- Email: [email protected]
Morphology of antennal sensilla of the brown spruce longhorn beetle, Tetropium fuscum (Fabr.) (Coleoptera: Cerambycidae)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54
ASD567_proof ■ 28 April 2014 ■ 1/7
Arthropod Structure & Development xxx (2014) 1e7
Contents lists available at ScienceDirect
Arthropod Structure & Development journal homepage: www.elsevier.com/locate/asd
Morphology of antennal sensilla of the brown spruce longhorn beetle, Tetropium fuscum (Fabr.) (Coleoptera: Cerambycidae) Q4
Colin A. MacKay a, b, *, Jon D. Sweeney b, N. Kirk Hillier a a b
Department of Biology, Acadia University, 33 Westwood Ave., Wolfville, Nova Scotia B4P 2R6, Canada Natural Resources Canada, Canadian Forest Service-Atlantic Forestry Centre, PO Box 4000, Fredericton, New Brunswick E3B 5P7, Canada
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 December 2013 Received in revised form 11 April 2014 Accepted 11 April 2014
The antennal sensilla of the brown spruce longhorn beetle, Tetropium fuscum (Fabr.) (Coleoptera: Cerambycidae) were examined with particular focus on the sensilla present on the apical flagellomere. T. fuscum antennae are composed of 11 segments, namely the scape, pedicel, and nine flagellomeres. Nine types of sensilla were observed: three types of sensilla chaetica, sensilla trichodea, two types of sensilla basiconica, grooved peg sensilla, thick-walled sensilla, and Böhm bristles. Seven of these types were present on the apical flagellomere, the exceptions were sensilla chaetica type 3 and Böhm bristles. There were no significant differences in the distribution or density of sensilla present on the ninth flagellomere of males and females, except that males had significantly more sensilla chaetica type 1, which are put forward as the putative contact chemoreceptors for T. fuscum. Ó 2014 Published by Elsevier Ltd.
Keywords: Ultrastructure Morphology Olfactory Gustatory Sensillum
1. Introduction Longhorn beetles respond to olfactory cues such as plant volatiles (Ikeda et al., 1980; Chénier and Philogène, 1989; Hanks, 1999; Allison et al., 2004; Pajares et al., 2004) and pheromones (Lacey et al., 2004, 2007; Ray et al., 2009; Pajares et al., 2010; Hanks and Millar, 2013) when foraging for food, oviposition sites or mates. Tetropium fuscum (F.), as well as congeners Tetropium cinnamopterum Kirby in Richardson and Tetropium castaneum (L.), are attracted to a synthetic blend of spruce volatiles (racemic a-pinene, ()-bpinene, (þ)-3-carene, (þ)-limonene, and a-terpinolene), and that attraction is synergized by the addition of ethanol, which is emitted at greater rates from stressed trees (Sweeney et al., 2004, 2006, 2010). Males of T. fuscum and T. cinnamopterum emit the same aggregation pheromone (fuscumol) that, when combined with spruce volatiles, synergizes attraction of both males and females of those two species (Silk et al., 2007) and T. castaneum (Sweeney et al., 2010). Fuscumol was the first homoterpenoid alcohol to be
Abbreviations: EAG, electroantennogram; GP, grooved peg sensilla; SB, sensilla basiconica; ORN, olfactory receptor neuron; SC, sensilla chaetica; SEM, scanning electron microscopy/micrograph; SSR, single sensillum recording; ST, sensilla tricodea; TEM, transmission electron microscopy/micrograph; TW, thick walled sensilla. * Corresponding author. Department of Biology, Acadia University, 33 Westwood Ave., Wolfville, Nova Scotia B4P 2R6, Canada. E-mail address: [email protected] (C.A. MacKay).
discovered from cerambycids and the first pheromone to be described from the subfamily Spondylidinae (Silk et al., 2007). Traps baited with fuscumol and spruce volatiles have been used in operational surveys to determine the distribution of T. fuscum in Nova Scotia (NS), Canada for regulatory purposes since 2007 (Sweeney et al., 2010), and pheromone-based tactics such as mass trapping and mating disruption may have potential use for suppression of T. fuscum populations. The more fundamentally we understand how insects, particularly longhorn beetles such as T. fuscum, use smell, taste and other senses to locate hosts and mates, the more likely we will develop effective semiochemical-based methods for managing those species that are pests. To this end, our objectives were to describe the morphology of antennal sensilla on T. fuscum, following the methods of Crook et al. (2008a,b) in their examination of the antennal sensilla of the woodwasp Sirex noctilio Fabr. (Hymenoptera: Siricidae), and the emerald ash borer, Agrilus planipennis Fairmaire. We describe the external and internal structure of different types of antennal sensilla observed on T. fuscum, and compare the density of each type of sensillum on the ninth flagellomere of male and female T. fuscum for evidence of sexual dimorphism. We predicted that males would have more contact chemoreceptors at the tips of their antennae than females because males of many cerambycid species, including T. fuscum, recognize conspecific females by antennal contact with specific cuticular hydrocarbons on the surface of the female elytra (Ginzel and Hanks, 2003; Ginzel et al., 2003, 2006; Silk et al., 2011). We also predicted
http://dx.doi.org/10.1016/j.asd.2014.04.005 1467-8039/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: MacKay, C.A., et al., Morphology of antennal sensilla of the brown spruce longhorn beetle, Tetropium fuscum (Fabr.) (Coleoptera: Cerambycidae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.04.005
55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
ASD567_proof ■ 28 April 2014 ■ 2/7
2
C.A. MacKay et al. / Arthropod Structure & Development xxx (2014) 1e7
that females would possess a greater number of olfactory sensilla than males because, although both sexes are synergistically attracted to the combination of host volatiles and fuscumol (the male-emitted pheromone), there is often a significant female bias in trap catches (Sweeney et al., 2010). 2. Methods 2.1. Beetles Sections of the main stem (i.e., bolts about 35 cm long by 18e 25 cm diam.) were cut from red spruce trees in Hemlock Ravine Park, Bedford, NS (44.690278 N, 63.668056 W) in the winter of 2009e2010, incubated at 20e22 C, 40e65% RH, in PlexiglasÒ cages in containment facilities at the Atlantic Forestry Centre, Natural Resources Canada e Canadian Forest Service, Fredericton, New Brunswick (NB), Canada, and checked 5 d per week for emergence of adult T. fuscum. Adults that emerged in the spring of 2010 were examined for antennal deformities before selection, and the largest pieces of frass and other debris were removed with a paintbrush. 2.2. Scanning electron microscopy Ten beetles (five males and five females) were killed by placing them in a 18 C freezer for 4e8 h. Beetles were soaked overnight in a 4% Triton X-100 solution to remove debris and lipids and then dehydrated for 5 min each in 70% ethanol and 95% ethanol (Ray et al., 2006). Following dehydration, specimens were soaked in hexane for 2 h, sonicated (ultrasonic bath) (Branson 5200, Branson Ultrasonic Corp., Danbury, CT) for 30 s in hexane to remove any remaining lipids, and then allowed to air dry. Using a clean, new razor blade, the heads were removed and cut in half. Both halves of each head were mounted on the same aluminum stub using double-sided carbon tape so that antennae were as perpendicular as possible to the stub (Fig. 1a). They were then fixed in place with conductive colloidal graphite with an isopropanol base, coated with carbon using carbon evaporation (Edwards E12E carbon evaporation unit, Crawley, England) and sputter coated with gold (Edwards S150A Sputter coating unit, Crawley, England). Scanning electron microscopy (SEM) was performed using a scanning electron microscope (JEOL JSL 6400,
University of New Brunswick (UNB), Fredericton, NB) at 5 kV. For each specimen, at least three micrographs of each antennal segment were taken around the circumference of the left antenna (right halves reserved as spares) to enable a full 360-degree view. When subsequent examination of images revealed gaps in the circumferential view of a given specimen, additional SEM was performed in the Acadia Centre for Microstructural Analysis (ACMA) lab at Acadia University using a JEOL JSM 5900 LV at 25 kV. Of the 10 beetles that were micrographed using SEM, two antennae of each sex were completely analyzed in a preliminary examination (MacKay, 2010). To increase efficiency and provide comparisons among multiple beetles, data from six other beetles, three male and three female, were collected only from the most distal flagellomere (F9). 2.3. Transmission electron microscopy Antennae from two male and two female T. fuscum were prepared for transmission electron microscopy (TEM) by injecting 0.1 M glutaraldehyde in 0.05 M Na cacodylate buffer, pH 7.4 with 0.1 M sucrose directly into live beetles before antennae were removed and submerged in fixative for 3 h at 20 C (Lucarotti, 2000). Antennae were post-fixed in 1% OsO4 in 0.05 M Na cacodylate buffer pH 7.4, dehydrated and embedded in Epon-Araldite as described by Lucarotti (2000). Serial sections 1 mm thick were cut from the tip of one antenna using a Diatome Histo knife on an RMC MT-7000 ultra microtome. Cuts were made prior to sectioning to expose areas of interest identified from SEM images. Sections were placed on clean, glass slides and stained with toluidine blue (EMS) (Lucarotti et al., 2012). Preliminary TEM work was done using a JEOL 2001 STEM (UNB, Fredericton, NB) at 200 kV. Micrographs were taken at three different locations along F9 (tip, “sensillar field”, midpoint) as well as from the apical face of F8. Further sectioning and TEM was done using a Phillips 301 TEM (ACMA, Acadia University, Wolfville, NS). Sections were taken from the “sensillar field” present on the ninth flagellomere, as well as from the distal face of F8. 2.3.1. Sensilla classification Q1 Sensilla were classified as chaetic, trichoid, or basiconic types based on external morphology from SEM images (Altner and
Fig. 1. aeb. Scanning electron micrographs of (a) mounted male Tetropium fuscum head, lateral view, and (b) female T. fuscum antenna showing scape (S), pedicel (P) and all nine flagellomeres with arrowhead indicating pseudo-segment on distal ninth flagellomere. Orientation bar, A ¼ anterior, V ¼ ventral.
