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Morphology and ultrastructure of antennal sensilla of Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) and their probable functions
Accepted Manuscript Title: Morphology and ultrastructure of antennal sensilla of Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) and their probable functions Author: Tofael Ahmed Tian-tao Zhang Zhen-ying Wang Kang-lai He Shu-xiong Bai PII: DOI: Reference:
S0968-4328(13)00064-4 http://dx.doi.org/doi:10.1016/j.micron.2013.04.003 JMIC 1948
To appear in:
Micron
Received date: Revised date: Accepted date:
9-11-2012 24-3-2013 4-4-2013
Please cite this article as: AHMED, T., ZHANG, T.-t., WANG, Z.-y., HE, K.-l., BAI, S.-x., Morphology and ultrastructure of antennal sensilla of Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) and their probable functions, Micron (2013), http://dx.doi.org/10.1016/j.micron.2013.04.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Morphology and ultrastructure of antennal sensilla of Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) and their probable functions
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Tofael AHMED1, 2, Tian-tao ZHANG1, Zhen-ying WANG1*, Kang-lai HE1, Shu-xiong BAI1
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1.State Key Laboratory for the Biology of the Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
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2. Bangladesh Sugarcane Research Institute Ishurdi-6620, Pabna, Bangladesh;
*Correspondence to:
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Zhen-ying WANG Institute of Plant Protection Chinese Academy of Agricultural Sciences No.2 West Yuanmingyuan Road
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Beijing 100193, CHINA Phone: 86-10-62815945
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Fax: 86-10-62896114 E-mail: [email protected]
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Tofael AHMED and Tian-tao ZHANG contributed equally to this work.
Running Title: Morphology and ultrastructure of antennal sensilla
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Morphology and ultrastructure of antennal sensilla of Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) and their probable functions Abstract
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Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) is a polyembryonic endoparasitoid of the Asian corn borer, Ostrinia furnacalis and the European corn borer, O. nubilalis. To better understand the host location mechanism, we examined the external morphology and ultrastructure of the antennal sensilla of this parasitoid by using scanning and transmission electron microscopy. Antennae of male and female of the M. cingulum are filiform in shape, 5.90 to 6.64 mm in length and consist of scape, pedicel, and flagellum with 39 and 40 flagellomeres, respectively. Cuticular pore and nine types of morphologically distinct sensilla were identified in both sexes, including two types of sensilla chaetica (nonporous), s. trichodea (nonporous), s. basiconica I (nonporous blunt tip), s. basiconica II (porous wall) and basiconica III (nonporous wall) with branched blunt tip, s. coeloconica with finger-like projections, protruded s. campaniform with central tip pore, and plate-like s. placodea (porous). We compared number, morphology, and distribution of sensilla between sexes. S. campaniform and non-porous basiconica type I may play a role in gustatory functions whereas type II, and s. placodea may play a function in detecting odor stimuli due to their pores wall. The sensilla chaetica and s. trichodea may be involved in mechnosensation. S. coeloconica probably plays a role as thermohygro receptor, whilst cuticular pores may detect odor stimuli. No differences in antenna shape and basic structure in the males and females, but male antennae length and width were significantly greater than those of females. Furthermore, males had more placodea than females. The sensilla types, morphology, and structure of both sexes were compared to those found in other parasitic hymenoptera, especially braconid wasps.
1. Introduction
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Kew Words: Macrocentrus cingulum; antennal sensilla; morphology; SEM; TEM; ultrastructure
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The parasitic wasp Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) is a polyembryonic endoparasitoid of the Asian corn borer, Ostrinia furnacalis Guenée (Lepidoptera: Crambidae) and the European corn borer, O. nubilalis Hübner (Edwards and Hopper, 1999; Hu et al., 2003) and is widely distributed in Europe, Japan, Korea and China (Watanabe, 1967). Parasitic effectiveness of M. cingulum depends on location and infection by pathogens and other factors like temperature and humidity. Female M. cingulum oviposits in all instars but prefers second to fourth instars of the larvae. Through polyembryony, each egg can produce an average of 25-26 wasps (Parker, 1931). After parasitization the host larvae consume less maize tissue than unparasitized counterparts, thus causing reduced plant injury (Zhou et al., 2011). Insect antennae detect odor, sound, heat, cold, humidity and tactile information by means of numerous sensilla, which typically occur in the form of hairs, pegs, or plates (Altner and Prillinger, 1980). Parasitic hymenopterans are equipped with specialized antennal sensilla to facilitate their biological functions (Li et al., 2011). Antennae of female hymenoptera are important sensory receptors involved in various behaviors including habitat searching, host
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location, host detection, host recognition, host acceptance, oviposition, mating, and host discrimination (Bin and Vinson, 1986; Isidoro et al., 2001; Vinson, 1976). Several studies have characterized antennal sensilla of braconid wasps by using scanning and transmission electron microscopic techniques. However, similar studies on polyembryonic parasitoids are lacking.
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Parasitoids are directed to their host through a series of physical and chemical cues that narrow the search area (Vinson, 1976). Parasitoids locate suitable hosts by detecting the green leaf volatiles of their preferred plants as chemical cues (Ochieng et al., 2000). In areas closely surrounding the host, parasitoids are reported to use host kairomones and damaged-plant synomones for finally tracking their target (Eller et al., 1988; Turlings et al., 1995). M. cingulum are stimulated by host larval frass in stalk tunnels (Parker, 1931). The aim of this research is to better understand the host locating mechanism of M. cingulum. We investigated antennal morphology, ultrastructure, and distribution of antennal sensilla in male and female parasitoids by using scanning and transmission electron microscopy. The types of antennal sensilla of both sexes of M. cingulum are compared with those of other parasitic hymenoptera and putative functions are discussed with reference to their morphology, distribution, and ultrastructure.
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2. Materials and methods
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2.1. Insects The parasitoid, M. cingulum was collected from O. furnacalis larvae living on corn plants at the Langfang Experiment Station of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, China and reared on O. furnacalis larvae from a laboratory colony. Parasitoid wasps were fed a 20% honey solution and host O. furnacalis were reared on an artificial diet as described by Zhou et al. (1980). They were maintained in a room at 25 ºC with a 16:8, light: dark photoperiod. Parasitism by M. cingulum was carried out as described by Hu et al. (2003).
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2.2. Scanning electron microscope (SEM) Antennae of male and female wasps were excised from heads of freshly emerged parasitoids with fine forceps. The antennae were immersed overnight in CCl4 (Aldrich, Milwaukee, WI) at room temperature and subsequently boiled three times with new CCl4, each time 1 minute. Antennae were dehydrated through a graded ethanol series and then put into a holder with double-sided adhesive tape (dorsal, ventral and profile orientations). Finally, the antennae were sputter-coated with gold/palladium (40:60). Specimens were examined using a scanning electron microscope (FEI Quanta-200F) with accelerating voltage set at 6.0 kV. Images were recorded digitally and stored on a computer. 2.3. Transmission electron microscope (TEM) Antennae were fixed in 2% glutaraldehyde and 1% sucrose in 0.1 M cacodylate buffer for 3 h, briefly washed in 0.1 M cacodylate buffer, and then post-fixed in 1% osmium tetraoxide in 0.1 M cacodylate buffer for 2 h, all at pH 7.0 and room temperature. Sample antennae were embedded in epon after dehydration in a graded series of ethanol. Epon was polymerized at 60ºC for 48 h. Specimens were then transferred to embedding molds with Spur‟s resin and polymerized in an oven set at 60ºC for 24 h. Ultra-thin serial sections of 80 nm were cut on a Leica Super Nova ultramicrotome with a diamond knife and collected on pioloform-coated grids. Sections were contrasted with uranylacetate and stained with 1% toluidine blue. The sample
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grids were observed using a Hitachi H-7500 (Tokyo, Japan) TEM at an accelerating voltage of 6.0 kV. Images were recorded digitally and stored on a computer. 2.4. Terminology of sensilla Identification and classification of the sensilla types and the terminology used in this work is based on the methodology described by Keil (1999) and Zacharuk (1985).
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2.5. Data analysis Sensilla numbers for each segment were counted directly from the computer screen. The antenna length and diameter were measured from digital images by using Adobe Photoshop version CS3. At least six antennae were investigated per sex, and 18 sensilla of each type per segment were examined to calculate means. Data obtained on the distribution and abundance of different types of sensilla on male and female antennae were performed using a software package SPSS Version 16 (http:// www.spss.com). Mean numbers of the different types of sensilla found in male and female wasps were calculated before the independent sample T test was used to determine any significant sexual differences occurred. 3. Results
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3.1. General morphology of the antenna Antennae of both male and female M. cingulum are filiform in shape. For both sexes, the proximal part is composed of a cylindrical scape (length 220.06 ± 0.79 μm in male and 206.24 ± 0.35 μm in female), with a basal radicel fitting into the antennal socket and the barrel shaped pedicel (length 59.14 ± 0.42 μm in male and 56.64 ± 0.24 μm in female). The distal part is the flagellum. There is a sexual dimorphism in the number of flagellomeres; the male flagellum has 39 flagellomeres while that of females has 40. The length of the individual antennomeres in males are somewhat longer than those of females (72.79 to 368.47 um versus 47.44 to 354.04 um, respectively; Table 1). Also, there is a slight sexual difference in flagellar length as that of females measures 5.90 ± 0.05 mm and that of males 6.64 ± 0.01 mm. However, the individual antennomeres of males are longer than those of females (72.79 to 368.47 μm versus 47.44 to 354.04 μm, respectively; Table 1).
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3.2. Sensilla on the antenna Nine types of morphologically distinct sensilla were identified on the antennae of male and female M. cingulum, including two types of sensilla chaetica (CHA), s. trichodea (TRI), three types of s. basiconica (BAS), s. coeloconica (COE), s. campaniform (CAM), and s. placodea (PLA). In addition, cuticular pores were found on the antennae. Below, each type of sensilla, plus the cuticular pore structure is described. 3.2.1. Sensilla chaetica (CHA) Sensilla chaetica I (CHA I) have a smooth surface. These sensilla occur mainly on the basal radicel in both sexes, with a few occurring on the pedicle. They are blunt tipped barrel-shaped and are inserted into the cuticular depression (Fig. 1 inset). The length of these sensilla type ranges from 2.89 to 5.70 μm whereas the basal diameter ranges from 1.18 to 1.51 μm. CHA II has a smooth surface and is distributed on the basal radicel and pedicel for both males and females. They also have a blunt but elongated tip and are positioned in cuticular socket (Fig. 1
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inset). CHA II is relatively longer as compared to CHA I, ranging from 7.44 to 9.89 μm in length and with a narrow basal diameter of 1.1 to 1.30 μm.
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3.2.2. Sensilla basiconica (BAS) These sensilla occur on all flagellomeres of both sexes. They are mostly distributed on the dorsal and latero-ventral side of the antenna. Based on their different morphologies, they are classified into three types, BAS I, II, and III. BAS I is differentiated by from BAS II, and also from s. trichodea, the grooved surface that projects slightly more perpendicular to the antennae than the last mentioned types (Fig. 2 and 3). They gradually curve distally toward the apex of the segment ending with a blunt tip and are fitted with a cuticular socket that ranges from 16.81 to 24.29 μm in length and 2.17 to 2.67 μm in basal diameter. BAS I are characterized by thick non-porous cuticular walls with an inner lumen that is innervated by a various number of dendritic branches, one of which forms a tubular body at the base of the hair shaft (Fig. 4).
