ANTENNAL MORPHOLOGY AND SENSILLUM DISTRIBUTION IN PISSODES VALIDIROSTRIS (COLEOPTERA: CURCULIONIDAE)

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1 Indian J. Agric. Res.., 46 (1) : 7-74, 212 AGRICULTURAL RESEARCH COMMUNICATION CENTRE / indianjournals.com ANTENNAL MORPHOLOGY AND SENSILLUM DISTRIBUTION IN PISSODES VALIDIROSTRIS (COLEOPTERA: CURCULIONIDAE) Hongying Zhao, Zhiying Wang, Yu Gao 1, and Shilin Ge Northeast Forestry University, Harbin, Heilongjiang, 15 4, China Received : Accepted : ABSTRACT The antenna and antennal sensilla of Scotts pine cone weevil Pissodes validirostris were examined with scanning electron microscopy. The antenna of adult cone weevil is geniculate, composed of a scape, a pedicle and a flagellum. The flagellum has seven segments. Six types of antennal sensilla were found in an antenna: trichodea, chaetica, scolopalia, palmate, basiconica and Böhm bristles. Sensilla palmate are most abundant. Sensilla scolopalia () have three subtypes: I and II were found verticulate in pedicle and flagellum, while III are found in scape. Sensilla palmate, sensilla chaetica and sensilla scolopalia are found in the distal end of the seventh segment of flagella. Sensilla basiconica and Böhm bristles are found at the base of pedicles. There were no sexual differences in sensillum morphology, type and distribution. Key words: Scotts pine, Cone weevil, Antennal sensilla, Scanning electron microscopy. INTRODUCTION Cone weevil (Pissodes validirostris) is the major cone pest of Scotts pine (Pinus sylvestris var. mongolica). Overwintering adults of cone weevils feed on young branches. They also consume biannual elongating cones when they are nutrient and water rich. Adult weevils lay eggs on biannual cones. Larvae of stages 1-5 consume and damage cone tissue (Yue et al., 199). Weevil infestation is usually controlled by pesticides and weevil natural enemies (Wang et al., 199). Sometimes entrapping methods based on chemotaxis of the pests towards plant volatile products are also used. During searching for food, partners and breeding places, cone weevils rely on antennae to feel, distinguish and select environmental chemical signals (Yan et al.,23). Therefore, it is of practical interest to know the structure, type and function of antennal sensilla. Here, the structure, number and distribution of sensilla from cone weevils of Scotts pine are described. MATERIALS AND METHODS Insects: Cones of Scotts pine were collected from an Inner Mongolian Forest Corporation field. Pupae of weevils were collected by dissecting the cone, cultured at 25-2 C with % humidity until emergence of adults. Sample preparation for scanning electron microscopy: Antennae of male and females were removed under stereoscope, washed in distilled water with ultrasound. After drying, antennae were fixed with 2.5% glutaric dialdehyde in phosphate buffer (ph 7.4) for 12 h, washed 3 times with phosphate buffer, and briefly rinsed with distilled water, dehydrated in ethanol series (3%, 5%, 7%, %, 9%, 95%), for 1 minutes in each dilution. The antennae were finally washed 2 times for 2 minutes. After drying at room temperature, they were mounted with conducting resin, sprayed with gold on magnetron sputtering system. Electron microscopy and measurement: Samples were examined and photographed under FEI- QUANTA-2 and KYKY-EM32 scanning electron microscope at 2 KV. Sensilla were identified according to Schneider (1964) and Keil (1999). Sizes of antenna and sensilla were measured with image analysis software V5.2. The number of sensilla was counted manually. Data were analyzed with SPSS. Images were processed with Photoshop.. 1 Tea Institute of Chinese Agriculture Science Academy, Hangzhou, Zhejiang, 31, China.

