The Antennal Sensilla of Zamagiria dixolophella Dyar (Lepidoptera: Pyralidae)

Abstract

The number, external morphology, and distribution of antennal sensilla of Zamagiria dixolopella Dyar were determined by light and scanning electron microscopy. The antennal flagellum of both sexes has six types of sensilla: trichodea (subtypes I and II), chaetica, coeloconica, styloconica, auricillica, and squamiformia. There is sexual dimorphism in the antennae of Z. dixolophella. The male antenna has a tuft of hairs or modified scales at its base and one protuberance for each segment in the first seven segments, below the tuft of hairs or modified scales. These structures are not present in the female antenna. We believe that these structures have not been reported previously in Pyralidae. Sexual dimorphism was also observed in the subtypes of sensilla trichoidea. One subtype of long sensilla trichoidea (subtype I) occurs only on male antennae, whereas the other subtype of short sensilla trichoidea (subtype II) occurs mainly on female antennae.

The moth, Zamagiria dixolophella Dyar, occurs in Mexico, Central America, and possibly South America (Heinrich 1956, Iruegas et al. 2002). This moth species has been reported to attack sapodilla, Manilkara zapota Van Royen, in southern Mexico and is considered by local farmers as an important pest of this fruit (Iruegas et al. 2002). Z. dixolophella oviposits on the buds of sapodilla. Larvae feed on the ovaries and petals of the flowers but frequently bore into tender young shoots. Larvae are sometimes also found inside sapodilla fruits. Insect infestations persist throughout the year; however, the highest populations coincide with the peak of blooming (Iruegas et al. 2002, unpublished data).

We are currently studying the chemical ecology of Z. dixolophella; specifically, we are identifying the semiochemicals mediating sexual and host-finding behavior of this species. In this paper, we describe the antennal sensory organs of both sexes of this insect to provide a morphological basis for future behavioral and electrophysiological studies. The olfactory system of Pyralidae has been studied in only a few cases, despite the pest status of many species of this family (Hallberg et al. 1994).

Materials and Methods

Insects.

Larvae of last instar Z. dixolophella were collected from sapodilla trees at Rancho Cazanares (farm) (14°44′40″ N and 92°24′20″ W), located between Tapachula City and Puerto Madero, Chiapas, Mexico. In the laboratory, larvae were kept on a 16:8 (D:L)-h cycle, at 26 ± 2°C and 60 ± 10% RH until pupation. The pupae were sexed and kept separate until adults emerged. The adults were decapitated, and their heads were stored in 70% ethanol until required. Insects between 1 and 3 d old were used.

Scanning Electron Microscopy.

The antennae of 32 males and 32 females were separately placed in a solution of 70% ethanol and 2% formaldehyde for 24 h. They were washed with 76% ethanol and dehydrated in 80, 90, and 100% (two changes each) ethanol for 1 h each. They were dried at the critical point of CO₂ and gold-palladium coated in a Denton-vacuum desk II ion sputter (Denton Vaccum, Moorestown, NJ) (20 nm). The specimens were viewed using a TOPCON SM-510 microscope (Top Con Corp., Tokyo, Japan) at 5 kV (Wall 1978, Valdez 1991). The average length and basal diameter of the external part of each sensillum were calculated from 15 measurements taken from photomicrographs (Faucheux 1991).

Light Microscopy.

The antennae of both sexes were soaked in 10% KOH at 80°C until they cleared (≈30 min). They were washed with distilled water at the same temperature and time described above. The scales were removed using a 60-Hz ultrasonic cleaner (Branson Sonic, Danbury, CT) for 20 s, and the antennae were dehydrated in 70 and 100% alcohol for 30 and 60 min, respectively. They were cleared with xylol and mounted in Canada balsam. Observations were made using a Zeiss (Carl Zeiss, Mexico City, Mexico) microscope (40×). The sensilla were counted on each flagellar segment of 10 antennae for sensilla trichoidea and 20 antennae for all other sensilla of each sex (Faucheux 1991). The length and width of each flagellar segment were measured with a graduated ocular.