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BAS II sensilla have grooved cuticular surfaces penetrated by numerous pores and are slightly enlarged at the base; they curve gradually and have a blunt tip. These sensilla are distributed in a circular arrangement along the distal end of each antennomere (Fig. 3) and range from 31.47 to 55.84 μm in length and 3.03 to 4.51 μm in basal diameter. The porous cuticular walls surrounded numerous dendritic branches within the lumen (Fig. 4). Females had significantly higher number of BAS I than males except for segment 25, 26, 28, and 39 (Table 2). Similarly, females had significantly higher BAS II than males for all flagellar sub-segments except for sub-segments 16 (Table 2). These two types of sensilla are distributed on all flagellar sub-segments in both sexes and most commonly on the mid- and distal region of each sub-segment (Fig. 2 and 3).
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The sensilla BAS III occur only on female antennae. Similar to BAS I, they have a longitudinally grooved shaft that is inserted into a small ear-like socket. They are more perpendicular than BAS I and other sensilla and have apices with two branches (Fig. 2 and 3).
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3.2.3. Sensilla trichodea (TRI) Sensilla trichodea (TRI) are the most abundant type of sensilla, found on all antennal segments of both sexes. These long sensilla have a grooved surface, are tapered in shape, and are widely distributed on the scape, pedicel, and frontal part of the flagella. The base of this sensilla type is fitted into a slightly flexible socket, which is elevated above the cuticle. The sensilla possess a sharp tip that is slightly curved toward the apex of the segment. The non-porous cuticle walls with longitudinal grooves can be observed under the high magnification image in figure 2. This sensillium has a relatively thick wall and a lumen that is devoid of dendrites as revealed from TEM (Fig. 5). They are innervated by a single dendrite terminating in a tubular body that is surrounded by a dendritic sheath at the base of the hair shaft (Fig. 5). 3.2.4. Coeloconica sensilla (COE) Sensilla coeloconica (COE) are found on the mid dorsal surface of all flagellomeres for both sexes except for the most proximal and the last three distal flagellomeres (Table 3). One to four COEs occur on each sub-segment. They are peg shaped and protrude from a pit and range from 1.95 to 2.24 μm in diameter and 3.01 to 4.46 μm in length (Fig. 6). The tip of the peg stalk protrudes like a finger and is surrounded by a donut-shaped groove with an inner and outer diameter of 3.1 and 6.6 μm, respectively (Fig. 6). The number of COE sensilla is significantly higher in male than females for all flagellar sub-segments except 25 and 32 (Table 3).
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3.2.5. Sensilla placodea (PLA) Sensilla placodea (PLA) are long narrow structures that are slightly elevated above the antennal surface. They are smooth and surrounded by grooved gap and cuticular ridges and range from 64.89 to 91.42 μm in length (Fig.2). Generally they are aligned in parallel with the antennal axes and are located between rows of s. tricodea and basiconica. They are unevenly distributed on most flagellar sub-segments and are not found on the basal and apex sub-segments of males. In females, they are absent on the basal and last four proximal sub-segments. There are fewer PLA in proximal compared with mid to distal part of the sub-segments. Females had significantly more PLAs than males for segments 2, 3, and 7, but males had significantly more PLAs for segments 8-16, 18-24, and 26-36 (Table 4).
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3.2.6. Sensilla campaniform (CAM) Sensilla campaniform (CAM) were found on odd number flagellomere (except 1 and 39) in both sexes and flagellomere 37 in females (Table 5). CAM has a dome-shaped structure, fitted into a cuticular socket that protrudes from the center of a disc. The CAM has a pore at the center of the tip (Fig. 7). This dome-shaped disc ranges 0.51 to 0.67μm in width and 0.44 to 0.55 μm in length, and width of the stalk measures 0.70 to 0.93 μm. Male antennae have significantly more CAMs than those of females (Table 4).
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3.2.7. The cuticular pore (CP) Cuticular pores (CP) are found on the flagellar sub-segments of both sexes (Fig. 8) and look like small holes on the epidermis. These structures are more often found at the sprout of the cuticle. Each pore ranged from 0.63 to 0.75 μm in width.
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4. Discussion
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The external morphology and distribution pattern of nine antennal sensilla types, plus one pore structure, was described in male and female M. cingulum recorded in the current study. Prior to this investigation, no ultrastructural information on antennal sensilla was reported neither for M. cingulum nor for any other polyembryonic parasitoid. Antennae of insects typically have three basic segments, i.e. scape with radicel, pedicel, and flagellum (Chapman, 1998; Isidoro et al., 1996). The antenna of M. cingulum is filiform in shape and has a large scape with a radicle inserted into the antennal socket, a shorter pedicel, and a flagellum composed of 39 (male) and 40 (female) flagellomeres. Based on this study, it can be concluded that there are nine types of morphologically distinct sensilla on the antennae of both sexes, which correspond to those described in another closely related species, Apanteles cypris (Zhou et al., 2011). Six of these types correspond to those described in other parasitic hymenoptera (Bleeker et al., 2004; Bourdais et al., 2006; Cônsoli et al., 1999; Dweck and Gadallah, 2008; Gao et al., 2007; Ochieng et al., 2000; Roux et al., 2005). Chemosensory and tactile antennal sensilla have been described in various hymenoptera parasitoids (Barlin and Vinson, 1981; Norton and Vinson, 1974; Ochieng et al., 2000). The two types of s. chaetica observed in this study assumingly correspond with similar types found in other braconid wasps (Ochieng et al., 2000; Zhou et al., 2011). In certain species of Cotesia, however, they are absent (Bleeker et al., 2004; Roux et al., 2005). Due to their location and structure CHAs probably serve a mechanical function (Gao et al., 2007). In particular, it has been suggested that they may act as proprioceptors perceiving antennal movement (Zhou et al., 2011).
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Nonporous thick walled s. trichodea have been reported in other parasitic wasps through TEM studies (Amornsak et al., 1998; Baaren et al., 1996; Cônsoli et al., 1999; Gao et al., 2007; Ochieng et al., 2000; Zhou et al., 2011). The socket-like connection with the cuticle and the spatial arrangement of these sensilla suggest a mechanosensitive function in M. cingulum. A previous study reported about TRI lacking cuticular pores, probably serving an olfactory function (Zhou et al., 2011).
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The s. basiconica types I and II, present in both sexes of M. cingulum, are similar to those previously found in Microplitis croceipes (Ochieng et al., 2000), M. pallidipes (Gao et al., 2007), and A. cypris (Zhou et al., 2011). In the former studies the BAS I was described as fluted basiconic sensilla (Norton and Vinson, 1974), trichodea TP (TP = tip pore) (Bleeker et al., 2004), or curved trichoid formations with an apical pore (Barbarossa et al., 1998) whereas the BAS II category was described as trichodea WP (WP = wall pore) (Bleeker et al., 2004) or as a curved non-fluted BAS (Norton and Vinson, 1974). Differences between BAS I and II in M. cingulum not only concern shape but also dendritic innervations. BAS I has grooves in the surface and an inner lumen surrounded by a thick nonporous wall (Fig. 4), similar to that previously described in M. croceipes (Ochieng et al., 2000) and M. pallidipes (Gao et al., 2007). BAS II on the other hand is encircled by a thick wall with multiple pores, similar to that found by Gao et al. (2007) and Ochieng et al. (2000). BAS I may have a gustative function, since it has four to six dendrites inside the wall (Fig. 4), whereas BAS II presumably have an olfactory function (Baaren et al., 1996; Gao et al., 2007; Ochieng et al., 2000; Zhou et al., 2011). Hansson et al. (1991) confirmed the olfactory function of sensilla basiconica type II in Neodiprion sertifer Geoffroy (Hymenoptera: Diprionidae) by using electrophysiological recordings with sex pheromones.
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Both sexes of M. cingulum have only one type of coeloconica sensillum, occurring in a varying number, i.e. from one to four on each flagellomere. The morphological features of sensilla coeloconica are similar to those found with M. pallidipes (Gao et al., 2007), M. croceipes (Navasero and Elzen, 1991; Ochieng et al., 2000), and within braconids, type I of Cotesia spp. and A. cypris (Bleeker et al., 2004; Zhou et al., 2011). Sensilla coeloconica were categorized as types I and II in the Cotesia spp. and A. cypris in the braconid family (Bleeker et al., 2004; Gao et al., 2007; Roux et al., 2005; Zhou et al., 2011). Other researchers have described coeloconica sensilla using particular terminology, e.g. “small sub-terminal sensilla” (Udayagiri and Jones, 1993), „„bulb sensilla‟‟ (Cave and Gaylor, 1987), and “smooth basiconic sensilla” (Norton and Vinson, 1974). COEs are suggested to have an olfactory function (Altner et al., 1983; Gao et al., 2007; Keil, 1999; Ochieng et al., 2000; Olson and Andow, 1993; Roux et al., 2005; Steinbrecht, 1997). The elongated sensilla placodea are found throughout most parts of the antennae in almost all parasitic hymenoptera (Amornsak et al., 1998; Barlin and Vinson, 1981; Dweck and Gadallah, 2008; Gao et al., 2007; Ochieng et al., 2000; Roux et al., 2005; Zhou et al., 2011). The PLA category is probably involved in olfactory reception used to detect host derived semiochemicals because they have multiple cuticular pores. Electroantennography (EAG) performed from single s. placodea in M. croceipes is reported to respond in a dose dependent manner to plant volatiles (Ochieng et al., 2000). The PLA in M. cingulum have a multiporous cuticular structure similar to that found by Bleeker et al. (2004) and Gao et al. (2007). They likely have an olfactory function.
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One type of campaniform sensilla was found in the current study, present on each of the odd numbered flagellomeres on the antennae of both sexes. The CAM sensilla have previously been found on antennae of several insects, e.g. ground beetles (Must et al., 2006) and four Australian spittlebug species (Liang and Fletcher, 2002). In A. cypris, two types of CAM have been reported, CAM I and CAM II (Zhou et al., 2011). In general, CAM are considered to be mechanoreceptors (Ågren, 1977; Zacharuk, 1985). However, Ochieng et al. (2000) reported that CAM may serve a gustatory role due to the presence of a central porous tip. Dietz et al (1971) actually reported that the porous central tip in the CAM is involved in a gustatory function and is highly susceptible to humidity. The cuticular pores (CP) resemble a small hole on the epidermis. Zhou et al., (2011) described as cuticular pores in A. cypris and suggested that act as olfactory receptors and highly responsiveness to sex pheromone.