2 RESULTS AND DISCUSSION Antenna morphology: Cone weevil antennae were geniculate, composed of a scape, a pedicel, and a flagellum with 7 segments (Fig 1). There were no differences in length and diameter. An antenna was about 1.3 mm in length (Table 1). The scape is 511 um in length and 42 m in diameter at the base. The pedicle is 15 m in length and m in diameter at the base. The flagella are 7 m in length. The last segment was enlarged, forming a club, with a diameter of 213 m. Antenna sensillum types and distribution: Six types of sensilla were observed: trichodea, chaetica, palmate, scolopalia, basiconica, and Böhm bristles. Except the membrane between segments of flagella, all other areas are full of sensilla of various types. The density of sensilla increases from proximal to distal ends. The last segment of flagella has the most sensillum types and numbers. No clear sexual differences were observed in the type, number and density of sensilla. Sensilla trichodea (St): Sensilla trichodea are found at the end of the seventh segment. They have smooth surfaces, a sharp tip and arc-like morphology. They appear from a socket with a smooth mouth (Fig 2A). Sensilla trichodea are m in length, with a diameter of 2 m at the base. In an area of 1 m diameter, there were 1 sensilla trichodea (Table 2, 3). Length (μm) Vol. 46, No. 1, Sensilla chaetica, (Sc): Sensilla chaetica are found on the club of flagellum. They are 1-36 m in length and 3 m in diameter protruding from mortar-like cavity. These slim structures are arclike with a blunt tip and longitudinal grooves on the surface (Fig 2B). On average, three sensilla chaetica may be found within an area of 1 m in diameter (Table 2, 3). Sensilla scolopalia (): Sensilla scolopalia are found along an entire antenna. attached to sockets, parallel to antennal surface with tip pointing to the distal ends. The surface of sensilla scolopalia looks like scales, but narrower, with obvious longitudinal grooves. These sensillla have three subtypes. I whorl around the proximal of the last flagellum segment and the distal end of pedicel or scape. They are m in length and 4 m at the proximal end but wider at the distal end with tooth protrusions (Fig. 2C). There are 3 I in an area of 1 m in diameter (Table 2, 3). II appear verticillately in the proximal and middle of flagella. They are ìm in length and 3 m in diameter, topologically similar to I but slim and short (Fig. 2C, D). Twelve II are usually found in an area of 1 m diameter (Table 2, 3). III are distributed in the middle and distal of pedicle, sickle looking, wider in the middle with a sharp tip. III are 2-4 m long and 3 m in diameter at the base (Fig. 2E). Four III are usually found in an area of 1 m diameter (Table 3, 4). TABLE 1: Measurements of antennae from Scotts pine cone weevil (n=1) a. Scape ±2.56 Base diameter (μm) b 42.15±.46 Distal diameter (μm) c 11.5 ±.32 Pedicle ± ± ±.3 Flagellum F1 F2 F3 F4 F5 F6 F ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±.52 NOTE: a Data shown were mean ±S.E. b Base diameter. Except the last segment, all other segments were measured at the narrowest position. c Values were the diameter at the widest position, except segments of flagella, where distal diameters were shown. TABLE 2: Base diameter and density of antennal sensilla in cone weevils of Scotts pine (n=12) a. Sensillum type Sc St Sp Sb Bs!c a!c b! Base diameter ( m) 2.49±.2 1.6±.1 1.1±.1 1.± ±. 4.4± ± ±.1 Density b 3.41± ± ± ± ± ± ±. 4.42±.15 NOTE: a Data shown are mean±s.e. b The number of sensilla in a area of 1 m in diameter. c n =4.

3 72 INDIAN JOURNAL OF AGRICULTURAL RESEARCH Scape Pedicle Sensilla basiconica (Sb): Sensilla basiconica are found in the junction between scape and pedicle, growing out of sockets, perpendicular to the surface of antenna. Sb are burly, straight and smooth with blunt tip (Fig. 2G). They are 6 m in length and 2 m in diameter. Usually 2 Sb can be found in an area of 1 m diameter (Table 2, 3). Sensilla palmate (Sp): Sensilla palmate are found in the distal of the club. They appeared from sockets, verticillate, parallel to the antennal surface, pointing to the distal end of antenna. They grew wider from the base to the tip, with longitudinal grooves, and 4-5 fingers at the end (Fig. 2F). Sp were 3-59 m long and 2 m in diameter. There are usually 96 Sp in a area of 1 m diameter (Table 2, 3). Böhm bristles (Bb): Böhm bristles grow out of sockets. They looked like thorns with smooth surface, appearinf in clusters at the junction of scape and pedicle, perpendicular to the surface of antenna (Fig. 2H). Sb were 7 m long and 2 m in diameter. The average density of (Fig G) Sb was three per 1 m diameter area (Table 2, 3). Discussion: Six types of antennal sensilla are found in Scotts pine cone weevil: trichodea, chaetica, scolopalia, palmate, basiconica and Böhm bristles. The types and topology of antennal sensilla in Scotts pine cone weevil were very different from those in other insects of hymenoptera and lepidoptera reported by others, but are very similar to those found in coleopteran (Bland, 191; Sald et al., 23). They Flagellum St Sc Sp ? I -? II -? III 1.9±. 59 TABLE 3: Distribution of antennal sensilla in Scotts pine cone weevil (n=1) a. 13.6±1. 4.1±.3 5.6± ± ±. 4.±. 6.±. 7.1±.2 6.±. 46.4±.4 3.4± ± ± ± ± ± ± ± ± Total 64.± ± ± ± ± ±.5 9 Sb - 2.4± ±.27 Böh m - 2.± ±.39 NOTE: a Data shown are mean ±S.E. - Corresponding sensilla do not exist. are more similar to those found in Possides nitidus. Sensilla palmate are unique structure, which were only found in weevils of the same genus (Peng, 29). These observations indicate that not only the sibling species have similar morphology and behavior, but also similar sensillum types and distribution. Sensilla in Scotts pine cone weevils are morphologically and structurally similar to that in pine weevils, the hosts of these weevils were also closely related. Further studies such as electrophysiological responses were required to establish whether their functions were also identical or not. Sensillum types and structures have been reported in many species. It was clear that there were species difference and sexual difference in the types of sensilla. For example, sexual dimorphism of sensilla has been reported in Holotrichia diomphalia (Su, 27) provide space of coleopteran. In cone weevils of Scotts pine, there were no clear difference between males and females in antennal topology, sensillum types, sizes, diameters and numbers. This was similar to the observation in pine weevils (Peng, 29). The structure and function of sensilla trichodea, chaetica, scolopalia and Böhm s bristles have been reported widely (Ma and Du, 2;). But there were few reports about sensilla scolopalia. Sensilla scolopalia were believed to be typical sound sensors (Schneider, 1964). Cone weevils of Scotts pine were very sensitive to sound. This might be the