Statistical Analysis.

All data were analyzed using the paired samples t-test in the statistical program SPSS, version 10.0 (SPSS Inc., Chicago, IL). For all comparisons between male and female antennae, the level of P = 0.05 was considered significant.

Results

General Morphology of the Antenna.

The antenna of Z. dixolophella is filiform and segmented, and the flagellum is spindle-shaped. The antenna of the male is ≈7.18 ± 0.04 mm long, whereas that of the female antenna measured 7.30 ± 0.07 mm (Table 1). “Böhm” bristles are located on the dorsal side of the scape-pedicel joint and consist of a group of tapering spines with a smooth surface that arise, more or less perpendicularly, out of small cuticular pits.

Table 1.

Length, width, and number of segments (Mean ± SEM) in the antennal flagellum of Z. dixolophella

The distal part, the flagellum, consisted of many subsegments. The number of segments is significantly greater in the flagellum of the female than in that of the male (t = −5.761, df = 31, P = 0. 001; Table 1). However, the segments are significantly longer in male than in female antennae (t = 4.24, df = 31, P = 0.001). The width of the segments did not differ between the sexes (t = 0.571, df = 31, P = 0.572; Table 1). The flagellar segments diminish in length and diameter from the base to the apex of the antenna and have the same general organization and pattern of sensory structures. A typical flagellar segment is cylindrical in cross-section and divided into two main areas, the dorsal and ventral surfaces. The dorsal surface has two rows of scales; the second row overlaps the first row of the following segment. The only obvious type of sensillum on the dorsal surface is the squamiform type (Figs. 1C and 2C). The antennal ventral surface has a sculpture of small ridges forming a pattern resembling a honeycomb mesh (Fig. 2, A–D). The ventral surface possesses most of the sensilla (Fig. 1D).

Fig. 1.

Scanning electron micrographs showing (A) male antenna. Hair tufts or modified scales occur on the base of the antenna. The discontinuous arrow shows the beginning of the antenna. (B) Male antenna, showing peaks on the first seven segments (1–7) and long trichoidea sensilla (type I). (C) Female antenna. The short sensilla (type II). (D) Examples of most sensilla types present on the antenna of both sexes of Z. dixolophella. Hf, hairs tuft or modified scales; Lt, long trichoidea sensilla; St, short trichoidea sensilla; Co, coeloconicum sensillum; Bb, Böhm bristles; Sq, squamiform sensillum; Au, auricillica sensillum; S, scales

Fig. 2.

Scanning electron micrographs showing (A) styloconic sensillum in the middle part of the distal edge of each segment in both sexes. (B) The terminal segment always has two sensilla joined. (C) Auricillica, squamiformia, chaetica, and short trichoidea sensilla. (D) Sensilla coeloconica are surrounded by cuticular spines. Lt, long trichoidea sensilla; St, short trichoidea sensilla; Co, coeloconicum sensillum; Sp, spines; Sq, squamiform sensilla; Au, auricillicum sensillum. Double arrows point the helicoidal pattern on the long trichoidea sensilla

Sexual Dimorphism.

There is sexual dimorphism in Z. dixolophella antennae. The male antenna has a tuft of hairs or modified scales on its base (Fig. 1A) and one protuberance for each flagellar segment in the first seven segments, below the tuft of hairs (Fig. 1B). The female antenna does not present the tuft of hairs or the other protuberances. We believe this is the first time that these structures have been reported in Pyralidae. One subtype of long sensilla trichoidea occurs only on male antennae, whereas another subtype of short sensilla trichoidea occurs mainly on female antennae (Fig. 1, B and C, respectively).

Distribution of Antennal Sensilla.

There are six types of sensilla on the flagellum: trichoidea, chaetica, coeloconica, styloconica, auricillica, and squamiformia. The chemoreceptive sensilla trichoidea are the most abundant type in Z. dixolophella. They can be divided into two subtypes according to their length. Subtype I (82.19 μm in length and 3.27 μm in diameter at their base) is present only on male antennae (Fig. 1B; Table 2). We estimated total number of these sensilla on the male antenna to be 1364.20 ± 38.75 (Table 3). Subtype II (39.81 μm in length and 2.32 μm in diameter) is mainly present on female antennae (Fig. 1C; Table 2). The estimated mean number of these sensilla on the female antenna was 1819.10 ± 52.83 (Table 3). Some subtype II sensilla trichoidea were also present on male antennae, but it was not possible to make a reliable count because subtype I sensilla trichoidea covered them.

Table 2.

Dimensions of the sensilla on the antenna of Z. dixolophella

Table 3.

Estimated mean number of sensilla on the antennae of Z. dixolophella

There are 3–5 sensilla chaetica present on each flagellar segment in both sexes, except in the apical segment, where there are 12–15 of these sensilla. They are stubby and curved apically, and the tip is truncated in the majority of sensilla (Fig. 2, B and C). The base is inserted into a socket that consists of a cuticular structure (Fig. 2, B and C). These sensilla are significantly longer in male than in female antennae (t = 4.77, df = 14, P = 0.001), but they are similar in width (Table 2). The total number of these sensilla is significantly greater in female than in male antennae (t = −5.26, df = 19, P = 0.001; Table 3).

The styloconica sensilla are poreless (96 dpi), lack a flexible base, and are located on a cylindrical protrusion from the antennal surface, the stylus. There is a single sensillum styloconic (from the 7th to 11th segment) in the middle part of the distal edge of each segment in males and females (Fig. 2A). On the terminal segment, there are two sensilla joined (Fig. 2B). The average length and width of the complete structure of sensilla styloconica are shown in Table 2. The number of sensilla styloconica is significantly greater on female than on male antennae (t = −7.55, df = 19, P = 0.001; Table 3).

Sensilla coeloconica are distributed on the ventral surface of the antennae. Zero to eight sensilla occur per segment of males and females. Each sensillum consists of a depression surrounded by 14–18 cuticular “spines” and a porous peg with longitudinal striations on its surface, arising from the center of the depression (Fig. 2D). The diameter of sensilla coeloconica in males is larger than in females (t = 3.49, df = 14, P = 0.004; Table 2). The number of sensilla is significantly greater in the female than in the male flagellum (t = −4.14, df = 19, P = 0.001; Table 3).

Sensilla auricillica are found on the lateral part of the antennal segments, close to the scale-bearing dorsal surface. These sensilla usually have a typical rabbit-ear shape, but sometimes are simply dorsoventrally flattened or biforked at the tip (Figs. 1D and 2C). There are 4–10 of these sensilla per segment in both sexes, but they were not precisely counted. The length and width of these sensilla are shown in Table 2. They are longer on female than on male antennae (t = −3.66, df = 14, P = 0.003), but they do not differ in width (t = 0.873, df = 14, P = 0.397; Table 2).

The only evident sensillum present on the dorsal part of the antenna, between the scales, is the sensillum squamiformia (Fig. 1D). Squamiformia structures are found on the scape, pedicel, and the first three to five segments of the flagellum. They are shorter and finer than the scales. The base is inserted into a socket and is pointed distally (Fig. 2C). These sensilla were not counted. The sensillum squamiformia is longer (t = 5.14, df = 14, P = 0.001) and wider (t = 3.26, df = 14, P = 0.006) on male than on female antennae (Table 2).

Discussion

The general structure of the antenna of Z. dixolophella is similar to that in other pyralid moths, such as Chilo partellus (Swinhoe) (Hansson et al. 1995), Ostrinia nubilalis Hübner (Cornford et al. 1973, Hallberg et al. 1994), Homoeosoma nebulella Den. and Shiff (Faucheux 1991), Conegethes punctiferalis (Guenée), and Conegethes sp (Honda and Hanyu 1989). The Böhm hairs of Z. dixolophella are morphologically similar to those on the scape and pedicel of O. nubilalis (Cornford et al. 1973), C. punctiferalis and Conegethes sp (Honda and Hanyu 1989), and H. nebulella (Faucheux 1991). Schneider (1964) suggested that these hairs may have a mechano-sensitive function, whereas Cuperus (1983) reported that they may have a mechano-receptor function in the scape-pedicel union.

Typically, scales occur along with sensilla on the surface of the antenna. Van der Pers et al. (1980) considered that the function of scales is not to protect the sensilla from mechanical damage, but that this disposition of sensilla and scales on the antenna favors the insect’s ability to detect the direction of a stimulus. Wall (1978) speculated that perhaps the function of the scales is not to protect a zone of the antenna of little importance, but that they may be a mechanism to trap and concentrate odorous molecules.

To our knowledge, the tuft of hairs observed at the base of antenna and the protuberances of the first seven segments found on male antennae of Z. dixolophella have not been reported for other pyralids and possibly for other moths. We do not know the function of these structures in male antennae. However, because they are only present in one sex, these structures may have a function related to sexual behavior.

In this study, we have identified only one type of long sensilla trichoidea on male antennae that is absent on female antennae. The presence of these sensilla has also been reported in other pyralids, such as C. partellus (Hansson et al. 1995), O. nubilalis (Cornford et al. 1973, Hallberg et al. 1994), H. nebulella (Faucheux 1991), and C. punctiferalis and Conegethes sp (Honda and Hanyu 1989). For example, in O. nubilalis, three subtypes of sensilla trichoidea were found on male antennae that differ in length and number of sensory cells (Hallberg et al. 1994). In several moth species, it has been demonstrated that the long sensilla trichoidea on male antennae may respond to the female sex pheromone (Boekh et al. 1965, Schneider and Steinbrecht 1968, Van der Pers and Den Otter 1978, Kaissling 1979, Zacharuk 1985, Hansson et al. 1995). Of the three types of sensilla trichoidea found on O. nubilalis male antennae, one contained receptor cells that responded to the two pheromonal components produced by the female in addition to a behavioral antagonist. Another sensillum had cells that responded only to the major pheromonal component. The third type had receptor cells that did not respond to the sex pheromone compounds (Hallberg et al. 1994, Hansson et al. 1994). We do not know the function of the sensilla trichoidea subtype II on female antennae of Z. dixolophella, but it is possible that they detect its own sex pheromone. Studies using the single-cell recording technique would be needed to prove this suggestion. Other studies have documented that antennae on females of Spodoptera littoralis (Boisduval) (Ljunberg et al. 1993) and Spodoptera frugiperda (J. E. Smith) (Malo et al., personal communication) respond to conspecific sex pheromones, although the behavioral significance of this finding is unknown. Females of Trichoplusia ni (Hübner) detect their own sex pheromone, and in response, aggregate to increase the chance of mating (Birch 1977). Females of Heliothis armigera (Hübner) and Helicoverpa zea Boddie detect conspecific sex pheromones, moving away from calling females (Saad and Scott 1981). When Choristoneura fumiferana (Clemens) females detect sex pheromone, they are stimulated to oviposit (Palanaswamy and Seabrook 1978).

Sensilla chaetica of Z. dixolophella are similar in structure to those reported for other pyralids (Cornford et al. 1973, Honda and Hanyu 1989, Anderson and Hallberg 1990, Faucheux 1991, Hallberg et al. 1994, Hansson et al. 1995). Hallberg et al. (1994) reported that these sensilla function as contact chemoreceptors based on their structure. In other families of Lepidoptera, it has been suggested that they function as contact chemoreceptors as well (Schneider 1964), but Altner et al. (1977) reported that this sensillum has a bimodal taste/tactile function in the cockroach Periplaneta americana (L.).

Sensilla styloconica, as observed in Z. dixolophella, have also been reported in H. nebulella (Faucheux 1991), O. nubilalis (Cornford et al. 1973, Hallberg et al. 1994), and C. punctiferalis and Conegethes sp (Honda and Hanyu 1989). The apical part of this sensillum is different from that reported for other moths (Jefferson et al. 1970, Albert and Seabrook 1973, Cuperus 1983). With the technique used in the current study, it was not possible to observe the apical pore of sensilla styloconica as reported with H. nebulella antennae (Faucheux 1991). Sensilla styloconica have been shown to be contact chemoreceptors in Noctuidae and Tortricidae (Jefferson et al. 1970, Albert and Seabrook 1973) and thermo-/higro-sensitive receptors in Pyralidae (Hallberg et al. 1994). However, in Yponomeutidae, Van der Pers et al. (1980) have suggested that these sensilla may have some other sensory function because they are located under scales, where a contact chemoreceptive function is not possible.

Sensilla coeloconic described here for Z. dixolophella are similar to those reported for H. nebulella, C. punctiferalis and Conegethes sp, C. partellus, and O. nubilalis (Cornford et al. 1973, Honda and Hanyu 1989, Faucheux 1991, Hallberg et al. 1994, Hansson et al. 1995). However, in the current study, only one morphological type of this sensillum was found on Z. dixolophella antennae, in contrast to C. partellus antennae, where two morphological types of sensillum coeloconicum were described (Hansson et al. 1995). The number of sensilla coeloconica reported here for Z. dixolophella is greater than reported for H. nebulella (Faucheux 1991) and O. nubilalis (Cornford et al. 1973). This sensillum has been considered to house olfactory-receptor cells in C. partellus (Hansson et al. 1995), H. nebulella (Faucheux 1991), and O. nubilalis (Cornford et al. 1973, Hallberg et al. 1994), possibly sensitive to plant volatiles (Van der Pers 1981). In insects other than moths, these sensilla have been considered to have humidity and temperature receptor cells (Davis and Sokolove 1975, Altner et al. 1977).

The “ear-shaped” sensilla auricillica present in Z. dixolophella are similar in shape to those found on O. nubilalis, H. nebulella, and C. punctiferalis and Conegethes sp antennae (Honda and Hanyu 1989, Faucheux 1991, Hallberg et al. 1994). However, the sensilla auricillica in O. nubilalis are longer than those occurring on Z. dixolophella. These sensilla were considered to be olfactory receptors for plant volatiles (Boekh et al. 1965, Kaissling 1971).

Sensilla squamiforma have been reported more frequently in other families than in Pyralidae (Cuperus 1983, Lavoie and McNeil 1987, Honda and Hanyu 1989). The sensilla squamiforma found here are similar in shape to those reported in H. nebulella (Faucheux 1991), but differ in length. Those found in Z. dixolophella are longer. These sensilla are located on the scape, the pedicel, and the first segment of the flagellum in both sexes of Z. dixolophella. A similar distribution for these sensilla has been reported in other moth species (Cuperus 1983).

From this study, we know the different types of sensilla present on the antennae of both sexes of Z. dixolophella. This knowledge will allow us to better understand results obtained in behavioral and electrophysiological bioassays aimed at identifying the semiochemicals mediating sexual and host-finding behavior of Z. dixolophella.

Acknowledgments

V.C.G. wishes to thank the Instituto Politécnico Nacional (Grant COTEPABE 295) and COFAA and CONACYT (Grant 91489) for financial support, A. Virgen (ECOSUR) for his help in collecting biological material, and A. Gonzalez of Rancho Cazanares for allowing us to collect insects from his farm. We also thank the bee laboratory (ECOSUR) for microscope facilities.