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The present finding that male M. cingulum adults have more sensilla placodea on their antennae than females which is a common trend found in other parasitic hymenoptera (Bleeker et al., 2004; Dweck and Gadallah, 2008; Gao et al., 2007; Navasero and Elzen, 1991; Ochieng et al., 2000; van Baaren et al., 1999; Zhou et al., 2011). This trend may indicate a role of the current sensillum type in mediating information about sex pheromones (Zhou et al., 2011). However, the s. placodea are also suggested to serve a role in detecting host plant odors (Barlin and Vinson, 1981; Bleeker et al., 2004; Chapman, 1982).
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The finding that M. cingulum males have significantly longer antennae than females agrees with former findings in other hymenopteran parasitic wasp such as, M. croceipes (Das et al., 2011; Navasero and Elzen, 1991; Ochieng et al., 2000), A. cypris (Zhou et al., 2011), M. pallidipes (Gao et al., 2007), Habrobracon hebetor (Dweck and Gadallah, 2008), and two Cotesia wasps (Bleeker et al., 2004). Interestingly, in this study M. cingulum females had more flagellomeres than males (male 39 and female 40) but males still had longer antennae than females due to the longer length of each flagellomeres. The adaptive significance of this phenomenon might be attributed to a larger surface area enabling more sensory receptors (Dweck, 2009; Schneider, 1964).
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The contact mechano-, chemo-, thermo-, and hygroreceptive sensilla are abundantly distributed on the antenna of M. cingulum in both sexes. Previous studies, suggest that insect behavior including habitat searching, host location, host detection, host recognition, host acceptance, oviposition, mating, and host discrimination essentially depend on the function of the antennal sensilla (Whitman and Eller, 1990). Further studies are required to understand the mechanisms involved in these behavioral responses. Future studies on functional morphology of antennal sensilla of M. cingulum using electrophysiological recordings are necessary to confirm the functions of the sensilla identified in this study. Acknowledgement This work was supported by China Agriculture Research System (CARS-02).The authors gratefully acknowledge Dr. Hellmich, R.L, USDA-ARS, Corn Insects and Crop Genetics Research Unit for comments and suggestions on this manuscript.
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Dweck, H.K.M., 2009. Antennal sensory receptors of Pteromalus puparum female (Hymenoptera: Pteromalidae), a gregarious pupal endoparasitoid of Pieris rapae. Micron 40, 769-774. Edwards, O.R., Hopper, K.R., 1999. Using superparasitism by a stem borer parasitoid to infer a host refuge. Ecol. Entom. 24, 7-12. Eller, F.J., Tumlinson, J.H., Lewis, W.J., 1988. Beneficial arthropod behavior mediated by airborne semiochemicals: source of volatiles mediating the host-location flight behavior of Microplitis croceipes (Cresson) (Hymenoptera: Braconidae), a parasitoid of Heliothis zea (Boddie) (Lepidoptera: Noctuidae). Environ. Entom. 17, 745-753. Gao, Y., Luo, L.Z., Hammond, A., 2007. Antennal morphology, structure and sensilla distribution in Microplitis pallidipes (Hymenoptera: Braconidae). Micron 38, 684-693. Hu, J., Zhu, X.X., Fu, W.J., 2003. Passive evasion of encapsulation in Macrocentrus cingulum Brischke (Hymenoptera: Braconidae), a polyembryonic parasitoid of Ostrinia furnacalis Guenée (Lepidoptera: Pyralidae). J. Insect Physiol. 49, 367-375. Isidoro, N., Bin, F., Colazza, S.,Vinson, S.B., 1996. Morphology of antennal gustatory sensilla and glands in some parasitoid Hymenoptera with hypothesis on their role in sex and host recognition. J. Hym. Res. 5, 206-239. Isidoro, N., Romani, R., Bin, F., 2001. Antennal multiporous sensilla: Their gustatory features for host recognition in female parasitic wasps (Insecta, Hymenoptera: Platygastroidea). Micros. Res. Tech. 55, 350-358. Keil, T.A., 1999. Morphology and development of the peripheral olfactory organs. In: Hansson, B.S. (Ed.), Insect Olfaction. Springer, Heidelberg, pp 5-47. Li, X., Lu, D., Liu, X., Zhang, Q., Zhou, X., 2011. Ultrastructural characterization of olfactory sensilla and immunolocalization of odorant binding and chemosensory proteins from an ectoparasitoid Scleroderma guani (Hymenoptera: Bethylidae) Int. J. Biol. Sci. 7, 848-868. Liang, A.P., Fletcher, M.J., 2002. Morphology of the antennal sensilla in four Australian spittlebug species (Hemiptera: Cercopidae) with implications for phylogeny. Aus. J. Entom. 41, 39-44. Must, A., Merivee, E., MÄNd, M., Luik, A., Heidemaa, M., 2006. Electrophysiological responses of the antennal campaniform sensilla to rapid changes of temperature in the ground beetles Pterostichus oblongopunctatus and Poecilus cupreus (Tribe Pterostichini) with different ecological preferences. Physiol. Entom. 31, 278-285. Navasero, R.C., Elzen, G.W., 1991. Sensilla on the antennae, foretarsi and palpi of Microplities croceipes (Cresson) (Hymenoptera: Braconodae). Proc. Entomol. Soc. Wash. 93, 737-747. Norton, W.N., Vinson, S.B., 1974. A comparative ultrastructural and behavioral study of the antennal sensory sensilla of the parasitoid Cardiochiles nigriceps (Hymenoptera:Braconidae). J. Morph. 142, 329-349. Ochieng, S.A., Park, K.C., Zhu, J.W., Baker, T.C., 2000. Functional morphology of antennal chemoreceptors of the parasitoid Microplitis croceipes (Hymenoptera: Braconidae). Arthropod Struct. Dev. 29, 231-240. Olson, D.M., Andow, D.A., 1993. Antennal sensilla of female Trichogramma nubilale (Ertle and Davis) (Hymenoptera : Trichogrammatidae) and comparisons with other parasitic Hymenoptera. Int. J. Insect Morphol. Embryol. 22, 507-520. Parker, H.L., 1931. Macrocentrus gifuensis Ashmead, a polyembrinic braconid parasite in the European corn borer. Tech. Bull. 230. Roux, O., van Baaren, J., Gers, C., Arvanitakis, L.Legal, L., 2005. Antennal structure and oviposition behavior of the Plutella xylostella specialist parasitoid: Cotesia plutellae. Micros. Res. Tech. 68, 36-44.
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Schneider, D., 1964. Insect antennae. Annu. Rev. Entomol. 9, 103-122. Steinbrecht, R.A., 1997. Pore structures in insect olfactory sensilla: A review of data and concepts. Int. J. Insect Morphol. Embryol. 26, 229-245. Turlings, T.C., Loughrin, J.H., McCall, P.J., Röse, U.S., Lewis, W.J., Tumlinson, J.H., 1995. How caterpillar-damaged plants protect themselves by attracting parasitic wasps. Proc.Natl. Acad. Sci. 92, 4169-4174. Udayagiri, S., Jones, R., 1993. Variation in flight response of the specialist parasitoid, Macrocentrus grandii Goidanich to odours from food plants of its european corn borer host. Entomol. Exp. Appl. 69, 183-193. van Baaren, J., Boivin, G., Le Lannic, J., Nénon, J.P., 1999. Comparison of antennal sensilla of Anaphes victus and A. listronoti (Hymenoptera, Mymaridae), egg parasitoids of Curculionidae. Zoomorphol. 119, 1-8. Vinson, S.B., 1976. Host selection by insect parasitoids. Annu. Rev. Entomol. 21, 109-133. Watanabe, C., 1967. Further revision of the genus Macrocentrus Curtis in Japan, with descriptions of two new species (Hymenoptera,Braconidae). Insecta Matsumurana 30, 1-16. Whitman, D., Eller, F., 1990. Parasitic wasps orient to green leaf volatiles. Chemoecol.1, 69-75. Zacharuk, R.Y., 1985. Antennae and sensilla. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive insect physiology biochemistry and pharmacology, sensory. Oxford,UK. Pergamon, pp. 1-69. Zhou, D.R., Wang, Y.Y., Liu, B.L., Ju, Z.L., 1980. Research on the reproduction of O. furnacalis in a large quantity: an artificial diet and its improvement. Acta Phytophyl. Sin. 7, 113-122.7, 113-122. Zhou, H., Wu, W.-J., Zhnag, Z.-F., Zhnag, Y., 2011. Antennal sensilla of Apanteles cypris Nixon (Hymenoptera: Braconidae), a larval endoparasitoid of Cnaphalocrocis medinalis Guenée (Lepidoptera: Pyralidae). Micros. Res. Tech. 74, 1199-1208.
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Table
Table 1: Morphological characteristics the antenna of male and female M. cingulum*. Female
Mean length of antenna (mm)
6.64 ± 0.01a
5.90 ± 0.05b
Length of antennomere (μm)**
72.79 to 368.47
47.44 to 354.04
Mean diameter of antennomere (μm)
44.85 ± 1.60b
49.91 ± 1.28a
Mean length of scape (μm)
220.06 ± 0.79a
Mean length of pedicel (μm)
59.14 ± 0.17a
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206.24 ± 0.35b 56.64 ± 0.24a
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*Data are presented as mean ± SD, n = 6. Means in rows followed by same letter are not significantly different in independent-sample T test (P >0.05). ** Range of antennomere length; data are not analyzed.
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Table 2: The number of sensilla basiconica types I and II on flagellomeres in male and female antenna of M. cingulum*. No. of Flagellomeres
Basiconica I Male
Basiconica II Female
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17.17 ± 0.75a 17.83 ± 0.75a 17.67 ± 0.52a 17.17 ± 0.41a 17.67 ± 0.52a 17.33 ± 0.82a 17.67 ± 0.52a 18.17 ± 0.75a 18.17 ± 0.41a 18.17 ± 0.41a 17.50 ± 0.84a 17.17 ± 0.98a 18.50 ± 0.55a 18.17 ± 0.75a 18.33 ± 0.52a 18.00 ± 0.63a 20.83 ± 0.98a 20.33 ± 0.82a 22.33 ± 0.52a 22.50 ± 0.84a 24.33 ± 0.82a 20.33 ± 0.82a 20.50 ± 0.55a 20.83 ± 0.98a 20.67 ± 0.82a 18.17 ± 0.41a 16.83 ± 0.75a 17.17 ± 0.75a 18.67 ± 0.82a 18.33 ± 0.52a 17.33 ± 0.82a 17.17 ± 0.98a 18.17 ± 0.41a 17.17 ± 0.98a 17.50 ± 0.55a 17.67 ± 0.52a 18.17 ± 0.75a 17.83 ± 0.75a 17.67 ± 0.82a 14.83 740.00 ± 3.85a followed by same
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1 7.67 ± 0.52b 9.33 ± 0.52a 15.67 ± 0.52b nd 2 7.67 ± 0.52b 9.50 ± 0.55a 15.83 ± 0.40b 3rd 7.83 ± 0.41b 9.67 ± 0.52a 16.33 ± 0.52b th 4 7.67 ± 0.82b 9.33 ± 0.52a 14.67 ± 0.52b th 5 7.83 ± 0.41b 9.83 ± 0.41a 14.83 ± 0.98b 6th 7.83 ± 0.98b 9.50 ± 0.84a 15.67 ± 0.52b th 7 7.67 ± 0.52b 9.33 ± 0.52a 16.33 ± 0.52b 8th 7.67 ± 0.52b 9.50 ± 0.84a 15.67 ± 0.52b th 9 7.83 ± 0.41b 10.33 ± 0.52a 14.83 ± 0.41b 10th 7.67 ± 0.51b 10.50 ± 0.55a 15.67 ± 0.52b th 11 7.83 ± 0.41b 10.33 ± 0.82a 15.33 ± 0.52b th 12 7.83 ± 0.41b 10.50 ± 0.84a 15.67 ± 0.52b 13th 8.17 ± 0.41b 10.67 ± 1.03a 16.33 ± 0.52b th 14 7.83 ± 0.41b 10.50 ± 0.84a 14.33 ± 0.52b 15th 8.83 ± 0.98b 12.50 ± 0.83a 16.33 ± 0.52b th 16 8.33 ± 0.52b 12.67 ± 1.03a 17.83 ± 0.75a 17th 8.67 ± 0.82b 13.17 ± 0.98a 17.83 ± 0.41b 18th 8.83 ± 0.41b 13.50 ± 0.84a 15.67 ± 0.52b th 19 10.33 ± 0.82b 12.83 ± 0.98a 16.67 ± 0.82b 20th 10.5 ± 0.84b 12.67 ± 0.82a 15.67 ± 0.82b st 21 10.33 ± 0.52b 12.83 ± 0.75a 16.83 ± 0.75b 22nd 7.83 ± 0.41b 12.50 ± 0.84a 15.33 ± 1.03b rd 23 7.83 ± 0.41b 10.83 ± 0.75a 15.33 ± 0.52b 24th 7.67 ± 0.82b 10.67 ± 0.75a 15.33 ± 0.82b th 25 10.33 ± 0.82a 10.33 ± 1.03a 15.67 ± 0.52b th 26 8.67 ± 0.82a 9.33 ± 0.82a 16.33 ± 0.52b 27th 8.83 ± 0.41b 9.67 ± 1.03a 15.33 ± 0.52b th 28 8.67 ± 0.52a 9.33 ± 0.52a 15.33 ± 0.82b 29th 8.17 ± 0.41b 9.33 ± 0.52a 15.67 ± 0.52b th 30 8.33 ± 0.52b 9.50 ± 0.52a 15.83 ± 0.41b st 31 7.67 ± 0.82b 9.33 ± 0.55a 14.33 ± 0.52b 32nd 7.33 ± 0.52b 9.67 ± 0.52a 14.83 ± 0.41b rd 33 7.67 ± 0.52b 9.83 ± 0.52a 15.50 ± 0.55b 34th 7.67 ± 0.52b 9.67 ± 0.75a 15.33 ± 0.82b th 35 7.83 ± 0.41b 9.50 ± 0.52a 14.50 ± 0.55b 36th 7.33 ± 0.82b 9.33 ± 0.55a 14.33 ± 0.82b th 37 7.33 ± 0.82b 9.50 ± 0.82a 14.83 ± 0.75b th 38 7.67 ± 0.52b 9.17 ± 0.84a 14.50 ± 0.84b 39th 8.67 ± 0.52a 9.33 ± 0.41a 13.67 ± 0.52b th 40 10.67 Total 320.33 ± 2.66b 416.50 ± 6.63a 606.00 ± 5.40b *Data are presented as mean ± SD, n = 6. Means in rows of each sensilla type letter are not significantly different in independent-sample T test (P > 0.05).
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Table 3: The number of sensilla coeloconica on flagellomeres on antenna of male and female M. cingulum*.
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No. of Male Female Flagellomeres 2nd 2.17 ± 0.41a 1.17 ± 0.41b 3rd 3.17 ± 0.41a 2.17 ± 0.41b th 4 3.33 ± 0.52a 2.17 ± 0.41b 5th 3.83 ± 0.41a 2.17 ± 0.41b th 6 3.67 ± 0.52a 1.83 ± 0.41b th 7 3.83 ± 0.41a 2.17 ± 0.41b 8th 3.83 ± 0.52a 1.83 ± 0.75b th 9 3.17 ± 0.41a 1.83 ± 0.41b 10th 3.83 ± 0.41a 1.67 ± 0.52b th 11 3.17 ± 0.41a 1.33 ± 0.52b th 12 3.67 ± 0.52a 1.33 ± 0.52b 13th 3.67 ± 0.52a 1.33 ± 0.52b th 14 3.67 ± 0.52a 1.17 ± 0.41b 15th 3.33 ± 0.52a 1.33 ± 0.52b th 16 3.67 ± 0.52a 1.33 ± 0.52b 17th 3.33 ± 0.52a 1.67 ± 0.52b th 18 3.67 ± 0.52a 1.67 ± 0.52b th 19 3.17 ± 0.41a 1.67 ± 0.52b 20th 3.17 ± 0.41a 1.67 ± 0.52b st 21 3.17 ± 0.41a 1.33 ± 0.52b 22nd 2.67 ± 0.52a 1.33 ± 0.52b rd 23 2.67 ± 0.52a 1.17 ± 0.41b 24th 2.67 ± 0.52a 1.17 ± 0.41b th 25 2.33 ± 0.52a 1.67 ± 0.52a th 26 2.67 ± 0.52a 1.33 ± 0.52b 27th 2.83 ± 0.41a 1.17 ± 0.41b th 28 3.17 ± 0.41a 1.33 ± 0.52b 29th 3.17 ± 0.41a 1.67 ± 0.52b th 30 2.83 ± 0.41a 1.83 ± 0.41b 31st 2.83 ± 0.41a 1.67 ± 0.52b nd 32 2.17 ± 0.41a 1.67 ± 0.52a rd 33 2.17 ± 0.41a 1.17 ± 0.41b 34th 2.17 ± 0.41a 1.17 ± 0.41b th 35 2.33 ± 0.52a 1.33 ± 0.52b 36th 2.17 ± 0.41a 1.17 ± 0.41b th 37 0 1.17 ± 0.41a Total 107.33 ± 6.38a 54.83 ± 4.58b *Data are presented as mean ± SD n = 6. Means in rows followed by same letter are not significantly different (P > 0.05) in independent-sample T test.
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Table 4: The Number of sensilla placodea on flagellomeres on antenna of male and female M. cingulum*.
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No. of Male Female Flagellomeres 2nd 6.33 ± 0.52b 7.83 ± 0.41a rd 3 5.83 ± 0.41b 9.83 ± 0.41a 4th 9.83 ± 0.41a 8.18 ± 0.41b 5th 7.67 ± 0.52a 8.17 ± 0.41a 6th 7.67 ± 0.52a 8.33 ± 0.52a th 7 8.33 ± 0.52b 9.83 ± 0.41a th 8 9.67 ± 0.52a 7.83 ± 0.41b 9th 9.67 ± 0.52a 7.83 ± 0.41b th 10 9.67 ± 0.52a 7.83 ± 0.41b th 11 10.17 ± 0.41a 8.33 ± 0.52b 12th 9.83 ± 0.41a 7.83 ± 0.41b th 13 8.33 ± 0.52a 6.33 ± 0.82b 14th 8.17 ± 0.41a 6.17 ± 0.41b th 15 8.17 ± 0.41a 6.17 ± 0.41b 16th 9.83 ± 0.41a 5.83 ± 0.41b th 17 6.17 ± 0.41a 6.83 ± 0.75a th 18 9.67 ± 0.52a 5.83 ± 0.41b 19th 7.83 ± 0.41a 6.17 ± 0.41b th 20 7.83 ± 0.41a 5.17 ± 0.98b 21st 5.83 ± 0.41a 4.83 ± 0.41b nd 22 9.83 ± 0.41a 5.17 ± 0.41b rd 23 8.17 ± 0.41a 6.17 ± 0.41b 24th 9.67 ± 0.82a 4.33 ± 0.52b th 25 5.67 ± 0.52a 4.83 ± 0.98a th 26 6.33 ± 0.52a 4.67 ± 0.82b 27th 5.83 ± 0.41a 4.17 ± 0.41b th 28 7.67 ± 0.52a 4.17 ± 0.41b 29th 5.83 ± 0.41a 3.83 ± 0.41b th 30 5.83 ± 0.41a 3.83 ± 0.41b 31st 6.17 ± 0.41a 3.83 ± 0.41b nd 32 5.83 ± 0.41a 2.33 ± 0.52b rd 33 6.17 ± 0.41a 2.17 ± 0.41b 34th 5.83 ± 0.41a 2.17 ± 0.41b th 35 5.67 ± 0.52a 2.17 ± 0.41b 36th 5.17 ± 0.41a 2.17 ± 0.41b th 37 3.67 ± 0.52a 0 th 38 3.67 ± 0.52a 0 Total 273.51 ± 0.1.87a 201.17 ± 4.31b *Data are presented as mean ± SD., n = 6. Means in rows followed by same letter are not significantly different in independent-sample T test (P > 0.05).
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TABLE 5: The Number of Campaniform Sensilla on Flagellomeres in Male and Female M. cingulum*.
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No. of Male Female Flagellomeres 3rd 0.83 ± 0.41a 0.83 ± 0.41a 5th 0.83 ± 0.41a 0.67 ± 0.52a th 7 0.83 ± 0.41a 0.67 ± 0.52a th 9 0.83 ± 0.41a 0.67 ± 0.52a 11th 0.83 ± 0.41a 0.67 ± 0.52a th 13 1.00 ± 0.00a 0.67 ± 0.52a th 15 1.00 ± 0.00a 1.00 ± 0.00a 17th 1.00 ± 0.41a 0.83 ± 0.41a th 19 0.83 ± 0.41a 0.83 ± 0.00a 21st 0.83 ± 0.41a 0.67 ± 0.52a rd 23 0.83 ± 0.41a 0.67 ± 0.52a 25th 0.83 ± 0.00a 0.67 ± 0.52a th 27 0.83 ± 0.41a 0.67 ± 0.52a th 29 0.83 ± 0.41a 0.50 ± 0.55a 31st 0.83 ± 0.41a 0.67 ± 0.52a rd 33 0.83 ± 0.41a 0.50 ± 0.55a 35th 0.83 ± 0.41a 0.50 ± 0.55a th 37 0.67 ± 0.52a 0 Total 15.33 ± 1.03a 11.67 ± 0.82b *Data are presented as mean ± SD., n = 6. Means in rows followed by same letter are not significantly different in independent-sample T test (P > 0.05).
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Figure 5
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Figure 8
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Fig.1 Electromicrograph of sensilla chaetica (CHA) on antenna of M. cingulum; A. Sensilla chaetica I (CHA I) and sensilla chaetica II (CHA II) at the basal radicel. B. CHA I and CHA II at the basal pedicel. High magnification pictures of CHA I and CHA II are shown in the inset.
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Fig.2 The distal part of the 20th antennomere of a female M. cingulum; showing sensilla basiconica I (white arrow), sensilla basiconica II (cross), sensilla basiconica III (open arrow), sensilla trichodea (triangles), sensilla placodea (star), and sensilla coeloconica (lightingbolt).