4 Vol. 46, No. 1, St Sc (A) (B) (C) (D) Sp? (E) (F) Sb Bb (G) (H) FIG 2: Electron micrograph of antennal sensilla in cone weevils of Scotts pine. (A) Sensilla trichodea(st). (B) Sensilla chaetica(sc). (C)Sensilla scolopalia(i,aii). (D)Sensilla scolopalia(ai). (E)Sensilla scolopalia(biii). (F) Sensilla palmate(sp). (G)Sensilla basiconica (Sb). (H)Böhm bristles (Bb).

5 74 INDIAN JOURNAL OF AGRICULTURAL RESEARCH (A) (B) FIG 1: Electron micrograph of Scotts pine cone weevil antenna. (A) The dorsal view of a female weevil antenna. (B) The ventral view of a male weevil antenna. results of dense presence of sensilla scolopalia. Sensilla palmate were not found in other species except pine weevils. Sensilla palmate have clear longitudinal grooves, which were typical of olfactory function (Zacharuk, 19). Sensilla palmate were most abundant in cone weevils of Scotts pine. Therefore it could be speculate that sensilla palmate were olfactory sensilla, important in host localization. But their exact function requires further studies into the structure and electromagnetic physiology. There were substantial differences in sensillum types, structure and functions among species. Novel sensillum types of insects were being continuously discovered. But studies on electrophysiology of sensilla were not sufficient. The function of many sensillum types awaits further studies. There was no report about antennal sensilla in Scotts pine cone weevils. It is of interest to study the ultrastructure, electrophysiology response, mating and localization mechanism. ACKNOWLEDGMENTS The study was sponsored by the National Forestry Special Project of Scientific Research of Public Benefit Sector of China (No ). REFERENCES Bland, R. G. (191). Antennal sensilla of the adult alfalfa weevil, Hypera postiea (Gyllenhal) (Coleoptera: Curculionidae). Int. J. Insect Morphol.Embryol., 1 : Keil, T. A. (1999). Morphology and development of the peripheral olfactory organs. In: Insect Olfaction. B.S. (Ed.), Springer, Hansson, Heidelberg, Ma, R. Y. and Du, J. W. (2). Sensilla on insect antenna. Chin. J. Entomol Knowl, 37(3): Peng, L. (29). Study on the olfactory location and its chemical Control of Pissodes nitidus. M. D. Thesis. China Said, I., et al. (23). Structure and function of the antennal sensilla of the Palm weevil Rhynchophorus palmarum (Coleoptera: Curculionidae). J. Insect Physiol., 49(9) : Schneider, D. (1964). Insect antennae. Annu. Rev. Entomol., 9 : Su, F., et al. (27). Ultrastructure of olfactory sensilla of Holotrichia diomphalia Bates (Coleoptera: Melolonthidae). Chin. J. Acta Entomol Sin., 5(7): Wang, A. C, et al. (199). Study on the Expert System (PPPES) for the Control and Decision-making of Pissodes validirostris Gyll. Chin. J. Northeast Forestry University, 26(2): Yan, S. C., et al. (23). Advances of studies on the effects of plant volatiles on insect behavior. Chin. J. Appl Ecol., 14(2): Yue, S. K., et al. (199). Biological characteristic of Pissodes validirostris. Cone pest of Pinus sylvestris var (1), Chin Zacharuk, R. Y. (19). Ultrastructure and function of insect chemo sensilla. Ann Rev Ent., 25: