus AM Uncultured Flavobacterium FJ Flavobacterium sp EF Ochrobactrum guangzhouense FJ Pseudochrobactrum sp EF Brucellaceae bacterium DQ Ochrobactrum sp AM Ochrobactrum sp AM Ochrobactrum oryzae EU Ochrobactrum sp AJ Ochrobactrum shiyianus HM Pseudochrobactrum sp GQ Phyllobacteriaceae bacterium AM Phyllobacteriaceae bacterium FJ542910_Uncultured Defluvibacter sp EU Defluvibacter lusatiensis AM Phyllobacteriaceae bacterium FJ982919_Defluvibacter lusatiensis AM Phyllobacteriaceae bacterium HM Uncultured Pseudomonas sp AM Uncultured Pseudomonas sp EU Pseudomonas sp FJ Pseudomonas putida FJ Pseudomonas putida AM Pseudomonas sp AM Pseudomonas putida DQ Pseudomonas putida AY Pseudomonas putida HM Pseudomonas geniculata Note: Pair wise genetic distance (below diagonal) inferred using maximum likelihood method, each sequence was bootstrapped (500 replicates) to determine standard error (above diagonal). Distances are in the units of the number of base substitution per site. All positions containing gaps and missing data were eliminated from the dataset

88 HM Uncultured Pseudomonas sp. France EU Pseudomonas sp. China FJ Pseudomonas putida China FJ Pseudomonas putida China AM Pseudomonas sp. Germany AM Pseudomonas putida China DQ Pseudomonas putida Korea HQ Pseudomonas putida India AY Pseudomonas putida Korea AM Uncultured Pseudomonas sp. Germany HM Pseudomonas geniculata India HQ Delftia acidovorans India AF Delftia acidovorans Belgium AF Delftia acidovorans Germany AM Uncultured Delftia acidovorans HQ Delftia acidovorans Canada FR Delftia sp. Belgium EF Delftia sp. Uruguav FJ Delftia sp. France GQ Delftia acidovorans Turkey AM Phyllobacteriaceae bacterium Germany FJ Uncultured Defluvibacter sp. USA HQ Defluvibacter sp. India GQ Phyllobacteriaceae bacterium China EU Defluvibacter lusatiensis China FJ Defluvibacter lusatiensis Spain AM Phyllobacteriaceae bacterium Germany AM Phyllobacteriaceae bacterium Germany FJ Pseudochrobactrum sp. India HM Pseudochrobactrum sp. China HQ Ochrobactrum sp. India EF Ochrobactrum guangzhouense China AM Ochrobactrum sp. Germany EF Brucellaceae bacterium China AM Ochrobactrum oryzae India DQ Ochrobactrum sp. china EU Ochrobactrum sp. China AJ Ochrobactrum shiyianus China AM Uncultured Flavobacterium Germany GQ Myroides sp. Korea AJ Myroides odoratimimus Germany HQ Flavobacterium sp. india FJ Flavobacterium sp. India AB Myroides odoratus Japan D14019 Flavobacterium odoratum Japan GU Myroides sp. China M58777 Myroides odoratus Fig 4.8. Phylogenetic tree based on 16S rrna gene sequences showing the relationships between five gut bacterial isolates of Bactrocera tau. Number above the branches are bootstrap values calculated by UPGMA (500 replicates). Phylogenetic analyses were conducted in software MEGA 4.1.

89 139 sequence data for 16S rrna loci and other robust markers, allows the precise identification of many associated species, even those that resist cultivation (Stevenson et al. 2004). Over 200,000 bacterial entries exist currently for 16S rrna, and 16S sequences can place most surveyed bacterial taxa securely into genera, if not species (Rupp 2004; Ueda et al. 2004). Delftia acidovorans was isolated from B. tau for the first time, though its association has been reported with cotton boll worm, Helicoverpa armigera (Hubner) (Xiang et al. 2006); wood borer, Saperda vestita (Say) (Delalibera et al. 2005), tobacco caterpillar Manduca sexta (Brinkmann et al. 2008) and mosquito, Aedes albopictus Skuse (Zouache et al. 2009). Whereas, Pseudomonas putida and member of genera Pseudomonas have been reported from many insects species including fruit flies as well as from B. tau (Gupta et al. 1982b; Kuzina et al. 2001; Sood and Nath 2002; Belcari et al. 2003; Delalibera et al. 2005; Brinkmann et al. 2008). The presence of Flavobacterium sp., Defluvibacter sp. and Ochrobactrum sp. have already been reported in other insect species viz. Flavobacterium sp. from ant, Tetraponera binghami Forel (van Borm et al. 2002); Honey bees (Mohr and Tebbe 2006; 2007) and tobacco caterpillar, Manduca sexta (Brinkmann et al. 2008), however, their association with fruit fly has been observed for the first time. Defluvibacter sp. is a member of bacterial family Phyllobacteriaceae from the class α- Proteobacteria and has not been reported from gut of any insect species. But an unassigned bacterium (member Phyllobacteriaceae) has been reported from the gut content of asian longhorned beetle, Anoplophora glabripennis Motschulsky (Geib et al. 2009). Phyllobacteriaceae is a bacterial family closely related with the family Bradyrhizobiaceae, Methylobacteriaceae and Rhizobiaceae. Bacteria from the family Methylobacteriaceae and Rhizobiaceae have been reported from different insect species viz. Rhizobium and Methylobacterium from the gut of Tetraponera ants (van Borm et al. 2002) and Rhizobium reported from the gut content of asian longhorned beetle, Anoplophora glabripennis (Geib et al. 2009).

90 140 Ochrobactrum sp. belongs to the α-2 subclass of the Proteobacteria (De Ley, 1992). This genus was first described by Holmes et al. (1988). The phylogenetic position of Ochrobactrum sp. was defined by De Ley (1992) and Yanagi & Yamasato (1993) on the basis of DNA±rRNA hybridization and 16S rdna homology studies. Its closest known relative is Brucella (De Ley 1992; Moreno 1992; Yanagi and Yamasato 1993; Velasco et al. 1998). Ochrobactrum sp. was reported from the insect gut (Asian longhorned beetle, Anoplophora glabripennis) by Geib et al. (2009). Whereas, its closest relative Brucella sp. was isolated from the gut of wood borer Saperda vestita by Delalibera et al. (2005) and identified by 16S rdna typing. A wide range of bacteria belonging to different genera viz. Acetobacter, Agrobacterium, Arthrobacter, Listeria, Enterobacter, Pantoea, Pectobacterium, Klebsiella, Citrobacter, Erwinia, Bacillus, Lactobacillus, Kluyvera, Micrococcus, Pseudomonas, Staphylococcus, Streptococcus, Proteus, Providencia, Hafnia, Serratia and Xanthomonas have been isolated and characterized from the fruit fly gut. (Lloyd et al. 1986; Drew and Lloyd 1987; Jang and Nishijima 1990; Lauzon et al. 1998; 2000; Zinder and Dworkin 2000; Bergey et al. 2001; Kuzina et al. 2001; Marchini et al. 2002; Sood and Nath 2002; Belcari et al. 2003; Behar et al. 2005; 2008; 2009; Capuzzo et al. 2005; Sacchetti et al. 2008; Kounatidis et al. 2009; Prabhakar et al. 2009b). However, this needs further in-depth investigation as many workers identified spectrum of bacteria from fruit fly gut. Still, detailed investigations are needed to establish the taxonomic positions of Flavobacterium sp., Defluvibacter sp. and Ochrobactrum sp. upto species level using chemotaxonomic and molecular approaches. 4.7 Gut bacteria as attractants to fruit flies The data recorded on adult fruit fly visits per 30 minutes for the comparative efficacy of promising gut bacteria with sugar as negative and protein hydrolyzate as positive control is presented in Table The perusal of data revealed that the maximum number of B. tau females (14.17) and males (12.50)

91 141 were attracted towards protein hydrolyzate and minimum number of B. tau females (4.50) and males (3.33) were attracted towards sugar. Whereas, among different gut bacteria, P. putida attracted maximum number of B. tau females (11.17) and males (8.17) followed by D. acidovorans (10.17 females and 7.33 males). However, these two bacteria were found statistically at par with each other but, inferior to protein hydrolyzate and superior to other treatments. All the bacterial attractants were significantly superior over negative control (sugar) for both the sexes, while more number of females of B. tau were attracted as compared to males in all the treatments (Table 4.13). Table 4.13: Attractancy of promising gut bacteria isolates to B. tau (Walker) Sr. No. Treatments Fruit flies visit /30 min Female* Male* Adult* 1 Deftia acidovorans P1B (2 ml) Pseudomonas putida P3A (2 ml) Flavobacterium sp. P10A (2 ml) Defluvibacter sp. B4A (2 ml) Ochrobacter sp. B10B (2 ml) Control (Sugar, 2 ml 10%) Control (ProteinX, 2 ml 10%) CD *Mean of six replication Gut bacteria were highly attractive to adult fruit flies when compared to control (sugar). The number of females attracted to different bacterial isolates was significantly higher as compared to the males. These findings are similar to those obtained in a laboratory experiment by Jang and Nishijima (1990). They studied the attractancy of bacteria and PIB-7 (protein hydrolyzate) and observed

92 142 significantly higher response of flies to the bacteria in the absence of PIB-7, but relatively lower response of flies to bacteria alone when PIB-7 was also a treatment. The attractancy of gut bacteria to fruit flies can be exploited as an important link in its effective management. Drew (1987) also proposed that bacterial volatiles are important attractants in Dacini and serve as a feeding attractant to females and a sex attractant to mature males. Present findings draw considerable support from earlier observations of Sood and Nath (1998) and Sood et al. (2010), who also reported the attractiveness of gut bacteria to adult flies (both males and females). 4.8 GCMS analysis of Gut bacterial metabolite Five promising gut bacteria of fruit fly B. tau were subjected to Gas Chromatography and Mass Spectrometry for indentifying the volatile chemicals formed after bacterial growth in culture media. Overall, 22 volatile chemicals were identified from five bacterial isolates (Table 4.14 and Plate 4.24). The main volatile components were cedrol, caryophyllene oxide and (Z) 9-tricosene. The former two were produced by D. acidovorans (P1B), Flavobacterium sp. (P10A) and Defluvibacter sp. (B4A) in culture media however, the area of detection varied from 4.26 to 7.40 per cent in different bacterial cultures. (Z) 9-tricosene was produced solely by P. putida (P3A) with per cent area of detection. The volatile chemicals (Z-(9)-tricosene, cedrol and chryophllene oxide) are known to be associated with insect chemical communication behavior in Musca domestica, Cryptomeria bark borer and Compoletis sonorensis, respectively. The chemical characteristics of these three chemical compounds along with associated insects are given in Table Certain components of bacterial odours serve as either feeding or ovipositional stimulants (Drew and Lloyd, 1987). Under laboratory conditions, flies frequently return to the same spot, regurgitate and reingest several times (Lloyd, 1988). This behaviour is probably involved in some form of host marking the help of bacterial odours. Robacker and Flath (1995) identified ammonia, trimethylamine, isoamylamine, 2-methyl-

93 143 Table 4.14 : Identified Chemicals in promising gut bacterial culture of B. tau Sr No Retention Index Chemical identified Delftia acidovorans (P1B) Pseudomonas putida (P3A) Flavobacterium sp. (P10A) Defluvibacter sp. Ochrobactrum sp. (B4A) (B10B) Detection Area Detection Area Detection Area Detection Area Detection Area Detection Area Dioxo-bis (pyridine) [tretrafluoro-1,2-ethanediolato] d nd - nd - nd - nd - nd - osmium* N-benzylidene-dimethylammonium chloride d 4.56 nd - nd - nd - nd - nd Stationary Phase* nd - nd - nd - d 2.82 nd - nd Propene, 3-propoxy, allyl n-propyl ether* nd - nd - nd - nd - d 7.26 nd H-indene, 1-methylene* nd - nd - nd - d 4.39 d 9.4 nd Cyclohexasiloxane, dodecamethyl* nd - nd - d 8.57 nd - nd - d Stationary Phase* nd - nd - nd - d 2.73 nd - nd Cycloheptasiloxane, tetradecamethyl d d 1.34 d 9.47 d d d Cyclooctasiloxane, hexadecamethyl d 6.57 d 0.71 d 5.69 d 9.11 d 6.89 d Octadecamethylcyclonanasilioxane nd - d 3.11 nd - d 5.38 nd - d ,1,1,5,7,7,7-Heptamethyl-3,3-bis (trimethylsiloxy) d 3.98 d 1.64 d 3.84 nd - d 4.46 d 0.44 tetrasiloxane Cyclodecasiloxane, Eicosamethyl d 5.39 nd - nd - d 6.24 nd - nd Butyl-2-methyl propyl phthalate d 5.12 nd - nd - nd - nd - nd Triacontane nd - d 4.63 nd - nd - nd - d , 2-Benzenedicarboxylic acid, bis (2-methylpropyl) nd - nd - d 3.33 d 3.25 d 3.42 nd - ester H-purin-6-amine* d 4.22 d 2.72 d 4.39 d 9.73 d 5.4 d Silikonfett SE30* nd - nd - d nd - nd - nd (Z) 9-Tricosene** nd - d nd - nd - nd - nd Tetracosamethylcyclododecasiloxane nd - nd - d 5.71 nd - d 6.82 nd Tetratriacontane nd - nd - nd - nd - nd - d Silikonfett SE30* d 8.16 nd - nd - nd - nd - nd Octadecane* d 8.83 nd - nd - nd - nd - d Cedrol, Caryophyllene oxide *** d 4.26 nd - d 7.40 d 5.33 nd - nd Matairesinol nd - nd - nd - d 3.88 nd - nd GC septum Bleed* nd - d 1.02 nd - nd - nd - d Octadecenamide nd - d 4.66 nd - nd - nd - nd Silikonfett SE30 d 4.32 nd - nd - nd - nd - nd H-purin-6-amine* d 7.36 d 1.92 d 7.25 d d 8 d ,2,2-2H(3)-4-Methoxyphenylethene, (-)- nd - nd - nd - nd - d nd - Nortrachelogenin Dodecanamide nd - d 1.46 nd - nd - nd - nd Behenyl alcohol nd - d 0.78 nd - nd - nd - d Canophyllal nd - nd - nd - d 3.09 nd - nd Cyclohexadecanolide nd - d 1.8 nd - nd - nd - d Hexadecanoic acid, (3-bromoprop-2-ynyl) nd - nd - d 3.8 nd - nd - d n-tetracosane* d 6.96 d d 6.21 d d 8.42 d *GCMS contaminant, **Insect female sex pheromone, ***Insect allomone, d-detected, nd-not detected Control

94 144 Table 4.15: Properties of insect related chemicals identified in GCMS analysis Sr. No 1 Common name Caryophyllene oxide IUPAC name 4,12,12-trimethyl-9- methylene-5- oxatricyclo[ ]4, 6)]dodecane Chemical formula Molecular weight Chemical structure Chemical reported from insect species C 15H 24O Campoletis sonorensis (Cameron) (Hymenoptera: Ichneumonidae) Elzen et al. (1984) European grapevine moth (Lobesia botrana Denis & Schiffermüller) (Lepidoptera: Tortricidae) Katerinopoulos et al. (2005) Tasin et al. (2006) 2 Cedrol 2,6,6,8- Tetramethyltricyclo[ ]undecan-8-ol Allomone C 15H 26O Cryptomeria bark borer (Semanotus japonicus Lacordaire) (Coleoptera: Cerambycidae) Yatagai et al. (2002) Allomone 3 Muscalure (Z)-9-Tricosene C 23H House fly ( Musca domestica Linnaeus) (Diptera: Muscidae) Chapman et al. (1998) Female sex pheromone