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Fig.3 SEM micrograph of the last flagellomere on a male antenna of M. cingulum showing sensilla basiconica. A. The distal tip of the flagellum includes sensilla basiconica I (BAS I) and sensilla basiconica II (BAS II). B. Sensilla basiconica III (BAS III) in the distal part of the 20th subsegment. C. As demonstrated BAS I has a smooth wall in the proximal end of the 21st sub-segment. D. BAS II with a blunt tip in the distal region of last flagellars.
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Fig.4 TEM micrograph of basiconica sensilla (BAS). A. Cross section of BAS type II showing dendritic branches (D), sensillium lymph (L), a porous cuticular wall (arrow), and the sensillium wall (W). B. Cross section of BAS type I demonstrating a thick and groove non-porous sensillum wall (W) and the sensillum lymph (L), which is innervated by dendrites (D).
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Fig.5 TEM micrograph of sensilla trichodea (TRI) and sensilla placodea (PLA) of M. cingulum; A. TEM of sensilla tricodea showing sensillium lymph (L) surrounded by a non-porous thick wall. B. Transverse section of sensilla placodea showing dendritic branches (DB) within the median channel and elevated grooves (G), plus the porous sensillum wall (arrow) and the septum (S). Fig.6 SEM micrograph of sensilla coeloconica (COE) on antenna of M. cingulum. A. Distribution of COE (arrow) on the antennomere. B. The characteristic peg like stalk of the COE surrounded by a donut-shaped ring (asterisk) can be seen. In addition, fingerlike projections with a capitate peg are demonstrated (Inset). Fig.7 Electromicrograph of sensilla campaniform (CAM) of M. cingulum. A. The dome shaped CAM sensilla protrude from the center of the disc in the distal region of 9th subsegment. B. The pore (arrow) at the center of the sprout of the CAM can be seen. The CAM pore at high magnification is shown in the inset. Fig.8 SEM image of the socket like structure enveloping the sensilla trichodea on the antennae (A) and the cuticular pores present on the dorsal side of one sub-segment (B).
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Highlights
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SEM and TEM of antennal sensilla of Macrocentrus cingulum Brischke. Cuticular pore and nine types of morphologically distinct sensilla were identified. These sensilla may act as mechano-, chemo-, thermo-, and hygroreceptor in both sexes. The male antennae length and width were significantly greater than those of females.
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S0968-4328(13)00064-4 http://dx.doi.org/doi:10.1016/j.micron.2013.04.003 JMIC 1948
To appear in:
Micron
Received date: Revised date: Accepted date:
9-11-2012 24-3-2013 4-4-2013
Please cite this article as: AHMED, T., ZHANG, T.-t., WANG, Z.-y., HE, K.-l., BAI, S.-x., Morphology and ultrastructure of antennal sensilla of Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) and their probable functions, Micron (2013), http://dx.doi.org/10.1016/j.micron.2013.04.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Morphology and ultrastructure of antennal sensilla of Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) and their probable functions
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Tofael AHMED1, 2, Tian-tao ZHANG1, Zhen-ying WANG1*, Kang-lai HE1, Shu-xiong BAI1
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1.State Key Laboratory for the Biology of the Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
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2. Bangladesh Sugarcane Research Institute Ishurdi-6620, Pabna, Bangladesh;
*Correspondence to:
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Zhen-ying WANG Institute of Plant Protection Chinese Academy of Agricultural Sciences No.2 West Yuanmingyuan Road
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Beijing 100193, CHINA Phone: 86-10-62815945
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Fax: 86-10-62896114 E-mail: [email protected]
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Tofael AHMED and Tian-tao ZHANG contributed equally to this work.
Running Title: Morphology and ultrastructure of antennal sensilla
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Morphology and ultrastructure of antennal sensilla of Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) and their probable functions Abstract
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Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) is a polyembryonic endoparasitoid of the Asian corn borer, Ostrinia furnacalis and the European corn borer, O. nubilalis. To better understand the host location mechanism, we examined the external morphology and ultrastructure of the antennal sensilla of this parasitoid by using scanning and transmission electron microscopy. Antennae of male and female of the M. cingulum are filiform in shape, 5.90 to 6.64 mm in length and consist of scape, pedicel, and flagellum with 39 and 40 flagellomeres, respectively. Cuticular pore and nine types of morphologically distinct sensilla were identified in both sexes, including two types of sensilla chaetica (nonporous), s. trichodea (nonporous), s. basiconica I (nonporous blunt tip), s. basiconica II (porous wall) and basiconica III (nonporous wall) with branched blunt tip, s. coeloconica with finger-like projections, protruded s. campaniform with central tip pore, and plate-like s. placodea (porous). We compared number, morphology, and distribution of sensilla between sexes. S. campaniform and non-porous basiconica type I may play a role in gustatory functions whereas type II, and s. placodea may play a function in detecting odor stimuli due to their pores wall. The sensilla chaetica and s. trichodea may be involved in mechnosensation. S. coeloconica probably plays a role as thermohygro receptor, whilst cuticular pores may detect odor stimuli. No differences in antenna shape and basic structure in the males and females, but male antennae length and width were significantly greater than those of females. Furthermore, males had more placodea than females. The sensilla types, morphology, and structure of both sexes were compared to those found in other parasitic hymenoptera, especially braconid wasps.
1. Introduction
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Kew Words: Macrocentrus cingulum; antennal sensilla; morphology; SEM; TEM; ultrastructure
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The parasitic wasp Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) is a polyembryonic endoparasitoid of the Asian corn borer, Ostrinia furnacalis Guenée (Lepidoptera: Crambidae) and the European corn borer, O. nubilalis Hübner (Edwards and Hopper, 1999; Hu et al., 2003) and is widely distributed in Europe, Japan, Korea and China (Watanabe, 1967). Parasitic effectiveness of M. cingulum depends on location and infection by pathogens and other factors like temperature and humidity. Female M. cingulum oviposits in all instars but prefers second to fourth instars of the larvae. Through polyembryony, each egg can produce an average of 25-26 wasps (Parker, 1931). After parasitization the host larvae consume less maize tissue than unparasitized counterparts, thus causing reduced plant injury (Zhou et al., 2011). Insect antennae detect odor, sound, heat, cold, humidity and tactile information by means of numerous sensilla, which typically occur in the form of hairs, pegs, or plates (Altner and Prillinger, 1980). Parasitic hymenopterans are equipped with specialized antennal sensilla to facilitate their biological functions (Li et al., 2011). Antennae of female hymenoptera are important sensory receptors involved in various behaviors including habitat searching, host
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location, host detection, host recognition, host acceptance, oviposition, mating, and host discrimination (Bin and Vinson, 1986; Isidoro et al., 2001; Vinson, 1976). Several studies have characterized antennal sensilla of braconid wasps by using scanning and transmission electron microscopic techniques. However, similar studies on polyembryonic parasitoids are lacking.
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Parasitoids are directed to their host through a series of physical and chemical cues that narrow the search area (Vinson, 1976). Parasitoids locate suitable hosts by detecting the green leaf volatiles of their preferred plants as chemical cues (Ochieng et al., 2000). In areas closely surrounding the host, parasitoids are reported to use host kairomones and damaged-plant synomones for finally tracking their target (Eller et al., 1988; Turlings et al., 1995). M. cingulum are stimulated by host larval frass in stalk tunnels (Parker, 1931). The aim of this research is to better understand the host locating mechanism of M. cingulum. We investigated antennal morphology, ultrastructure, and distribution of antennal sensilla in male and female parasitoids by using scanning and transmission electron microscopy. The types of antennal sensilla of both sexes of M. cingulum are compared with those of other parasitic hymenoptera and putative functions are discussed with reference to their morphology, distribution, and ultrastructure.
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2. Materials and methods
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2.1. Insects The parasitoid, M. cingulum was collected from O. furnacalis larvae living on corn plants at the Langfang Experiment Station of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, China and reared on O. furnacalis larvae from a laboratory colony. Parasitoid wasps were fed a 20% honey solution and host O. furnacalis were reared on an artificial diet as described by Zhou et al. (1980). They were maintained in a room at 25 ºC with a 16:8, light: dark photoperiod. Parasitism by M. cingulum was carried out as described by Hu et al. (2003).
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2.2. Scanning electron microscope (SEM) Antennae of male and female wasps were excised from heads of freshly emerged parasitoids with fine forceps. The antennae were immersed overnight in CCl4 (Aldrich, Milwaukee, WI) at room temperature and subsequently boiled three times with new CCl4, each time 1 minute. Antennae were dehydrated through a graded ethanol series and then put into a holder with double-sided adhesive tape (dorsal, ventral and profile orientations). Finally, the antennae were sputter-coated with gold/palladium (40:60). Specimens were examined using a scanning electron microscope (FEI Quanta-200F) with accelerating voltage set at 6.0 kV. Images were recorded digitally and stored on a computer. 2.3. Transmission electron microscope (TEM) Antennae were fixed in 2% glutaraldehyde and 1% sucrose in 0.1 M cacodylate buffer for 3 h, briefly washed in 0.1 M cacodylate buffer, and then post-fixed in 1% osmium tetraoxide in 0.1 M cacodylate buffer for 2 h, all at pH 7.0 and room temperature. Sample antennae were embedded in epon after dehydration in a graded series of ethanol. Epon was polymerized at 60ºC for 48 h. Specimens were then transferred to embedding molds with Spur‟s resin and polymerized in an oven set at 60ºC for 24 h. Ultra-thin serial sections of 80 nm were cut on a Leica Super Nova ultramicrotome with a diamond knife and collected on pioloform-coated grids. Sections were contrasted with uranylacetate and stained with 1% toluidine blue. The sample
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grids were observed using a Hitachi H-7500 (Tokyo, Japan) TEM at an accelerating voltage of 6.0 kV. Images were recorded digitally and stored on a computer. 2.4. Terminology of sensilla Identification and classification of the sensilla types and the terminology used in this work is based on the methodology described by Keil (1999) and Zacharuk (1985).
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2.5. Data analysis Sensilla numbers for each segment were counted directly from the computer screen. The antenna length and diameter were measured from digital images by using Adobe Photoshop version CS3. At least six antennae were investigated per sex, and 18 sensilla of each type per segment were examined to calculate means. Data obtained on the distribution and abundance of different types of sensilla on male and female antennae were performed using a software package SPSS Version 16 (http:// www.spss.com). Mean numbers of the different types of sensilla found in male and female wasps were calculated before the independent sample T test was used to determine any significant sexual differences occurred. 3. Results
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3.1. General morphology of the antenna Antennae of both male and female M. cingulum are filiform in shape. For both sexes, the proximal part is composed of a cylindrical scape (length 220.06 ± 0.79 μm in male and 206.24 ± 0.35 μm in female), with a basal radicel fitting into the antennal socket and the barrel shaped pedicel (length 59.14 ± 0.42 μm in male and 56.64 ± 0.24 μm in female). The distal part is the flagellum. There is a sexual dimorphism in the number of flagellomeres; the male flagellum has 39 flagellomeres while that of females has 40. The length of the individual antennomeres in males are somewhat longer than those of females (72.79 to 368.47 um versus 47.44 to 354.04 um, respectively; Table 1). Also, there is a slight sexual difference in flagellar length as that of females measures 5.90 ± 0.05 mm and that of males 6.64 ± 0.01 mm. However, the individual antennomeres of males are longer than those of females (72.79 to 368.47 μm versus 47.44 to 354.04 μm, respectively; Table 1).
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3.2. Sensilla on the antenna Nine types of morphologically distinct sensilla were identified on the antennae of male and female M. cingulum, including two types of sensilla chaetica (CHA), s. trichodea (TRI), three types of s. basiconica (BAS), s. coeloconica (COE), s. campaniform (CAM), and s. placodea (PLA). In addition, cuticular pores were found on the antennae. Below, each type of sensilla, plus the cuticular pore structure is described. 3.2.1. Sensilla chaetica (CHA) Sensilla chaetica I (CHA I) have a smooth surface. These sensilla occur mainly on the basal radicel in both sexes, with a few occurring on the pedicle. They are blunt tipped barrel-shaped and are inserted into the cuticular depression (Fig. 1 inset). The length of these sensilla type ranges from 2.89 to 5.70 μm whereas the basal diameter ranges from 1.18 to 1.51 μm. CHA II has a smooth surface and is distributed on the basal radicel and pedicel for both males and females. They also have a blunt but elongated tip and are positioned in cuticular socket (Fig. 1
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inset). CHA II is relatively longer as compared to CHA I, ranging from 7.44 to 9.89 μm in length and with a narrow basal diameter of 1.1 to 1.30 μm.
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3.2.2. Sensilla basiconica (BAS) These sensilla occur on all flagellomeres of both sexes. They are mostly distributed on the dorsal and latero-ventral side of the antenna. Based on their different morphologies, they are classified into three types, BAS I, II, and III. BAS I is differentiated by from BAS II, and also from s. trichodea, the grooved surface that projects slightly more perpendicular to the antennae than the last mentioned types (Fig. 2 and 3). They gradually curve distally toward the apex of the segment ending with a blunt tip and are fitted with a cuticular socket that ranges from 16.81 to 24.29 μm in length and 2.17 to 2.67 μm in basal diameter. BAS I are characterized by thick non-porous cuticular walls with an inner lumen that is innervated by a various number of dendritic branches, one of which forms a tubular body at the base of the hair shaft (Fig. 4).
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BAS II sensilla have grooved cuticular surfaces penetrated by numerous pores and are slightly enlarged at the base; they curve gradually and have a blunt tip. These sensilla are distributed in a circular arrangement along the distal end of each antennomere (Fig. 3) and range from 31.47 to 55.84 μm in length and 3.03 to 4.51 μm in basal diameter. The porous cuticular walls surrounded numerous dendritic branches within the lumen (Fig. 4). Females had significantly higher number of BAS I than males except for segment 25, 26, 28, and 39 (Table 2). Similarly, females had significantly higher BAS II than males for all flagellar sub-segments except for sub-segments 16 (Table 2). These two types of sensilla are distributed on all flagellar sub-segments in both sexes and most commonly on the mid- and distal region of each sub-segment (Fig. 2 and 3).
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The sensilla BAS III occur only on female antennae. Similar to BAS I, they have a longitudinally grooved shaft that is inserted into a small ear-like socket. They are more perpendicular than BAS I and other sensilla and have apices with two branches (Fig. 2 and 3).
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3.2.3. Sensilla trichodea (TRI) Sensilla trichodea (TRI) are the most abundant type of sensilla, found on all antennal segments of both sexes. These long sensilla have a grooved surface, are tapered in shape, and are widely distributed on the scape, pedicel, and frontal part of the flagella. The base of this sensilla type is fitted into a slightly flexible socket, which is elevated above the cuticle. The sensilla possess a sharp tip that is slightly curved toward the apex of the segment. The non-porous cuticle walls with longitudinal grooves can be observed under the high magnification image in figure 2. This sensillium has a relatively thick wall and a lumen that is devoid of dendrites as revealed from TEM (Fig. 5). They are innervated by a single dendrite terminating in a tubular body that is surrounded by a dendritic sheath at the base of the hair shaft (Fig. 5). 3.2.4. Coeloconica sensilla (COE) Sensilla coeloconica (COE) are found on the mid dorsal surface of all flagellomeres for both sexes except for the most proximal and the last three distal flagellomeres (Table 3). One to four COEs occur on each sub-segment. They are peg shaped and protrude from a pit and range from 1.95 to 2.24 μm in diameter and 3.01 to 4.46 μm in length (Fig. 6). The tip of the peg stalk protrudes like a finger and is surrounded by a donut-shaped groove with an inner and outer diameter of 3.1 and 6.6 μm, respectively (Fig. 6). The number of COE sensilla is significantly higher in male than females for all flagellar sub-segments except 25 and 32 (Table 3).
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3.2.5. Sensilla placodea (PLA) Sensilla placodea (PLA) are long narrow structures that are slightly elevated above the antennal surface. They are smooth and surrounded by grooved gap and cuticular ridges and range from 64.89 to 91.42 μm in length (Fig.2). Generally they are aligned in parallel with the antennal axes and are located between rows of s. tricodea and basiconica. They are unevenly distributed on most flagellar sub-segments and are not found on the basal and apex sub-segments of males. In females, they are absent on the basal and last four proximal sub-segments. There are fewer PLA in proximal compared with mid to distal part of the sub-segments. Females had significantly more PLAs than males for segments 2, 3, and 7, but males had significantly more PLAs for segments 8-16, 18-24, and 26-36 (Table 4).
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3.2.6. Sensilla campaniform (CAM) Sensilla campaniform (CAM) were found on odd number flagellomere (except 1 and 39) in both sexes and flagellomere 37 in females (Table 5). CAM has a dome-shaped structure, fitted into a cuticular socket that protrudes from the center of a disc. The CAM has a pore at the center of the tip (Fig. 7). This dome-shaped disc ranges 0.51 to 0.67μm in width and 0.44 to 0.55 μm in length, and width of the stalk measures 0.70 to 0.93 μm. Male antennae have significantly more CAMs than those of females (Table 4).
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3.2.7. The cuticular pore (CP) Cuticular pores (CP) are found on the flagellar sub-segments of both sexes (Fig. 8) and look like small holes on the epidermis. These structures are more often found at the sprout of the cuticle. Each pore ranged from 0.63 to 0.75 μm in width.
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4. Discussion
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The external morphology and distribution pattern of nine antennal sensilla types, plus one pore structure, was described in male and female M. cingulum recorded in the current study. Prior to this investigation, no ultrastructural information on antennal sensilla was reported neither for M. cingulum nor for any other polyembryonic parasitoid. Antennae of insects typically have three basic segments, i.e. scape with radicel, pedicel, and flagellum (Chapman, 1998; Isidoro et al., 1996). The antenna of M. cingulum is filiform in shape and has a large scape with a radicle inserted into the antennal socket, a shorter pedicel, and a flagellum composed of 39 (male) and 40 (female) flagellomeres. Based on this study, it can be concluded that there are nine types of morphologically distinct sensilla on the antennae of both sexes, which correspond to those described in another closely related species, Apanteles cypris (Zhou et al., 2011). Six of these types correspond to those described in other parasitic hymenoptera (Bleeker et al., 2004; Bourdais et al., 2006; Cônsoli et al., 1999; Dweck and Gadallah, 2008; Gao et al., 2007; Ochieng et al., 2000; Roux et al., 2005). Chemosensory and tactile antennal sensilla have been described in various hymenoptera parasitoids (Barlin and Vinson, 1981; Norton and Vinson, 1974; Ochieng et al., 2000). The two types of s. chaetica observed in this study assumingly correspond with similar types found in other braconid wasps (Ochieng et al., 2000; Zhou et al., 2011). In certain species of Cotesia, however, they are absent (Bleeker et al., 2004; Roux et al., 2005). Due to their location and structure CHAs probably serve a mechanical function (Gao et al., 2007). In particular, it has been suggested that they may act as proprioceptors perceiving antennal movement (Zhou et al., 2011).
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Nonporous thick walled s. trichodea have been reported in other parasitic wasps through TEM studies (Amornsak et al., 1998; Baaren et al., 1996; Cônsoli et al., 1999; Gao et al., 2007; Ochieng et al., 2000; Zhou et al., 2011). The socket-like connection with the cuticle and the spatial arrangement of these sensilla suggest a mechanosensitive function in M. cingulum. A previous study reported about TRI lacking cuticular pores, probably serving an olfactory function (Zhou et al., 2011).
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The s. basiconica types I and II, present in both sexes of M. cingulum, are similar to those previously found in Microplitis croceipes (Ochieng et al., 2000), M. pallidipes (Gao et al., 2007), and A. cypris (Zhou et al., 2011). In the former studies the BAS I was described as fluted basiconic sensilla (Norton and Vinson, 1974), trichodea TP (TP = tip pore) (Bleeker et al., 2004), or curved trichoid formations with an apical pore (Barbarossa et al., 1998) whereas the BAS II category was described as trichodea WP (WP = wall pore) (Bleeker et al., 2004) or as a curved non-fluted BAS (Norton and Vinson, 1974). Differences between BAS I and II in M. cingulum not only concern shape but also dendritic innervations. BAS I has grooves in the surface and an inner lumen surrounded by a thick nonporous wall (Fig. 4), similar to that previously described in M. croceipes (Ochieng et al., 2000) and M. pallidipes (Gao et al., 2007). BAS II on the other hand is encircled by a thick wall with multiple pores, similar to that found by Gao et al. (2007) and Ochieng et al. (2000). BAS I may have a gustative function, since it has four to six dendrites inside the wall (Fig. 4), whereas BAS II presumably have an olfactory function (Baaren et al., 1996; Gao et al., 2007; Ochieng et al., 2000; Zhou et al., 2011). Hansson et al. (1991) confirmed the olfactory function of sensilla basiconica type II in Neodiprion sertifer Geoffroy (Hymenoptera: Diprionidae) by using electrophysiological recordings with sex pheromones.
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Both sexes of M. cingulum have only one type of coeloconica sensillum, occurring in a varying number, i.e. from one to four on each flagellomere. The morphological features of sensilla coeloconica are similar to those found with M. pallidipes (Gao et al., 2007), M. croceipes (Navasero and Elzen, 1991; Ochieng et al., 2000), and within braconids, type I of Cotesia spp. and A. cypris (Bleeker et al., 2004; Zhou et al., 2011). Sensilla coeloconica were categorized as types I and II in the Cotesia spp. and A. cypris in the braconid family (Bleeker et al., 2004; Gao et al., 2007; Roux et al., 2005; Zhou et al., 2011). Other researchers have described coeloconica sensilla using particular terminology, e.g. “small sub-terminal sensilla” (Udayagiri and Jones, 1993), „„bulb sensilla‟‟ (Cave and Gaylor, 1987), and “smooth basiconic sensilla” (Norton and Vinson, 1974). COEs are suggested to have an olfactory function (Altner et al., 1983; Gao et al., 2007; Keil, 1999; Ochieng et al., 2000; Olson and Andow, 1993; Roux et al., 2005; Steinbrecht, 1997). The elongated sensilla placodea are found throughout most parts of the antennae in almost all parasitic hymenoptera (Amornsak et al., 1998; Barlin and Vinson, 1981; Dweck and Gadallah, 2008; Gao et al., 2007; Ochieng et al., 2000; Roux et al., 2005; Zhou et al., 2011). The PLA category is probably involved in olfactory reception used to detect host derived semiochemicals because they have multiple cuticular pores. Electroantennography (EAG) performed from single s. placodea in M. croceipes is reported to respond in a dose dependent manner to plant volatiles (Ochieng et al., 2000). The PLA in M. cingulum have a multiporous cuticular structure similar to that found by Bleeker et al. (2004) and Gao et al. (2007). They likely have an olfactory function.
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One type of campaniform sensilla was found in the current study, present on each of the odd numbered flagellomeres on the antennae of both sexes. The CAM sensilla have previously been found on antennae of several insects, e.g. ground beetles (Must et al., 2006) and four Australian spittlebug species (Liang and Fletcher, 2002). In A. cypris, two types of CAM have been reported, CAM I and CAM II (Zhou et al., 2011). In general, CAM are considered to be mechanoreceptors (Ågren, 1977; Zacharuk, 1985). However, Ochieng et al. (2000) reported that CAM may serve a gustatory role due to the presence of a central porous tip. Dietz et al (1971) actually reported that the porous central tip in the CAM is involved in a gustatory function and is highly susceptible to humidity. The cuticular pores (CP) resemble a small hole on the epidermis. Zhou et al., (2011) described as cuticular pores in A. cypris and suggested that act as olfactory receptors and highly responsiveness to sex pheromone.
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The present finding that male M. cingulum adults have more sensilla placodea on their antennae than females which is a common trend found in other parasitic hymenoptera (Bleeker et al., 2004; Dweck and Gadallah, 2008; Gao et al., 2007; Navasero and Elzen, 1991; Ochieng et al., 2000; van Baaren et al., 1999; Zhou et al., 2011). This trend may indicate a role of the current sensillum type in mediating information about sex pheromones (Zhou et al., 2011). However, the s. placodea are also suggested to serve a role in detecting host plant odors (Barlin and Vinson, 1981; Bleeker et al., 2004; Chapman, 1982).
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The finding that M. cingulum males have significantly longer antennae than females agrees with former findings in other hymenopteran parasitic wasp such as, M. croceipes (Das et al., 2011; Navasero and Elzen, 1991; Ochieng et al., 2000), A. cypris (Zhou et al., 2011), M. pallidipes (Gao et al., 2007), Habrobracon hebetor (Dweck and Gadallah, 2008), and two Cotesia wasps (Bleeker et al., 2004). Interestingly, in this study M. cingulum females had more flagellomeres than males (male 39 and female 40) but males still had longer antennae than females due to the longer length of each flagellomeres. The adaptive significance of this phenomenon might be attributed to a larger surface area enabling more sensory receptors (Dweck, 2009; Schneider, 1964).
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The contact mechano-, chemo-, thermo-, and hygroreceptive sensilla are abundantly distributed on the antenna of M. cingulum in both sexes. Previous studies, suggest that insect behavior including habitat searching, host location, host detection, host recognition, host acceptance, oviposition, mating, and host discrimination essentially depend on the function of the antennal sensilla (Whitman and Eller, 1990). Further studies are required to understand the mechanisms involved in these behavioral responses. Future studies on functional morphology of antennal sensilla of M. cingulum using electrophysiological recordings are necessary to confirm the functions of the sensilla identified in this study. Acknowledgement This work was supported by China Agriculture Research System (CARS-02).The authors gratefully acknowledge Dr. Hellmich, R.L, USDA-ARS, Corn Insects and Crop Genetics Research Unit for comments and suggestions on this manuscript.
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Schneider, D., 1964. Insect antennae. Annu. Rev. Entomol. 9, 103-122. Steinbrecht, R.A., 1997. Pore structures in insect olfactory sensilla: A review of data and concepts. Int. J. Insect Morphol. Embryol. 26, 229-245. Turlings, T.C., Loughrin, J.H., McCall, P.J., Röse, U.S., Lewis, W.J., Tumlinson, J.H., 1995. How caterpillar-damaged plants protect themselves by attracting parasitic wasps. Proc.Natl. Acad. Sci. 92, 4169-4174. Udayagiri, S., Jones, R., 1993. Variation in flight response of the specialist parasitoid, Macrocentrus grandii Goidanich to odours from food plants of its european corn borer host. Entomol. Exp. Appl. 69, 183-193. van Baaren, J., Boivin, G., Le Lannic, J., Nénon, J.P., 1999. Comparison of antennal sensilla of Anaphes victus and A. listronoti (Hymenoptera, Mymaridae), egg parasitoids of Curculionidae. Zoomorphol. 119, 1-8. Vinson, S.B., 1976. Host selection by insect parasitoids. Annu. Rev. Entomol. 21, 109-133. Watanabe, C., 1967. Further revision of the genus Macrocentrus Curtis in Japan, with descriptions of two new species (Hymenoptera,Braconidae). Insecta Matsumurana 30, 1-16. Whitman, D., Eller, F., 1990. Parasitic wasps orient to green leaf volatiles. Chemoecol.1, 69-75. Zacharuk, R.Y., 1985. Antennae and sensilla. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive insect physiology biochemistry and pharmacology, sensory. Oxford,UK. Pergamon, pp. 1-69. Zhou, D.R., Wang, Y.Y., Liu, B.L., Ju, Z.L., 1980. Research on the reproduction of O. furnacalis in a large quantity: an artificial diet and its improvement. Acta Phytophyl. Sin. 7, 113-122.7, 113-122. Zhou, H., Wu, W.-J., Zhnag, Z.-F., Zhnag, Y., 2011. Antennal sensilla of Apanteles cypris Nixon (Hymenoptera: Braconidae), a larval endoparasitoid of Cnaphalocrocis medinalis Guenée (Lepidoptera: Pyralidae). Micros. Res. Tech. 74, 1199-1208.
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Table
Table 1: Morphological characteristics the antenna of male and female M. cingulum*. Female
Mean length of antenna (mm)
6.64 ± 0.01a
5.90 ± 0.05b
Length of antennomere (μm)**
72.79 to 368.47
47.44 to 354.04
Mean diameter of antennomere (μm)
44.85 ± 1.60b
49.91 ± 1.28a
Mean length of scape (μm)
220.06 ± 0.79a
Mean length of pedicel (μm)
59.14 ± 0.17a
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206.24 ± 0.35b 56.64 ± 0.24a
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*Data are presented as mean ± SD, n = 6. Means in rows followed by same letter are not significantly different in independent-sample T test (P >0.05). ** Range of antennomere length; data are not analyzed.
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Table 2: The number of sensilla basiconica types I and II on flagellomeres in male and female antenna of M. cingulum*. No. of Flagellomeres
Basiconica I Male
Basiconica II Female
Male
Female
st
17.17 ± 0.75a 17.83 ± 0.75a 17.67 ± 0.52a 17.17 ± 0.41a 17.67 ± 0.52a 17.33 ± 0.82a 17.67 ± 0.52a 18.17 ± 0.75a 18.17 ± 0.41a 18.17 ± 0.41a 17.50 ± 0.84a 17.17 ± 0.98a 18.50 ± 0.55a 18.17 ± 0.75a 18.33 ± 0.52a 18.00 ± 0.63a 20.83 ± 0.98a 20.33 ± 0.82a 22.33 ± 0.52a 22.50 ± 0.84a 24.33 ± 0.82a 20.33 ± 0.82a 20.50 ± 0.55a 20.83 ± 0.98a 20.67 ± 0.82a 18.17 ± 0.41a 16.83 ± 0.75a 17.17 ± 0.75a 18.67 ± 0.82a 18.33 ± 0.52a 17.33 ± 0.82a 17.17 ± 0.98a 18.17 ± 0.41a 17.17 ± 0.98a 17.50 ± 0.55a 17.67 ± 0.52a 18.17 ± 0.75a 17.83 ± 0.75a 17.67 ± 0.82a 14.83 740.00 ± 3.85a followed by same
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1 7.67 ± 0.52b 9.33 ± 0.52a 15.67 ± 0.52b nd 2 7.67 ± 0.52b 9.50 ± 0.55a 15.83 ± 0.40b 3rd 7.83 ± 0.41b 9.67 ± 0.52a 16.33 ± 0.52b th 4 7.67 ± 0.82b 9.33 ± 0.52a 14.67 ± 0.52b th 5 7.83 ± 0.41b 9.83 ± 0.41a 14.83 ± 0.98b 6th 7.83 ± 0.98b 9.50 ± 0.84a 15.67 ± 0.52b th 7 7.67 ± 0.52b 9.33 ± 0.52a 16.33 ± 0.52b 8th 7.67 ± 0.52b 9.50 ± 0.84a 15.67 ± 0.52b th 9 7.83 ± 0.41b 10.33 ± 0.52a 14.83 ± 0.41b 10th 7.67 ± 0.51b 10.50 ± 0.55a 15.67 ± 0.52b th 11 7.83 ± 0.41b 10.33 ± 0.82a 15.33 ± 0.52b th 12 7.83 ± 0.41b 10.50 ± 0.84a 15.67 ± 0.52b 13th 8.17 ± 0.41b 10.67 ± 1.03a 16.33 ± 0.52b th 14 7.83 ± 0.41b 10.50 ± 0.84a 14.33 ± 0.52b 15th 8.83 ± 0.98b 12.50 ± 0.83a 16.33 ± 0.52b th 16 8.33 ± 0.52b 12.67 ± 1.03a 17.83 ± 0.75a 17th 8.67 ± 0.82b 13.17 ± 0.98a 17.83 ± 0.41b 18th 8.83 ± 0.41b 13.50 ± 0.84a 15.67 ± 0.52b th 19 10.33 ± 0.82b 12.83 ± 0.98a 16.67 ± 0.82b 20th 10.5 ± 0.84b 12.67 ± 0.82a 15.67 ± 0.82b st 21 10.33 ± 0.52b 12.83 ± 0.75a 16.83 ± 0.75b 22nd 7.83 ± 0.41b 12.50 ± 0.84a 15.33 ± 1.03b rd 23 7.83 ± 0.41b 10.83 ± 0.75a 15.33 ± 0.52b 24th 7.67 ± 0.82b 10.67 ± 0.75a 15.33 ± 0.82b th 25 10.33 ± 0.82a 10.33 ± 1.03a 15.67 ± 0.52b th 26 8.67 ± 0.82a 9.33 ± 0.82a 16.33 ± 0.52b 27th 8.83 ± 0.41b 9.67 ± 1.03a 15.33 ± 0.52b th 28 8.67 ± 0.52a 9.33 ± 0.52a 15.33 ± 0.82b 29th 8.17 ± 0.41b 9.33 ± 0.52a 15.67 ± 0.52b th 30 8.33 ± 0.52b 9.50 ± 0.52a 15.83 ± 0.41b st 31 7.67 ± 0.82b 9.33 ± 0.55a 14.33 ± 0.52b 32nd 7.33 ± 0.52b 9.67 ± 0.52a 14.83 ± 0.41b rd 33 7.67 ± 0.52b 9.83 ± 0.52a 15.50 ± 0.55b 34th 7.67 ± 0.52b 9.67 ± 0.75a 15.33 ± 0.82b th 35 7.83 ± 0.41b 9.50 ± 0.52a 14.50 ± 0.55b 36th 7.33 ± 0.82b 9.33 ± 0.55a 14.33 ± 0.82b th 37 7.33 ± 0.82b 9.50 ± 0.82a 14.83 ± 0.75b th 38 7.67 ± 0.52b 9.17 ± 0.84a 14.50 ± 0.84b 39th 8.67 ± 0.52a 9.33 ± 0.41a 13.67 ± 0.52b th 40 10.67 Total 320.33 ± 2.66b 416.50 ± 6.63a 606.00 ± 5.40b *Data are presented as mean ± SD, n = 6. Means in rows of each sensilla type letter are not significantly different in independent-sample T test (P > 0.05).
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Table 3: The number of sensilla coeloconica on flagellomeres on antenna of male and female M. cingulum*.
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No. of Male Female Flagellomeres 2nd 2.17 ± 0.41a 1.17 ± 0.41b 3rd 3.17 ± 0.41a 2.17 ± 0.41b th 4 3.33 ± 0.52a 2.17 ± 0.41b 5th 3.83 ± 0.41a 2.17 ± 0.41b th 6 3.67 ± 0.52a 1.83 ± 0.41b th 7 3.83 ± 0.41a 2.17 ± 0.41b 8th 3.83 ± 0.52a 1.83 ± 0.75b th 9 3.17 ± 0.41a 1.83 ± 0.41b 10th 3.83 ± 0.41a 1.67 ± 0.52b th 11 3.17 ± 0.41a 1.33 ± 0.52b th 12 3.67 ± 0.52a 1.33 ± 0.52b 13th 3.67 ± 0.52a 1.33 ± 0.52b th 14 3.67 ± 0.52a 1.17 ± 0.41b 15th 3.33 ± 0.52a 1.33 ± 0.52b th 16 3.67 ± 0.52a 1.33 ± 0.52b 17th 3.33 ± 0.52a 1.67 ± 0.52b th 18 3.67 ± 0.52a 1.67 ± 0.52b th 19 3.17 ± 0.41a 1.67 ± 0.52b 20th 3.17 ± 0.41a 1.67 ± 0.52b st 21 3.17 ± 0.41a 1.33 ± 0.52b 22nd 2.67 ± 0.52a 1.33 ± 0.52b rd 23 2.67 ± 0.52a 1.17 ± 0.41b 24th 2.67 ± 0.52a 1.17 ± 0.41b th 25 2.33 ± 0.52a 1.67 ± 0.52a th 26 2.67 ± 0.52a 1.33 ± 0.52b 27th 2.83 ± 0.41a 1.17 ± 0.41b th 28 3.17 ± 0.41a 1.33 ± 0.52b 29th 3.17 ± 0.41a 1.67 ± 0.52b th 30 2.83 ± 0.41a 1.83 ± 0.41b 31st 2.83 ± 0.41a 1.67 ± 0.52b nd 32 2.17 ± 0.41a 1.67 ± 0.52a rd 33 2.17 ± 0.41a 1.17 ± 0.41b 34th 2.17 ± 0.41a 1.17 ± 0.41b th 35 2.33 ± 0.52a 1.33 ± 0.52b 36th 2.17 ± 0.41a 1.17 ± 0.41b th 37 0 1.17 ± 0.41a Total 107.33 ± 6.38a 54.83 ± 4.58b *Data are presented as mean ± SD n = 6. Means in rows followed by same letter are not significantly different (P > 0.05) in independent-sample T test.
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Table 4: The Number of sensilla placodea on flagellomeres on antenna of male and female M. cingulum*.
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No. of Male Female Flagellomeres 2nd 6.33 ± 0.52b 7.83 ± 0.41a rd 3 5.83 ± 0.41b 9.83 ± 0.41a 4th 9.83 ± 0.41a 8.18 ± 0.41b 5th 7.67 ± 0.52a 8.17 ± 0.41a 6th 7.67 ± 0.52a 8.33 ± 0.52a th 7 8.33 ± 0.52b 9.83 ± 0.41a th 8 9.67 ± 0.52a 7.83 ± 0.41b 9th 9.67 ± 0.52a 7.83 ± 0.41b th 10 9.67 ± 0.52a 7.83 ± 0.41b th 11 10.17 ± 0.41a 8.33 ± 0.52b 12th 9.83 ± 0.41a 7.83 ± 0.41b th 13 8.33 ± 0.52a 6.33 ± 0.82b 14th 8.17 ± 0.41a 6.17 ± 0.41b th 15 8.17 ± 0.41a 6.17 ± 0.41b 16th 9.83 ± 0.41a 5.83 ± 0.41b th 17 6.17 ± 0.41a 6.83 ± 0.75a th 18 9.67 ± 0.52a 5.83 ± 0.41b 19th 7.83 ± 0.41a 6.17 ± 0.41b th 20 7.83 ± 0.41a 5.17 ± 0.98b 21st 5.83 ± 0.41a 4.83 ± 0.41b nd 22 9.83 ± 0.41a 5.17 ± 0.41b rd 23 8.17 ± 0.41a 6.17 ± 0.41b 24th 9.67 ± 0.82a 4.33 ± 0.52b th 25 5.67 ± 0.52a 4.83 ± 0.98a th 26 6.33 ± 0.52a 4.67 ± 0.82b 27th 5.83 ± 0.41a 4.17 ± 0.41b th 28 7.67 ± 0.52a 4.17 ± 0.41b 29th 5.83 ± 0.41a 3.83 ± 0.41b th 30 5.83 ± 0.41a 3.83 ± 0.41b 31st 6.17 ± 0.41a 3.83 ± 0.41b nd 32 5.83 ± 0.41a 2.33 ± 0.52b rd 33 6.17 ± 0.41a 2.17 ± 0.41b 34th 5.83 ± 0.41a 2.17 ± 0.41b th 35 5.67 ± 0.52a 2.17 ± 0.41b 36th 5.17 ± 0.41a 2.17 ± 0.41b th 37 3.67 ± 0.52a 0 th 38 3.67 ± 0.52a 0 Total 273.51 ± 0.1.87a 201.17 ± 4.31b *Data are presented as mean ± SD., n = 6. Means in rows followed by same letter are not significantly different in independent-sample T test (P > 0.05).
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TABLE 5: The Number of Campaniform Sensilla on Flagellomeres in Male and Female M. cingulum*.
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No. of Male Female Flagellomeres 3rd 0.83 ± 0.41a 0.83 ± 0.41a 5th 0.83 ± 0.41a 0.67 ± 0.52a th 7 0.83 ± 0.41a 0.67 ± 0.52a th 9 0.83 ± 0.41a 0.67 ± 0.52a 11th 0.83 ± 0.41a 0.67 ± 0.52a th 13 1.00 ± 0.00a 0.67 ± 0.52a th 15 1.00 ± 0.00a 1.00 ± 0.00a 17th 1.00 ± 0.41a 0.83 ± 0.41a th 19 0.83 ± 0.41a 0.83 ± 0.00a 21st 0.83 ± 0.41a 0.67 ± 0.52a rd 23 0.83 ± 0.41a 0.67 ± 0.52a 25th 0.83 ± 0.00a 0.67 ± 0.52a th 27 0.83 ± 0.41a 0.67 ± 0.52a th 29 0.83 ± 0.41a 0.50 ± 0.55a 31st 0.83 ± 0.41a 0.67 ± 0.52a rd 33 0.83 ± 0.41a 0.50 ± 0.55a 35th 0.83 ± 0.41a 0.50 ± 0.55a th 37 0.67 ± 0.52a 0 Total 15.33 ± 1.03a 11.67 ± 0.82b *Data are presented as mean ± SD., n = 6. Means in rows followed by same letter are not significantly different in independent-sample T test (P > 0.05).
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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Figure 6
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Figure 7
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Figure 8
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Fig.1 Electromicrograph of sensilla chaetica (CHA) on antenna of M. cingulum; A. Sensilla chaetica I (CHA I) and sensilla chaetica II (CHA II) at the basal radicel. B. CHA I and CHA II at the basal pedicel. High magnification pictures of CHA I and CHA II are shown in the inset.
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Fig.2 The distal part of the 20th antennomere of a female M. cingulum; showing sensilla basiconica I (white arrow), sensilla basiconica II (cross), sensilla basiconica III (open arrow), sensilla trichodea (triangles), sensilla placodea (star), and sensilla coeloconica (lightingbolt).
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Fig.3 SEM micrograph of the last flagellomere on a male antenna of M. cingulum showing sensilla basiconica. A. The distal tip of the flagellum includes sensilla basiconica I (BAS I) and sensilla basiconica II (BAS II). B. Sensilla basiconica III (BAS III) in the distal part of the 20th subsegment. C. As demonstrated BAS I has a smooth wall in the proximal end of the 21st sub-segment. D. BAS II with a blunt tip in the distal region of last flagellars.
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Fig.4 TEM micrograph of basiconica sensilla (BAS). A. Cross section of BAS type II showing dendritic branches (D), sensillium lymph (L), a porous cuticular wall (arrow), and the sensillium wall (W). B. Cross section of BAS type I demonstrating a thick and groove non-porous sensillum wall (W) and the sensillum lymph (L), which is innervated by dendrites (D).
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Fig.5 TEM micrograph of sensilla trichodea (TRI) and sensilla placodea (PLA) of M. cingulum; A. TEM of sensilla tricodea showing sensillium lymph (L) surrounded by a non-porous thick wall. B. Transverse section of sensilla placodea showing dendritic branches (DB) within the median channel and elevated grooves (G), plus the porous sensillum wall (arrow) and the septum (S). Fig.6 SEM micrograph of sensilla coeloconica (COE) on antenna of M. cingulum. A. Distribution of COE (arrow) on the antennomere. B. The characteristic peg like stalk of the COE surrounded by a donut-shaped ring (asterisk) can be seen. In addition, fingerlike projections with a capitate peg are demonstrated (Inset). Fig.7 Electromicrograph of sensilla campaniform (CAM) of M. cingulum. A. The dome shaped CAM sensilla protrude from the center of the disc in the distal region of 9th subsegment. B. The pore (arrow) at the center of the sprout of the CAM can be seen. The CAM pore at high magnification is shown in the inset. Fig.8 SEM image of the socket like structure enveloping the sensilla trichodea on the antennae (A) and the cuticular pores present on the dorsal side of one sub-segment (B).
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Highlights
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SEM and TEM of antennal sensilla of Macrocentrus cingulum Brischke. Cuticular pore and nine types of morphologically distinct sensilla were identified. These sensilla may act as mechano-, chemo-, thermo-, and hygroreceptor in both sexes. The male antennae length and width were significantly greater than those of females.
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