Please cite this article in press as: MacKay, C.A., et al., Morphology of antennal sensilla of the brown spruce longhorn beetle, Tetropium fuscum (Fabr.) (Coleoptera: Cerambycidae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.04.005
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
ASD567_proof ■ 28 April 2014 ■ 3/7
C.A. MacKay et al. / Arthropod Structure & Development xxx (2014) 1e7 Table 1 Mean numbers (SD) of different types of sensilla on the ninth flagellomere of five male and five female Tetropium fuscum, with statistical comparisons (Welch Twosample t-test for unequal variances, N ¼ 5 ea. sex), and pooled male and female mean sensillar lengths (SD) in mm (N ¼ 20 ea. type). Values in square brackets are number of sensilla per mm2 surface area. S. chaetica 3 (SC3) has no values as it was not present on F9. Length measurements were taken from sensilla present on F9 except for SC3, which were taken from the sensilla on the scape. Sensilla type
Number on F9 Males
S. chaetica 1
77.8 (14.38) [0.39] (0.08) S. chaetica 2 268.4 (45.94) [1.34] (0.21) S. chaetica 3 e S. trichodea 89.8 (31.02) [0.45] (0.16) S. basiconica 1 319.4 (16.92) [1.60] (0.14) S. basiconica 2 163.8 (36.95) [0.82] (0.21) Thick walled 9.8 (2.77) [0.05] (0.02) Total 929 (115.4) [4.65] (0.65) 1.4 (1.1) Bifid
Females
t-Value df
p
39.2 (13.16) [0.29] (0.05) 209.4 (66.16) [1.55] (0.38) e 52.6 (9.40) [0.41] (0.13) 255.4 (97.18) [1.83] (0.27) 139.2 (42.03) [1.03] (0.24) 6.2 (3.27) [0.05] (0.02) 702 (220.7) [5.15] (0.98) 2.6 (1.8)
4.43 [2.36] 1.64 [1.06] e 2.57 [0.43] 1.45 [1.70] 0.98 [1.46] 1.88 [0] 2.04 [0.96] e
0.0022 [0.051] 0.14 [0.33] e 0.053 [0.68] 0.22 [0.14] 0.36 [0.18] 0.098 [1] 0.087 [0.37] e
7.94 [6.92] 7.13 [6.31] e 4.73 [7.85] 4.24 [5.81] 7.87 [7.89] 7.79 [7.72] 6.04 [6.97] e
Length (sexes pooled) 64.8 (6.5) 31.6 (4.0) 153.9 (23.7) 34.3 (4.9) 9.1 (0.9) 17.0 (3.1) 2.8 (0.3) e e
3
Prillinger, 1980; Zacharuk, 1980; Keil, 1999), and their possible function (olfactory, gustatory, etc.) was suggested from ultrastructure observed in TEM images and compared with other Coleoptera (Hallberg, 1982; Isidoro et al., 1998; Bartlet et al., 1999; Sen and Mitchell, 2001; Lopes et al., 2002; Crook et al., 2008a,b). Based on ultrastructure, the three main types are: wall pore sensilla, which are typically olfactory; tip pore sensilla, which are typically gustatory or mechanosensory; and aporous sensilla, which are typically mechanoreceptors (Altner and Prillinger, 1980). 2.4. Analysis Scanning electron micrographs were analyzed, and each type of sensilla counted using the image processing software ImageJ (Rasband, 1997). Micrographs of each segment were first visually inspected for points of reference that could be used to delineate the different micrographs so that areas were not counted twice or missed. Color-coded dots were added to the different types of sensilla to avoid counting any sensilla more than once. Each time a dot was added, the program would save the coordinates of each point. The mean number and density of each sensillar type on F9 was compared between male and female T. fuscum using the Welch t-test (R Development Core Team, 2011). Surface area (mm2) of F9 was estimated using the formula for a cylinder (pD L) and a cone
Fig. 2. Ninth flagellomere of a female Tetropium fuscum antenna showing the distribution of s. chaetica types 1 and 2 (SC1, SC2), s. trichodea (ST), and s. basiconica types 1 and 2 (SB1, SB2). The high density SB1 sensillar field is outlined in white.
Please cite this article in press as: MacKay, C.A., et al., Morphology of antennal sensilla of the brown spruce longhorn beetle, Tetropium fuscum (Fabr.) (Coleoptera: Cerambycidae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.04.005
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
ASD567_proof ■ 28 April 2014 ■ 4/7
4
C.A. MacKay et al. / Arthropod Structure & Development xxx (2014) 1e7
(pr S), where D ¼ diameter, L ¼ length, r ¼ radius, and S ¼ length of cone side. 3. Results T. fuscum antennae have 11 segments: scape, pedicel, and nine flagellomeres. F9 appeared on initial inspection to be divided into two different sections (pseudo-segments) (Fig. 1b), but upon SEM examination, what appeared to be a division between flagellomeres was determined to be a grouping of chemoreceptors. Nine different morphological types of sensilla were found on the antennae of T. fuscum. On average, males had more of each type of sensilla than females, but the difference was significant only for SC1 and ST (Table 1). When sensillar density was expressed per mm2 of surface area to account for flagellomere size, results were similar, but only SC1 density was significantly different between males and females (Table 1). Mean surface area of F9 was greater on males than females but not significantly (Welch’s t ¼ 2.59, p ¼ 0.057). Statistical analyses, however, were limited by small sample size. 3.1. Sensilla chaetica type 1 (SC1) SC1 were long, robust, bristle-like sensilla, 64.8 6.5 mm in length (Table 1) that extended roughly perpendicular to the surface of the antennal body except at the tip, where they extended beyond the end of the antenna roughly parallel to the antennal body. On F9, SC1 were distributed sporadically around the circumference proximally, but were most concentrated at the distal tip of the flagellomere (Figs. 2 and 3a). SC1 had pore channels and more innervations than pure mechanoreceptors (Fig. 3b), suggesting they may have some gustatory function as well. 3.2. Sensilla chaetica type 2 (SC2) SC2 were, 31.6 4.0 mm long, robust, bristle-like sensilla (Table 1) and extended from the antenna at roughly a 20 angle (Fig. 3c). On F9, SC2 were distributed evenly around the circumference (Fig. 2). SC2 had no evidence of pore channels and limited innervation (Fig. 3d), suggesting they may be mechanoreceptors. 3.3. Sensilla chaetica type 3 (SC3) SC3 were 153.9 23.7 mm long, robust, bristle-like sensilla (Fig. 3e, Table 1). Based on preliminary data, SC3 were present on all antennomeres except F9, concentrated on the scape, pedicel and dorsal side (MacKay, 2010). The internal ultrastructure of SC3 was not examined.
Fig. 3. (a) Scanning electron micrograph of several sensilla chaetica type 1 (SC1) at the tip of a female Tetropium fuscum antenna. (b) Transmission electron micrograph of a SC1 from the ninth flagellomere of a female T. fuscum with pore channels indicated by white arrowheads. (c) Scanning electron micrograph of two sensilla chaetica type 2 (SC2) at the base of a SC1 on the seventh flagellomere of a female T. fuscum (white arrowheads). (d) Transmission electron micrograph of a SC2 on the ninth flagellomere of a female T. fuscum showing the sensillar lumen (SL) free of dendritic material. (e) Scanning electron micrograph of a sensilla chaetica type 3 (SC3) on the second flagellomere of a female T. fuscum.
be peg-like contact chemoreceptors like those found on the antennae of the emerald ash borer (Crook et al., 2008a). Internal ultrastructural analysis of the sensillar field of the eighth flagellomere revealed that they were in fact multiporous olfactory sensilla (Fig. 5b).
3.4. Sensilla trichodea (ST) ST were 34.3 4.9 mm long, hair-like sensilla that were pointed apically (Table 1), and lay relatively parallel to the body of the antenna (Figs. 2 and 4a). The ST were multiporous (Fig. 4b), suggesting they may be olfactory receptors. 3.5. Sensilla basiconica type 1 (SB1) SB1 were short, peg-like sensilla, 9.1 0.9 mm in length (Table 1). On F9, SB1 were patchily distributed over the surface but were concentrated on the “sensillar field” (Figs. 2 and 5a), a band of SB1 that appeared to create a pseudo-segment (Fig. 1b) as mentioned above. Based on preliminary data, SB1 were present on F1 and F3e9 and were typically concentrated on the distal portions of the ventral side of the segments (MacKay, 2010). During the preliminary morphological survey, SB1 were originally believed to
Fig. 4. (a) Scanning electron micrograph of a sensillum trichodea (ST) (white arrowhead) on the ninth flagellomere of a female Tetropium fuscum. (b) Transmission electron micrograph of a ST from the ninth flagellomere of a female T. fuscum showing pore channels (black arrowheads).
Please cite this article in press as: MacKay, C.A., et al., Morphology of antennal sensilla of the brown spruce longhorn beetle, Tetropium fuscum (Fabr.) (Coleoptera: Cerambycidae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.04.005
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
ASD567_proof ■ 28 April 2014 ■ 5/7
C.A. MacKay et al. / Arthropod Structure & Development xxx (2014) 1e7
5
Fig. 5. (a) Scanning electron micrograph of a field of sensilla basiconica type 1 (SB1) on the ninth flagellomere of a female Tetropium fuscum. (b) Transmission electron micrograph of a sensillum basiconica on the eighth flagellomere of a female T. fuscum with pore channels clearly visible around the circumference (black arrowheads). (c) Scanning electron micrograph of two sensilla basiconica type 2 (SB2) on the ninth flagellomere of a female T. fuscum.
3.6. Sensilla basiconica type 2 (SB2) SB2 were slightly longer (17.0 3.1 mm) than SB1 and less blunt at the tip (Table 1). Based on preliminary data, SB2 were present on F3e9 and were typically concentrated on the distal portions of the ventral side of the segments (MacKay, 2010). On F9, SB2 were patchily distributed, with fewer at the very tip of the flagellomere (Figs. 2 and 5c). SB2 have a similar highly branched dendritic ultrastructure and multiporous walls to SB1, suggesting they are also olfactory sensilla (Fig. 5b). 3.7. Grooved peg sensilla (GP) The distribution of GP sensilla is unknown, but they were confirmed on F8 and F9 from TEM sections (Fig. 6a). Only one GP was observed in the SEM micrographs (Fig. 6b) out of ten specimens.
the same number as Leptura arcuata Panzer and Leptura aethiops Poda (Zhang et al., 2011), and fewer than the yellow longicorn, Phoracantha recurva Newman, which had 12 types (Faucheux, 2011). Slightly more than half of all sensilla present on F9 appeared to be olfactory in nature (SB1, SB2, ST) in both females (64%) and males (62%). The proportion of all sensilla over the entire antenna that are olfactory may be lower because preliminary observations indicated that densities of olfactory sensilla were highest on the distal flagellomeres, specifically F9. Zhang et al. (2011) observed greater numbers of sensilla basiconica on apical than on basal flagellomeres in L. aethiops. T. fuscum had fewer of each type of sensilla than L. aethiops, but their apical flagellomeres are much shorter than those of L. aethiops. For each type of sensilla observed on F9 of T. fuscum, males had 1.2e2.0 times as many as females, but the difference was significant
3.8. Thick walled sensilla (TW) The distribution of TW sensilla is unknown, but they were observed on F8 in TEM sections (Fig 6c). TEM images could not be satisfactorily correlated with the SEM images, but a short (2.8 0.3 mm (Table 1)) nub-like candidate for TW sensilla was found on F9 (Fig 6d). 3.9. Other sensilla Böhm bristles (Fig. 7a) were found in the preliminary examination of the entire antenna at the junctions between the body and scape, and the scape and pedicel (MacKay, 2010), but they were not counted nor was their ultrastructure examined. Y-shaped bifid sensilla (Fig. 7b) were found on F9 of all females and four out of five males examined. Most specimens had only one or two of these sensilla, but one male had three relatively close together (Fig. 7c) and one female had five (Table 1). Due to their low frequency of occurrence, their unknown distribution on F3eF8, and because their ultrastructure was not determined, they are not included as one of the sensillar types described. 4. Discussion We observed nine different types of sensilla on the antenna of T. fuscum, based on morphology and ultrastructure from SEM and TEM imagery, respectively. Compared with other Cerambycidae, T. fuscum had more types of sensilla than the soybean stem borer, Dectes texanus texanus LeConte (which had five) (Crook et al., 2003),
Fig. 6. (a) Transmission electron micrograph of a grooved peg sensillum on the eighth flagellomere of a female Tetropium fuscum. (b) Scanning electron micrograph of a grooved peg sensillum on the ninth flagellomere of a female T. fuscum; (c) Transmission electron micrograph of a thick-walled sensillum on the eighth flagellomere of a female T. fuscum. (d) Scanning electron micrograph of the candidate for thick-walled sensillum on the ninth flagellomere of a female T. fuscum.
Please cite this article in press as: MacKay, C.A., et al., Morphology of antennal sensilla of the brown spruce longhorn beetle, Tetropium fuscum (Fabr.) (Coleoptera: Cerambycidae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.04.005
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
ASD567_proof ■ 28 April 2014 ■ 6/7
6
C.A. MacKay et al. / Arthropod Structure & Development xxx (2014) 1e7
Fig. 7. Scanning electron micrographs of (a) Böhm bristles (BB) present at the junction between the scape (S) and pedicel (P) of a female Tetropium fuscum; (b) Y-shaped bifid sensilla present on the ninth flagellomere of a female T. fuscum; and (c) three bifid sensilla (white arrows) present on the ninth flagellomere of a male T. fuscum.
only for SC1 and ST. When numbers of sensilla were expressed per mm2 surface area, the difference was significant only for SC1. As SC1 appear to be gustatory in function, this supports our hypothesis that males have more gustatory receptors than females. These sensilla may play a role in mate recognition and perception of specific hydrocarbons on the cuticle of female T. fuscum that elicit courtship behavior in T. fuscum (Silk et al., 2011). This type of sensilla chaetica has been observed in other beetles such as the cabbage stem flea beetle, Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae) (Isidoro et al., 1998). SC1 may also be sensitive to host plant compounds, as is the case with P. chrysocephala. SC2 may serve a protective function, as was suggested by Crook et al. (2003) in their examination of the antennae of D. texanus texanus, as well as the typical mechanosensory function of sensilla chaetica (Zhang et al., 2011). Females did not possess significantly more olfactory sensilla than males, contrary to our prediction based on a female bias in traps baited with fuscumol and host volatiles. This suggests that female T. fuscum may differ from males in the number or sensitivity of fuscumol-sensitive olfactory receptor neurons (ORNs) present in the olfactory sensilla or that the sexes differ in the way they process signals from fuscumol and/or host volatile ORNs. Grooved peg sensilla are present in other beetle species (Whitehead, 1981; Hallberg, 1982; Bartlet et al., 1999) and are comparable to what Zhang et al. (2011) described as SB3 in Leptura spp. Based on ultrastructure, they are double-walled s. basiconica, which are most often chemoreceptors, although some also exhibit a thermoreceptive function (Altner and Prillinger, 1980; Bartlet et al., 1999). The thick-walled sensilla candidate is comparable in external morphology to sensilla described on Leptura spp. and termed SB4 by Zhang et al. (2011), but the density of SB4 on Leptura was much greater than that of the thick-walled sensilla we observed on T. fuscum. Also, we observed thick-walled sensilla distributed sporadically on F9 on T. fuscum antennae, whereas Zhang et al. (2011) observed SB4 distributed only on F1 on Leptura spp. A Y-shaped bifid sensillum was observed on the ninth flagellomere of nine out of ten specimens in low, varying densities (1e 5), and its function is unknown. Saïd et al. (2003) found bifid sensilla on the antennae of the palm weevil, Rhynchophorus palmarum (L.) (Coleoptera: Curculionidae), but they were striated and did not show any sign of innervation, leading the authors to speculate a protective function. Bourdais et al. (2006) found morphological alterations, such as bifid sensilla, in individuals of the parasitoid Aphidius rhopalosiphi DeStefani-Peres (Hymenoptera: Braconidae) that were exposed to excessive cold or heat stresses while pupating. As the Y-shaped sensilla on T. fuscum typically resembled basiconic sensilla more than the striated chaetic or trichoid sensilla of R. palmarum, and also because these sensilla were
not observed on all samples, and in varying densities when they were present, it is likely that morphological alterations due to stress during development constitute a plausible explanation for their presence and that they are not a distinct sensillar type. SB1 were the most numerous sensilla present on F9. Along with SB2 and ST, their porous ultrastructure clearly indicates that they are olfactory receptors. Identification of the olfactory receptors of the beetle makes further physiological investigations of these receptors more feasible. Future work using single sensillum recording (SSR) will enable clarification of the role of these olfactory sensilla in processing pheromones, host volatiles, and non-host volatiles. Using electroantennograms (EAGs), Silk et al. (2010) proposed several relevant host and non-host volatiles that elicited strong antennal responses as potentially useful additions to the current trapping lures. Further physiological work should also focus on contact chemoreception, using elytral cuticular hydrocarbons identified by Silk et al. (2011). Acknowledgments We would like to thank Natural Science and Engineering Q2 Research Council, Natural Resources Canada e Canadian Forest Service, Canadian Food Inspection Agency, Canada Foundation for Innovation, Atlantic Canada Opportunities Agency e Atlantic Innovation Fund, and Acadia University for their generous funding. Also, the microscopists at University of New Brunswick Fredericton, S. Belfry and S. Cogswell, as well as H. Xu at ACMA for all their help getting such fantastic SEM and TEM images. We are also grateful to C. Lucarotti for much help with antennal fixing, embedding, antennal sectioning for preliminary light microscopy and TEM examinations, and comments on an earlier draft of this manuscript. We thank J. Simmons and Halifax Regional Municipality for granting us permission to fell beetle-infested trees in Hemlock Ravine Park. Many thanks to W. MacKay, L. Flaherty, C. Hughes, K. van Rooyen, A. Morrison, N. Harn for their assistance rearing beetles. Thanks also to P. Silk, T. Smith, A. Redden, and two anonymous reviewers for comments on an earlier draft of this manuscript and to C. Simpson for editorial review. Q3 References Allison, J.D., Borden, J.H., Seybold, S.J., 2004. A review of the chemical ecology of the Cerambycidae (Coleoptera). Chemoecology 14, 123e150. Altner, H., Prillinger, L., 1980. Ultrastructure of invertebrate chemo-, thermo-, and hygroreceptors and its functional significance. Int. Rev. Cytol. 67, 69e139. Bartlet, E., Romani, R., Williams, I.H., Isidoro, N., 1999. Functional anatomy of sensory structures on the antennae of Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae). Int. J. Insect Morphol. Embryol. 28, 291e300. Bourdais, D., Vernon, P., Krespi, L., Lannic, J.L., van Baaren, J.V., 2006. Antennal structure of male and female Aphidius rhopalosiphi DeStefani-Peres
Please cite this article in press as: MacKay, C.A., et al., Morphology of antennal sensilla of the brown spruce longhorn beetle, Tetropium fuscum (Fabr.) (Coleoptera: Cerambycidae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.04.005
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
ASD567_proof ■ 28 April 2014 ■ 7/7
C.A. MacKay et al. / Arthropod Structure & Development xxx (2014) 1e7 (Hymenoptera: Braconidae): description and morphological alterations after cold storage or heat exposure. Microsc. Res. Tech. 69, 1005e1013. Chénier, J.V.R., Philogène, B.J.R., 1989. Field responses of certain forest Coleoptera to conifer monoterpenes and ethanol. J. Chem. Ecol. 15, 1729e1745. Crook, D.J., Higgins, R.A., Ramaswamy, S.B., 2003. Antennal morphology of the soybean stemborer Dectes texanus texanus LeConte (Coleoptera: Cerambycidae). J. Kans. Entomol. Soc. 76, 397e405. Crook, D.J., Kerr, L.M., Mastro, V.C., 2008a. Distribution and fine structure of antennal sensilla in emerald ash borer (Coleoptera: Buprestidae). Ann. Entomol. Soc. Am. 101, 1103e1111. Crook, D.J., Kerr, L.M., Mastro, V.C., 2008b. Sensilla of the antennal flagellum of Sirex noctilio (Hymenoptera: Siricidae). Ann. Entomol. Soc. Am. 101, 1094e1102. Faucheux, M.J., 2011. Antennal sensilla of the yellow longicorn beetle Phoracantha recurva Newman, 1840: distribution and comparison with Phoracantha semipunctata (Fabricius, 1775) (Coleoptera: Cerambycidae). Bull. Inst. Sci. 33, 19e21. Ginzel, M.D., Blomquist, G., Millar, J.G., Hanks, L.M., 2003. Role of contact pheromones in mate recognition in Xylotrechus colonus. J. Chem. Ecol. 29, 533e545. Ginzel, M.D., Hanks, L.M., 2003. Contact pheromones as mate recognition cures of four species of longhorned beetles (Coleoptera: Cerambycidae). J. Insect Behav. 16, 181e187. Ginzel, M.D., Moreira, J.A., Ray, A.M., Millar, J.G., Hanks, L.M., 2006. (Z)-9-Nonacosenedmajor component of the contact sex pheromone of the beetle Megacyllene caryae. J. Chem. Ecol. 32, 435e451. Hallberg, E., 1982. Sensory organs in Ips typographus (Insecta: Coleoptera) e fine structure of antennal sensilla. Protoplasma 111, 206e214. Hanks, L.M., 1999. Influence of the larval host plant on reproductive strategies of cerambycid beetles. Annu. Rev. Entomol. 44, 483e505. Hanks, L.M., Millar, J.G., 2013. Field bioassays of cerambycid pheromones reveal widespread parsimony of pheromone structures, enhancement by host plant volatiles, and antagonism by components from heterospecifics. Chemoecology 23, 21e44. Ikeda, T., Enda, N., Yamane, A., Oda, K., Toyoda, T., 1980. Attractants for the Japanese pine sawyer, Monochamus alternatus Hope (Coleoptera: Cerambycidae). Appl. Entomol. Zool. 15, 358e361. Isidoro, N., Bartlet, E., Ziesmann, J., Williams, I.H., 1998. Antennal contact chemosensilla in Psylliodes chrysocephala responding to cruciferous allelochemicals. Physiol. Entomol. 23, 131e138. Keil, T.A., 1999. Morphology and development of the peripheral olfactory organs. In: Hansson, B.S. (Ed.), Insect Olfaction. Springer-Verlag, Berlin, pp. 5e47. Lacey, E.S., Ginzel, M.D., Millar, J.G., Hanks, L.M., 2004. Male-produced aggregation pheromone of the cerambycid beetle Neoclytus acuminatus acuminatus. J. Chem. Ecol. 30, 1493e1507. Lacey, E.S., Moreira, J.A., Millar, J.G., Ray, A.M., Hanks, L.M., 2007. Male-produced aggregation pheromone of the cerambycid beetle Neoclytus mucronatus mucronatus. Entomol. Exp. Appl. 122, 171e179. Lopes, O., Barata, E.N., Mustaparta, H., Araújo, J., 2002. Fine structure of antennal sensilla basiconica and their detection of plant volatiles in the eucalyptus woodborer, Phoracantha semipunctata Fabricius (Coleoptera: Cerambycidae). Arthrop. Struct. Dev. 31, 1e13. Lucarotti, C.J., 2000. Cytology of Leidyana canadensis (Apicomplexa: Eugregarinida) in Lambdina fiscellaria fiscellaria larvae (Lepidoptera: Geometridae). J. Invertebr. Pathol. 75, 117e125. Lucarotti, C.J., Whittome-Waygood, B.H., Levin, D.B., 2012. Histology of the larval Neodiprion abietis (Hymenoptera: Diprionidae) digestive tract. Psyche 2012, 10.
7
MacKay, C., 2010. A Survey of the Antennal Sensilla of Tetropium fuscum (Fabr.) (Coleoptera: Cerambycidae) (BScH thesis). Department of Biology, Acadia University, Wolfville, NS, Canada. Pajares, J., Álvarez, G., Ibeas, F., Gallego, D., Hall, D., Farman, D., 2010. Identification and field activity of a male-produced aggregation pheromone in the pine sawyer beetle, Monochamus galloprovincialis. J. Chem. Ecol. 36, 570e583. Pajares, J.A., Ibeas, F., Díez, J.J., Gallego, D., 2004. Attractive responses by Monochamus galloprovincialis (Col., Cerambycidae) to host and bark beetle semiochemicals. J. Appl. Entomol. 128, 633e638. R Development Core Team, 2011. R: a Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. Rasband, W.S., 1997. ImageJ. U.S. National Institutes of Health, Bethesda, Maryland, USA. Ray, A.M., Lacey, E.S., Hanks, L.M., 2006. Predicted taxonomic patterns in pheromone production by longhorned beetles. Naturwissenschaften 93, 543e550. Ray, A., Millar, J., McElfresh, J., Swift, I., Barbour, J., Hanks, L., 2009. Male-produced aggregation pheromone of the cerambycid beetle Rosalia funebris. J. Chem. Ecol. 35, 96e103. Saïd, I., Tauban, D., Renou, M., Mori, K., Rochat, D., 2003. Structure and function of the antennal sensilla of the palm weevil Rhynchophorus palmarum (Coleoptera, Curculionidae). J. Insect Physiol. 49, 857e872. Sen, A., Mitchell, B.K., 2001. Olfaction in the Colorado potato beetle: ultrastructure of antennal sensilla in Leptinotarsa sp. J. Biosci. 26, 233e246. Silk, P.J., Lemay, M.A., LeClaire, G., Sweeney, J., MaGee, D., 2010. Behavioural and electrophysiological responses of Tetropium fuscum to pheromone and spruce volatiles. Environ. Entomol. 39, 1997e2005. Silk, P.J., Sweeney, J., Wu, J., Price, J., Gutowski, J.M., Kettela, E.G., 2007. Evidence for a male-produced pheromone in Tetropium fuscum (F.) and Tetropium cinnamopterum (Kirby) (Coleoptera: Cerambycidae). Naturwissenschaften 94, 697e 701. Silk, P.J., Sweeney, J., Wu, J., Sopow, S., Mayo, P.D., Magee, D., 2011. Contact sex pheromones identified for two species of longhorned beetles (Coleoptera: Cerambycidae) Tetropium fuscum and T. cinnamopterum in the subfamily Spondylidinae. Environ. Entomol. 40, 714e726. Sweeney, J., De Groot, P., MacDonald, L., Smith, S., Cocquempot, C., Kenis, M., Gutowski, J.M., 2004. Host volatile attractants and traps for detection of Tetropium fuscum (F.), Tetropium castaneum L., and other longhorned beetles (Coleoptera: Cerambycidae). Environ. Entomol. 33, 844e854. Sweeney, J., Gutowski, J.M., Price, J., De Groot, P., 2006. Effect of semiochemical release rate, killing agent, and trap design on detection of Tetropium fuscum (F.) and other longhorn beetles (Coleoptera: Cerambycidae). Environ. Entomol. 35, 645e654. Sweeney, J.D., Silk, P.J., Gutowski, J.M., Wu, J., Lemay, M.A., Mayo, P.D., Magee, D.I., 2010. Effect of chirality, release rate, and host volatiles on response of Tetropium fuscum (F.), Tetropium cinnamopterum Kirby, and Tetropium castaneum (L.) to the aggregation pheromone, fuscumol. J. Chem. Ecol. 36, 1309e1321. Whitehead, A.T., 1981. Ultrastructure of sensilla of the female mountain pine beetle, Dendroctonus ponderosae hopkins (Coleoptera: Scolytidae). Int. J. Insect Morphol. Embryol. 10, 19e28. Zacharuk, R.Y., 1980. Ultrastructure and function of insect chemosensilla. Annu. Rev. Entomol. 25, 27e47. Zhang, J., Guan, L., Ren, B., 2011. Fine structure and distribution of antennal sensilla of longicorn beetles Leptura arcuata and Leptura aethiops (Coleoptera: Cerambycidae). Ann. Entomol. Soc. Am. 104, 778e787.
Please cite this article in press as: MacKay, C.A., et al., Morphology of antennal sensilla of the brown spruce longhorn beetle, Tetropium fuscum (Fabr.) (Coleoptera: Cerambycidae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.04.005
45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88
ASD567_proof ■ 28 April 2014 ■ 1/7
Arthropod Structure & Development xxx (2014) 1e7
Contents lists available at ScienceDirect
Arthropod Structure & Development journal homepage: www.elsevier.com/locate/asd
Morphology of antennal sensilla of the brown spruce longhorn beetle, Tetropium fuscum (Fabr.) (Coleoptera: Cerambycidae) Q4
Colin A. MacKay a, b, *, Jon D. Sweeney b, N. Kirk Hillier a a b
Department of Biology, Acadia University, 33 Westwood Ave., Wolfville, Nova Scotia B4P 2R6, Canada Natural Resources Canada, Canadian Forest Service-Atlantic Forestry Centre, PO Box 4000, Fredericton, New Brunswick E3B 5P7, Canada
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 December 2013 Received in revised form 11 April 2014 Accepted 11 April 2014
The antennal sensilla of the brown spruce longhorn beetle, Tetropium fuscum (Fabr.) (Coleoptera: Cerambycidae) were examined with particular focus on the sensilla present on the apical flagellomere. T. fuscum antennae are composed of 11 segments, namely the scape, pedicel, and nine flagellomeres. Nine types of sensilla were observed: three types of sensilla chaetica, sensilla trichodea, two types of sensilla basiconica, grooved peg sensilla, thick-walled sensilla, and Böhm bristles. Seven of these types were present on the apical flagellomere, the exceptions were sensilla chaetica type 3 and Böhm bristles. There were no significant differences in the distribution or density of sensilla present on the ninth flagellomere of males and females, except that males had significantly more sensilla chaetica type 1, which are put forward as the putative contact chemoreceptors for T. fuscum. Ó 2014 Published by Elsevier Ltd.
Keywords: Ultrastructure Morphology Olfactory Gustatory Sensillum
1. Introduction Longhorn beetles respond to olfactory cues such as plant volatiles (Ikeda et al., 1980; Chénier and Philogène, 1989; Hanks, 1999; Allison et al., 2004; Pajares et al., 2004) and pheromones (Lacey et al., 2004, 2007; Ray et al., 2009; Pajares et al., 2010; Hanks and Millar, 2013) when foraging for food, oviposition sites or mates. Tetropium fuscum (F.), as well as congeners Tetropium cinnamopterum Kirby in Richardson and Tetropium castaneum (L.), are attracted to a synthetic blend of spruce volatiles (racemic a-pinene, ()-bpinene, (þ)-3-carene, (þ)-limonene, and a-terpinolene), and that attraction is synergized by the addition of ethanol, which is emitted at greater rates from stressed trees (Sweeney et al., 2004, 2006, 2010). Males of T. fuscum and T. cinnamopterum emit the same aggregation pheromone (fuscumol) that, when combined with spruce volatiles, synergizes attraction of both males and females of those two species (Silk et al., 2007) and T. castaneum (Sweeney et al., 2010). Fuscumol was the first homoterpenoid alcohol to be
Abbreviations: EAG, electroantennogram; GP, grooved peg sensilla; SB, sensilla basiconica; ORN, olfactory receptor neuron; SC, sensilla chaetica; SEM, scanning electron microscopy/micrograph; SSR, single sensillum recording; ST, sensilla tricodea; TEM, transmission electron microscopy/micrograph; TW, thick walled sensilla. * Corresponding author. Department of Biology, Acadia University, 33 Westwood Ave., Wolfville, Nova Scotia B4P 2R6, Canada. E-mail address: [email protected] (C.A. MacKay).
discovered from cerambycids and the first pheromone to be described from the subfamily Spondylidinae (Silk et al., 2007). Traps baited with fuscumol and spruce volatiles have been used in operational surveys to determine the distribution of T. fuscum in Nova Scotia (NS), Canada for regulatory purposes since 2007 (Sweeney et al., 2010), and pheromone-based tactics such as mass trapping and mating disruption may have potential use for suppression of T. fuscum populations. The more fundamentally we understand how insects, particularly longhorn beetles such as T. fuscum, use smell, taste and other senses to locate hosts and mates, the more likely we will develop effective semiochemical-based methods for managing those species that are pests. To this end, our objectives were to describe the morphology of antennal sensilla on T. fuscum, following the methods of Crook et al. (2008a,b) in their examination of the antennal sensilla of the woodwasp Sirex noctilio Fabr. (Hymenoptera: Siricidae), and the emerald ash borer, Agrilus planipennis Fairmaire. We describe the external and internal structure of different types of antennal sensilla observed on T. fuscum, and compare the density of each type of sensillum on the ninth flagellomere of male and female T. fuscum for evidence of sexual dimorphism. We predicted that males would have more contact chemoreceptors at the tips of their antennae than females because males of many cerambycid species, including T. fuscum, recognize conspecific females by antennal contact with specific cuticular hydrocarbons on the surface of the female elytra (Ginzel and Hanks, 2003; Ginzel et al., 2003, 2006; Silk et al., 2011). We also predicted
http://dx.doi.org/10.1016/j.asd.2014.04.005 1467-8039/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: MacKay, C.A., et al., Morphology of antennal sensilla of the brown spruce longhorn beetle, Tetropium fuscum (Fabr.) (Coleoptera: Cerambycidae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.04.005
55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
ASD567_proof ■ 28 April 2014 ■ 2/7
2
C.A. MacKay et al. / Arthropod Structure & Development xxx (2014) 1e7
that females would possess a greater number of olfactory sensilla than males because, although both sexes are synergistically attracted to the combination of host volatiles and fuscumol (the male-emitted pheromone), there is often a significant female bias in trap catches (Sweeney et al., 2010). 2. Methods 2.1. Beetles Sections of the main stem (i.e., bolts about 35 cm long by 18e 25 cm diam.) were cut from red spruce trees in Hemlock Ravine Park, Bedford, NS (44.690278 N, 63.668056 W) in the winter of 2009e2010, incubated at 20e22 C, 40e65% RH, in PlexiglasÒ cages in containment facilities at the Atlantic Forestry Centre, Natural Resources Canada e Canadian Forest Service, Fredericton, New Brunswick (NB), Canada, and checked 5 d per week for emergence of adult T. fuscum. Adults that emerged in the spring of 2010 were examined for antennal deformities before selection, and the largest pieces of frass and other debris were removed with a paintbrush. 2.2. Scanning electron microscopy Ten beetles (five males and five females) were killed by placing them in a 18 C freezer for 4e8 h. Beetles were soaked overnight in a 4% Triton X-100 solution to remove debris and lipids and then dehydrated for 5 min each in 70% ethanol and 95% ethanol (Ray et al., 2006). Following dehydration, specimens were soaked in hexane for 2 h, sonicated (ultrasonic bath) (Branson 5200, Branson Ultrasonic Corp., Danbury, CT) for 30 s in hexane to remove any remaining lipids, and then allowed to air dry. Using a clean, new razor blade, the heads were removed and cut in half. Both halves of each head were mounted on the same aluminum stub using double-sided carbon tape so that antennae were as perpendicular as possible to the stub (Fig. 1a). They were then fixed in place with conductive colloidal graphite with an isopropanol base, coated with carbon using carbon evaporation (Edwards E12E carbon evaporation unit, Crawley, England) and sputter coated with gold (Edwards S150A Sputter coating unit, Crawley, England). Scanning electron microscopy (SEM) was performed using a scanning electron microscope (JEOL JSL 6400,
University of New Brunswick (UNB), Fredericton, NB) at 5 kV. For each specimen, at least three micrographs of each antennal segment were taken around the circumference of the left antenna (right halves reserved as spares) to enable a full 360-degree view. When subsequent examination of images revealed gaps in the circumferential view of a given specimen, additional SEM was performed in the Acadia Centre for Microstructural Analysis (ACMA) lab at Acadia University using a JEOL JSM 5900 LV at 25 kV. Of the 10 beetles that were micrographed using SEM, two antennae of each sex were completely analyzed in a preliminary examination (MacKay, 2010). To increase efficiency and provide comparisons among multiple beetles, data from six other beetles, three male and three female, were collected only from the most distal flagellomere (F9). 2.3. Transmission electron microscopy Antennae from two male and two female T. fuscum were prepared for transmission electron microscopy (TEM) by injecting 0.1 M glutaraldehyde in 0.05 M Na cacodylate buffer, pH 7.4 with 0.1 M sucrose directly into live beetles before antennae were removed and submerged in fixative for 3 h at 20 C (Lucarotti, 2000). Antennae were post-fixed in 1% OsO4 in 0.05 M Na cacodylate buffer pH 7.4, dehydrated and embedded in Epon-Araldite as described by Lucarotti (2000). Serial sections 1 mm thick were cut from the tip of one antenna using a Diatome Histo knife on an RMC MT-7000 ultra microtome. Cuts were made prior to sectioning to expose areas of interest identified from SEM images. Sections were placed on clean, glass slides and stained with toluidine blue (EMS) (Lucarotti et al., 2012). Preliminary TEM work was done using a JEOL 2001 STEM (UNB, Fredericton, NB) at 200 kV. Micrographs were taken at three different locations along F9 (tip, “sensillar field”, midpoint) as well as from the apical face of F8. Further sectioning and TEM was done using a Phillips 301 TEM (ACMA, Acadia University, Wolfville, NS). Sections were taken from the “sensillar field” present on the ninth flagellomere, as well as from the distal face of F8. 2.3.1. Sensilla classification Q1 Sensilla were classified as chaetic, trichoid, or basiconic types based on external morphology from SEM images (Altner and
Fig. 1. aeb. Scanning electron micrographs of (a) mounted male Tetropium fuscum head, lateral view, and (b) female T. fuscum antenna showing scape (S), pedicel (P) and all nine flagellomeres with arrowhead indicating pseudo-segment on distal ninth flagellomere. Orientation bar, A ¼ anterior, V ¼ ventral.
Please cite this article in press as: MacKay, C.A., et al., Morphology of antennal sensilla of the brown spruce longhorn beetle, Tetropium fuscum (Fabr.) (Coleoptera: Cerambycidae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.04.005
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
ASD567_proof ■ 28 April 2014 ■ 3/7
C.A. MacKay et al. / Arthropod Structure & Development xxx (2014) 1e7 Table 1 Mean numbers (SD) of different types of sensilla on the ninth flagellomere of five male and five female Tetropium fuscum, with statistical comparisons (Welch Twosample t-test for unequal variances, N ¼ 5 ea. sex), and pooled male and female mean sensillar lengths (SD) in mm (N ¼ 20 ea. type). Values in square brackets are number of sensilla per mm2 surface area. S. chaetica 3 (SC3) has no values as it was not present on F9. Length measurements were taken from sensilla present on F9 except for SC3, which were taken from the sensilla on the scape. Sensilla type
Number on F9 Males
S. chaetica 1
77.8 (14.38) [0.39] (0.08) S. chaetica 2 268.4 (45.94) [1.34] (0.21) S. chaetica 3 e S. trichodea 89.8 (31.02) [0.45] (0.16) S. basiconica 1 319.4 (16.92) [1.60] (0.14) S. basiconica 2 163.8 (36.95) [0.82] (0.21) Thick walled 9.8 (2.77) [0.05] (0.02) Total 929 (115.4) [4.65] (0.65) 1.4 (1.1) Bifid
Females
t-Value df
p
39.2 (13.16) [0.29] (0.05) 209.4 (66.16) [1.55] (0.38) e 52.6 (9.40) [0.41] (0.13) 255.4 (97.18) [1.83] (0.27) 139.2 (42.03) [1.03] (0.24) 6.2 (3.27) [0.05] (0.02) 702 (220.7) [5.15] (0.98) 2.6 (1.8)
4.43 [2.36] 1.64 [1.06] e 2.57 [0.43] 1.45 [1.70] 0.98 [1.46] 1.88 [0] 2.04 [0.96] e
0.0022 [0.051] 0.14 [0.33] e 0.053 [0.68] 0.22 [0.14] 0.36 [0.18] 0.098 [1] 0.087 [0.37] e
7.94 [6.92] 7.13 [6.31] e 4.73 [7.85] 4.24 [5.81] 7.87 [7.89] 7.79 [7.72] 6.04 [6.97] e
Length (sexes pooled) 64.8 (6.5) 31.6 (4.0) 153.9 (23.7) 34.3 (4.9) 9.1 (0.9) 17.0 (3.1) 2.8 (0.3) e e
3
Prillinger, 1980; Zacharuk, 1980; Keil, 1999), and their possible function (olfactory, gustatory, etc.) was suggested from ultrastructure observed in TEM images and compared with other Coleoptera (Hallberg, 1982; Isidoro et al., 1998; Bartlet et al., 1999; Sen and Mitchell, 2001; Lopes et al., 2002; Crook et al., 2008a,b). Based on ultrastructure, the three main types are: wall pore sensilla, which are typically olfactory; tip pore sensilla, which are typically gustatory or mechanosensory; and aporous sensilla, which are typically mechanoreceptors (Altner and Prillinger, 1980). 2.4. Analysis Scanning electron micrographs were analyzed, and each type of sensilla counted using the image processing software ImageJ (Rasband, 1997). Micrographs of each segment were first visually inspected for points of reference that could be used to delineate the different micrographs so that areas were not counted twice or missed. Color-coded dots were added to the different types of sensilla to avoid counting any sensilla more than once. Each time a dot was added, the program would save the coordinates of each point. The mean number and density of each sensillar type on F9 was compared between male and female T. fuscum using the Welch t-test (R Development Core Team, 2011). Surface area (mm2) of F9 was estimated using the formula for a cylinder (pD L) and a cone
Fig. 2. Ninth flagellomere of a female Tetropium fuscum antenna showing the distribution of s. chaetica types 1 and 2 (SC1, SC2), s. trichodea (ST), and s. basiconica types 1 and 2 (SB1, SB2). The high density SB1 sensillar field is outlined in white.
Please cite this article in press as: MacKay, C.A., et al., Morphology of antennal sensilla of the brown spruce longhorn beetle, Tetropium fuscum (Fabr.) (Coleoptera: Cerambycidae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.04.005
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
ASD567_proof ■ 28 April 2014 ■ 4/7
4
C.A. MacKay et al. / Arthropod Structure & Development xxx (2014) 1e7
(pr S), where D ¼ diameter, L ¼ length, r ¼ radius, and S ¼ length of cone side. 3. Results T. fuscum antennae have 11 segments: scape, pedicel, and nine flagellomeres. F9 appeared on initial inspection to be divided into two different sections (pseudo-segments) (Fig. 1b), but upon SEM examination, what appeared to be a division between flagellomeres was determined to be a grouping of chemoreceptors. Nine different morphological types of sensilla were found on the antennae of T. fuscum. On average, males had more of each type of sensilla than females, but the difference was significant only for SC1 and ST (Table 1). When sensillar density was expressed per mm2 of surface area to account for flagellomere size, results were similar, but only SC1 density was significantly different between males and females (Table 1). Mean surface area of F9 was greater on males than females but not significantly (Welch’s t ¼ 2.59, p ¼ 0.057). Statistical analyses, however, were limited by small sample size. 3.1. Sensilla chaetica type 1 (SC1) SC1 were long, robust, bristle-like sensilla, 64.8 6.5 mm in length (Table 1) that extended roughly perpendicular to the surface of the antennal body except at the tip, where they extended beyond the end of the antenna roughly parallel to the antennal body. On F9, SC1 were distributed sporadically around the circumference proximally, but were most concentrated at the distal tip of the flagellomere (Figs. 2 and 3a). SC1 had pore channels and more innervations than pure mechanoreceptors (Fig. 3b), suggesting they may have some gustatory function as well. 3.2. Sensilla chaetica type 2 (SC2) SC2 were, 31.6 4.0 mm long, robust, bristle-like sensilla (Table 1) and extended from the antenna at roughly a 20 angle (Fig. 3c). On F9, SC2 were distributed evenly around the circumference (Fig. 2). SC2 had no evidence of pore channels and limited innervation (Fig. 3d), suggesting they may be mechanoreceptors. 3.3. Sensilla chaetica type 3 (SC3) SC3 were 153.9 23.7 mm long, robust, bristle-like sensilla (Fig. 3e, Table 1). Based on preliminary data, SC3 were present on all antennomeres except F9, concentrated on the scape, pedicel and dorsal side (MacKay, 2010). The internal ultrastructure of SC3 was not examined.
Fig. 3. (a) Scanning electron micrograph of several sensilla chaetica type 1 (SC1) at the tip of a female Tetropium fuscum antenna. (b) Transmission electron micrograph of a SC1 from the ninth flagellomere of a female T. fuscum with pore channels indicated by white arrowheads. (c) Scanning electron micrograph of two sensilla chaetica type 2 (SC2) at the base of a SC1 on the seventh flagellomere of a female T. fuscum (white arrowheads). (d) Transmission electron micrograph of a SC2 on the ninth flagellomere of a female T. fuscum showing the sensillar lumen (SL) free of dendritic material. (e) Scanning electron micrograph of a sensilla chaetica type 3 (SC3) on the second flagellomere of a female T. fuscum.
be peg-like contact chemoreceptors like those found on the antennae of the emerald ash borer (Crook et al., 2008a). Internal ultrastructural analysis of the sensillar field of the eighth flagellomere revealed that they were in fact multiporous olfactory sensilla (Fig. 5b).
3.4. Sensilla trichodea (ST) ST were 34.3 4.9 mm long, hair-like sensilla that were pointed apically (Table 1), and lay relatively parallel to the body of the antenna (Figs. 2 and 4a). The ST were multiporous (Fig. 4b), suggesting they may be olfactory receptors. 3.5. Sensilla basiconica type 1 (SB1) SB1 were short, peg-like sensilla, 9.1 0.9 mm in length (Table 1). On F9, SB1 were patchily distributed over the surface but were concentrated on the “sensillar field” (Figs. 2 and 5a), a band of SB1 that appeared to create a pseudo-segment (Fig. 1b) as mentioned above. Based on preliminary data, SB1 were present on F1 and F3e9 and were typically concentrated on the distal portions of the ventral side of the segments (MacKay, 2010). During the preliminary morphological survey, SB1 were originally believed to
Fig. 4. (a) Scanning electron micrograph of a sensillum trichodea (ST) (white arrowhead) on the ninth flagellomere of a female Tetropium fuscum. (b) Transmission electron micrograph of a ST from the ninth flagellomere of a female T. fuscum showing pore channels (black arrowheads).
Please cite this article in press as: MacKay, C.A., et al., Morphology of antennal sensilla of the brown spruce longhorn beetle, Tetropium fuscum (Fabr.) (Coleoptera: Cerambycidae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.04.005
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
ASD567_proof ■ 28 April 2014 ■ 5/7
C.A. MacKay et al. / Arthropod Structure & Development xxx (2014) 1e7
5
Fig. 5. (a) Scanning electron micrograph of a field of sensilla basiconica type 1 (SB1) on the ninth flagellomere of a female Tetropium fuscum. (b) Transmission electron micrograph of a sensillum basiconica on the eighth flagellomere of a female T. fuscum with pore channels clearly visible around the circumference (black arrowheads). (c) Scanning electron micrograph of two sensilla basiconica type 2 (SB2) on the ninth flagellomere of a female T. fuscum.
3.6. Sensilla basiconica type 2 (SB2) SB2 were slightly longer (17.0 3.1 mm) than SB1 and less blunt at the tip (Table 1). Based on preliminary data, SB2 were present on F3e9 and were typically concentrated on the distal portions of the ventral side of the segments (MacKay, 2010). On F9, SB2 were patchily distributed, with fewer at the very tip of the flagellomere (Figs. 2 and 5c). SB2 have a similar highly branched dendritic ultrastructure and multiporous walls to SB1, suggesting they are also olfactory sensilla (Fig. 5b). 3.7. Grooved peg sensilla (GP) The distribution of GP sensilla is unknown, but they were confirmed on F8 and F9 from TEM sections (Fig. 6a). Only one GP was observed in the SEM micrographs (Fig. 6b) out of ten specimens.
the same number as Leptura arcuata Panzer and Leptura aethiops Poda (Zhang et al., 2011), and fewer than the yellow longicorn, Phoracantha recurva Newman, which had 12 types (Faucheux, 2011). Slightly more than half of all sensilla present on F9 appeared to be olfactory in nature (SB1, SB2, ST) in both females (64%) and males (62%). The proportion of all sensilla over the entire antenna that are olfactory may be lower because preliminary observations indicated that densities of olfactory sensilla were highest on the distal flagellomeres, specifically F9. Zhang et al. (2011) observed greater numbers of sensilla basiconica on apical than on basal flagellomeres in L. aethiops. T. fuscum had fewer of each type of sensilla than L. aethiops, but their apical flagellomeres are much shorter than those of L. aethiops. For each type of sensilla observed on F9 of T. fuscum, males had 1.2e2.0 times as many as females, but the difference was significant
3.8. Thick walled sensilla (TW) The distribution of TW sensilla is unknown, but they were observed on F8 in TEM sections (Fig 6c). TEM images could not be satisfactorily correlated with the SEM images, but a short (2.8 0.3 mm (Table 1)) nub-like candidate for TW sensilla was found on F9 (Fig 6d). 3.9. Other sensilla Böhm bristles (Fig. 7a) were found in the preliminary examination of the entire antenna at the junctions between the body and scape, and the scape and pedicel (MacKay, 2010), but they were not counted nor was their ultrastructure examined. Y-shaped bifid sensilla (Fig. 7b) were found on F9 of all females and four out of five males examined. Most specimens had only one or two of these sensilla, but one male had three relatively close together (Fig. 7c) and one female had five (Table 1). Due to their low frequency of occurrence, their unknown distribution on F3eF8, and because their ultrastructure was not determined, they are not included as one of the sensillar types described. 4. Discussion We observed nine different types of sensilla on the antenna of T. fuscum, based on morphology and ultrastructure from SEM and TEM imagery, respectively. Compared with other Cerambycidae, T. fuscum had more types of sensilla than the soybean stem borer, Dectes texanus texanus LeConte (which had five) (Crook et al., 2003),
Fig. 6. (a) Transmission electron micrograph of a grooved peg sensillum on the eighth flagellomere of a female Tetropium fuscum. (b) Scanning electron micrograph of a grooved peg sensillum on the ninth flagellomere of a female T. fuscum; (c) Transmission electron micrograph of a thick-walled sensillum on the eighth flagellomere of a female T. fuscum. (d) Scanning electron micrograph of the candidate for thick-walled sensillum on the ninth flagellomere of a female T. fuscum.
Please cite this article in press as: MacKay, C.A., et al., Morphology of antennal sensilla of the brown spruce longhorn beetle, Tetropium fuscum (Fabr.) (Coleoptera: Cerambycidae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.04.005
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
ASD567_proof ■ 28 April 2014 ■ 6/7
6
C.A. MacKay et al. / Arthropod Structure & Development xxx (2014) 1e7
Fig. 7. Scanning electron micrographs of (a) Böhm bristles (BB) present at the junction between the scape (S) and pedicel (P) of a female Tetropium fuscum; (b) Y-shaped bifid sensilla present on the ninth flagellomere of a female T. fuscum; and (c) three bifid sensilla (white arrows) present on the ninth flagellomere of a male T. fuscum.
only for SC1 and ST. When numbers of sensilla were expressed per mm2 surface area, the difference was significant only for SC1. As SC1 appear to be gustatory in function, this supports our hypothesis that males have more gustatory receptors than females. These sensilla may play a role in mate recognition and perception of specific hydrocarbons on the cuticle of female T. fuscum that elicit courtship behavior in T. fuscum (Silk et al., 2011). This type of sensilla chaetica has been observed in other beetles such as the cabbage stem flea beetle, Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae) (Isidoro et al., 1998). SC1 may also be sensitive to host plant compounds, as is the case with P. chrysocephala. SC2 may serve a protective function, as was suggested by Crook et al. (2003) in their examination of the antennae of D. texanus texanus, as well as the typical mechanosensory function of sensilla chaetica (Zhang et al., 2011). Females did not possess significantly more olfactory sensilla than males, contrary to our prediction based on a female bias in traps baited with fuscumol and host volatiles. This suggests that female T. fuscum may differ from males in the number or sensitivity of fuscumol-sensitive olfactory receptor neurons (ORNs) present in the olfactory sensilla or that the sexes differ in the way they process signals from fuscumol and/or host volatile ORNs. Grooved peg sensilla are present in other beetle species (Whitehead, 1981; Hallberg, 1982; Bartlet et al., 1999) and are comparable to what Zhang et al. (2011) described as SB3 in Leptura spp. Based on ultrastructure, they are double-walled s. basiconica, which are most often chemoreceptors, although some also exhibit a thermoreceptive function (Altner and Prillinger, 1980; Bartlet et al., 1999). The thick-walled sensilla candidate is comparable in external morphology to sensilla described on Leptura spp. and termed SB4 by Zhang et al. (2011), but the density of SB4 on Leptura was much greater than that of the thick-walled sensilla we observed on T. fuscum. Also, we observed thick-walled sensilla distributed sporadically on F9 on T. fuscum antennae, whereas Zhang et al. (2011) observed SB4 distributed only on F1 on Leptura spp. A Y-shaped bifid sensillum was observed on the ninth flagellomere of nine out of ten specimens in low, varying densities (1e 5), and its function is unknown. Saïd et al. (2003) found bifid sensilla on the antennae of the palm weevil, Rhynchophorus palmarum (L.) (Coleoptera: Curculionidae), but they were striated and did not show any sign of innervation, leading the authors to speculate a protective function. Bourdais et al. (2006) found morphological alterations, such as bifid sensilla, in individuals of the parasitoid Aphidius rhopalosiphi DeStefani-Peres (Hymenoptera: Braconidae) that were exposed to excessive cold or heat stresses while pupating. As the Y-shaped sensilla on T. fuscum typically resembled basiconic sensilla more than the striated chaetic or trichoid sensilla of R. palmarum, and also because these sensilla were
not observed on all samples, and in varying densities when they were present, it is likely that morphological alterations due to stress during development constitute a plausible explanation for their presence and that they are not a distinct sensillar type. SB1 were the most numerous sensilla present on F9. Along with SB2 and ST, their porous ultrastructure clearly indicates that they are olfactory receptors. Identification of the olfactory receptors of the beetle makes further physiological investigations of these receptors more feasible. Future work using single sensillum recording (SSR) will enable clarification of the role of these olfactory sensilla in processing pheromones, host volatiles, and non-host volatiles. Using electroantennograms (EAGs), Silk et al. (2010) proposed several relevant host and non-host volatiles that elicited strong antennal responses as potentially useful additions to the current trapping lures. Further physiological work should also focus on contact chemoreception, using elytral cuticular hydrocarbons identified by Silk et al. (2011). Acknowledgments We would like to thank Natural Science and Engineering Q2 Research Council, Natural Resources Canada e Canadian Forest Service, Canadian Food Inspection Agency, Canada Foundation for Innovation, Atlantic Canada Opportunities Agency e Atlantic Innovation Fund, and Acadia University for their generous funding. Also, the microscopists at University of New Brunswick Fredericton, S. Belfry and S. Cogswell, as well as H. Xu at ACMA for all their help getting such fantastic SEM and TEM images. We are also grateful to C. Lucarotti for much help with antennal fixing, embedding, antennal sectioning for preliminary light microscopy and TEM examinations, and comments on an earlier draft of this manuscript. We thank J. Simmons and Halifax Regional Municipality for granting us permission to fell beetle-infested trees in Hemlock Ravine Park. Many thanks to W. MacKay, L. Flaherty, C. Hughes, K. van Rooyen, A. Morrison, N. Harn for their assistance rearing beetles. Thanks also to P. Silk, T. Smith, A. Redden, and two anonymous reviewers for comments on an earlier draft of this manuscript and to C. Simpson for editorial review. Q3 References Allison, J.D., Borden, J.H., Seybold, S.J., 2004. A review of the chemical ecology of the Cerambycidae (Coleoptera). Chemoecology 14, 123e150. Altner, H., Prillinger, L., 1980. Ultrastructure of invertebrate chemo-, thermo-, and hygroreceptors and its functional significance. Int. Rev. Cytol. 67, 69e139. Bartlet, E., Romani, R., Williams, I.H., Isidoro, N., 1999. Functional anatomy of sensory structures on the antennae of Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae). Int. J. Insect Morphol. Embryol. 28, 291e300. Bourdais, D., Vernon, P., Krespi, L., Lannic, J.L., van Baaren, J.V., 2006. Antennal structure of male and female Aphidius rhopalosiphi DeStefani-Peres
Please cite this article in press as: MacKay, C.A., et al., Morphology of antennal sensilla of the brown spruce longhorn beetle, Tetropium fuscum (Fabr.) (Coleoptera: Cerambycidae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.04.005
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
ASD567_proof ■ 28 April 2014 ■ 7/7
C.A. MacKay et al. / Arthropod Structure & Development xxx (2014) 1e7 (Hymenoptera: Braconidae): description and morphological alterations after cold storage or heat exposure. Microsc. Res. Tech. 69, 1005e1013. Chénier, J.V.R., Philogène, B.J.R., 1989. Field responses of certain forest Coleoptera to conifer monoterpenes and ethanol. J. Chem. Ecol. 15, 1729e1745. Crook, D.J., Higgins, R.A., Ramaswamy, S.B., 2003. Antennal morphology of the soybean stemborer Dectes texanus texanus LeConte (Coleoptera: Cerambycidae). J. Kans. Entomol. Soc. 76, 397e405. Crook, D.J., Kerr, L.M., Mastro, V.C., 2008a. Distribution and fine structure of antennal sensilla in emerald ash borer (Coleoptera: Buprestidae). Ann. Entomol. Soc. Am. 101, 1103e1111. Crook, D.J., Kerr, L.M., Mastro, V.C., 2008b. Sensilla of the antennal flagellum of Sirex noctilio (Hymenoptera: Siricidae). Ann. Entomol. Soc. Am. 101, 1094e1102. Faucheux, M.J., 2011. Antennal sensilla of the yellow longicorn beetle Phoracantha recurva Newman, 1840: distribution and comparison with Phoracantha semipunctata (Fabricius, 1775) (Coleoptera: Cerambycidae). Bull. Inst. Sci. 33, 19e21. Ginzel, M.D., Blomquist, G., Millar, J.G., Hanks, L.M., 2003. Role of contact pheromones in mate recognition in Xylotrechus colonus. J. Chem. Ecol. 29, 533e545. Ginzel, M.D., Hanks, L.M., 2003. Contact pheromones as mate recognition cures of four species of longhorned beetles (Coleoptera: Cerambycidae). J. Insect Behav. 16, 181e187. Ginzel, M.D., Moreira, J.A., Ray, A.M., Millar, J.G., Hanks, L.M., 2006. (Z)-9-Nonacosenedmajor component of the contact sex pheromone of the beetle Megacyllene caryae. J. Chem. Ecol. 32, 435e451. Hallberg, E., 1982. Sensory organs in Ips typographus (Insecta: Coleoptera) e fine structure of antennal sensilla. Protoplasma 111, 206e214. Hanks, L.M., 1999. Influence of the larval host plant on reproductive strategies of cerambycid beetles. Annu. Rev. Entomol. 44, 483e505. Hanks, L.M., Millar, J.G., 2013. Field bioassays of cerambycid pheromones reveal widespread parsimony of pheromone structures, enhancement by host plant volatiles, and antagonism by components from heterospecifics. Chemoecology 23, 21e44. Ikeda, T., Enda, N., Yamane, A., Oda, K., Toyoda, T., 1980. Attractants for the Japanese pine sawyer, Monochamus alternatus Hope (Coleoptera: Cerambycidae). Appl. Entomol. Zool. 15, 358e361. Isidoro, N., Bartlet, E., Ziesmann, J., Williams, I.H., 1998. Antennal contact chemosensilla in Psylliodes chrysocephala responding to cruciferous allelochemicals. Physiol. Entomol. 23, 131e138. Keil, T.A., 1999. Morphology and development of the peripheral olfactory organs. In: Hansson, B.S. (Ed.), Insect Olfaction. Springer-Verlag, Berlin, pp. 5e47. Lacey, E.S., Ginzel, M.D., Millar, J.G., Hanks, L.M., 2004. Male-produced aggregation pheromone of the cerambycid beetle Neoclytus acuminatus acuminatus. J. Chem. Ecol. 30, 1493e1507. Lacey, E.S., Moreira, J.A., Millar, J.G., Ray, A.M., Hanks, L.M., 2007. Male-produced aggregation pheromone of the cerambycid beetle Neoclytus mucronatus mucronatus. Entomol. Exp. Appl. 122, 171e179. Lopes, O., Barata, E.N., Mustaparta, H., Araújo, J., 2002. Fine structure of antennal sensilla basiconica and their detection of plant volatiles in the eucalyptus woodborer, Phoracantha semipunctata Fabricius (Coleoptera: Cerambycidae). Arthrop. Struct. Dev. 31, 1e13. Lucarotti, C.J., 2000. Cytology of Leidyana canadensis (Apicomplexa: Eugregarinida) in Lambdina fiscellaria fiscellaria larvae (Lepidoptera: Geometridae). J. Invertebr. Pathol. 75, 117e125. Lucarotti, C.J., Whittome-Waygood, B.H., Levin, D.B., 2012. Histology of the larval Neodiprion abietis (Hymenoptera: Diprionidae) digestive tract. Psyche 2012, 10.
7
MacKay, C., 2010. A Survey of the Antennal Sensilla of Tetropium fuscum (Fabr.) (Coleoptera: Cerambycidae) (BScH thesis). Department of Biology, Acadia University, Wolfville, NS, Canada. Pajares, J., Álvarez, G., Ibeas, F., Gallego, D., Hall, D., Farman, D., 2010. Identification and field activity of a male-produced aggregation pheromone in the pine sawyer beetle, Monochamus galloprovincialis. J. Chem. Ecol. 36, 570e583. Pajares, J.A., Ibeas, F., Díez, J.J., Gallego, D., 2004. Attractive responses by Monochamus galloprovincialis (Col., Cerambycidae) to host and bark beetle semiochemicals. J. Appl. Entomol. 128, 633e638. R Development Core Team, 2011. R: a Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. Rasband, W.S., 1997. ImageJ. U.S. National Institutes of Health, Bethesda, Maryland, USA. Ray, A.M., Lacey, E.S., Hanks, L.M., 2006. Predicted taxonomic patterns in pheromone production by longhorned beetles. Naturwissenschaften 93, 543e550. Ray, A., Millar, J., McElfresh, J., Swift, I., Barbour, J., Hanks, L., 2009. Male-produced aggregation pheromone of the cerambycid beetle Rosalia funebris. J. Chem. Ecol. 35, 96e103. Saïd, I., Tauban, D., Renou, M., Mori, K., Rochat, D., 2003. Structure and function of the antennal sensilla of the palm weevil Rhynchophorus palmarum (Coleoptera, Curculionidae). J. Insect Physiol. 49, 857e872. Sen, A., Mitchell, B.K., 2001. Olfaction in the Colorado potato beetle: ultrastructure of antennal sensilla in Leptinotarsa sp. J. Biosci. 26, 233e246. Silk, P.J., Lemay, M.A., LeClaire, G., Sweeney, J., MaGee, D., 2010. Behavioural and electrophysiological responses of Tetropium fuscum to pheromone and spruce volatiles. Environ. Entomol. 39, 1997e2005. Silk, P.J., Sweeney, J., Wu, J., Price, J., Gutowski, J.M., Kettela, E.G., 2007. Evidence for a male-produced pheromone in Tetropium fuscum (F.) and Tetropium cinnamopterum (Kirby) (Coleoptera: Cerambycidae). Naturwissenschaften 94, 697e 701. Silk, P.J., Sweeney, J., Wu, J., Sopow, S., Mayo, P.D., Magee, D., 2011. Contact sex pheromones identified for two species of longhorned beetles (Coleoptera: Cerambycidae) Tetropium fuscum and T. cinnamopterum in the subfamily Spondylidinae. Environ. Entomol. 40, 714e726. Sweeney, J., De Groot, P., MacDonald, L., Smith, S., Cocquempot, C., Kenis, M., Gutowski, J.M., 2004. Host volatile attractants and traps for detection of Tetropium fuscum (F.), Tetropium castaneum L., and other longhorned beetles (Coleoptera: Cerambycidae). Environ. Entomol. 33, 844e854. Sweeney, J., Gutowski, J.M., Price, J., De Groot, P., 2006. Effect of semiochemical release rate, killing agent, and trap design on detection of Tetropium fuscum (F.) and other longhorn beetles (Coleoptera: Cerambycidae). Environ. Entomol. 35, 645e654. Sweeney, J.D., Silk, P.J., Gutowski, J.M., Wu, J., Lemay, M.A., Mayo, P.D., Magee, D.I., 2010. Effect of chirality, release rate, and host volatiles on response of Tetropium fuscum (F.), Tetropium cinnamopterum Kirby, and Tetropium castaneum (L.) to the aggregation pheromone, fuscumol. J. Chem. Ecol. 36, 1309e1321. Whitehead, A.T., 1981. Ultrastructure of sensilla of the female mountain pine beetle, Dendroctonus ponderosae hopkins (Coleoptera: Scolytidae). Int. J. Insect Morphol. Embryol. 10, 19e28. Zacharuk, R.Y., 1980. Ultrastructure and function of insect chemosensilla. Annu. Rev. Entomol. 25, 27e47. Zhang, J., Guan, L., Ren, B., 2011. Fine structure and distribution of antennal sensilla of longicorn beetles Leptura arcuata and Leptura aethiops (Coleoptera: Cerambycidae). Ann. Entomol. Soc. Am. 104, 778e787.
Please cite this article in press as: MacKay, C.A., et al., Morphology of antennal sensilla of the brown spruce longhorn beetle, Tetropium fuscum (Fabr.) (Coleoptera: Cerambycidae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.04.005
45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88