Introduction

An antenna is an important sensory organ in arthropods (Clarke 1973). Particularly the sensilla, or sensory hairs and sensory cavities, on the surface of the antenna serve as mechanical and chemical receptors in search of hosts suitable for oviposition and females suitable for mating. Understanding the morphology and distribution of antennal sensilla is therefore an important first step to study behavioral and ecological mechanisms in arthropods.

Beetles of the subfamily Bruchinae, especially Callosobruchus, damage post-harvest dry fabaceous crop seeds and reduce their commercial value (Southgate 1979). Their various ecologies and behaviors have been well studied for pest species (Tuda 2007). The antennae of bruchine beetles carry various sensilla with different functions (Hu et al. 2009; Rup 1988) and play an important role in searching/discriminating mates (Shimomura et al. 2010c), detecting already-laid eggs on seeds (Messina et al. 1987) and deciding to lay eggs on host seeds and/or seed pods (Adhikary et al. 2014; Parr et al. 1998). The total number of antennal sensilla of Bruchinae have been studied and compared between sexes in Callosobruchus maculatus (F.) (Hu et al. 2009; Rup 1988) as in other insects (Zacharuk 1985) and between Callosobruchus chinensis (L.) and C. maculatus (Hu et al. 2009). However, sexual differences in antennal sensillum size and per-area-density has rarely been studied (but see Awad et al. 2014, 2015 for the sexual difference in the peach fruit fly).

Callosobruchus rhodesianus (Pic) (Coleoptera: Chrysomelidae: Bruchinae) is a stored bean pest beetle that damages dry leguminous seeds, particularly cowpeas, and inhabits subtropical/temperate Central through Southern Africa and the Middle East (Delobel 2012; Giga and Smith 1983, 1987; Giga et al. 1993; Tuda et al. 2005). Male C. rhodesianus respond specifically to conspecific female sex pheromone (Shimomura et al. 2010a, b, c) while males of other congeneric species respond not only to conspecific females’ pheromone but also to congeneric females’ (Shimomura et al. 2010c). Female C. rhodesianus sense oviposition-repellent chemicals of the same and other species on host seeds and restrain themselves from laying eggs (Giga and Smith 1985; see Sakai et al. 1986 for the chemical secreted). The previous report (Giga and Smith 1985) suggests there must be sexual differences in antennal sensilla. To date, antennal sensilla have not been studied in this species.

Here, we aim to clarify sexual differences in antennal sensilla and segments in C. rhodesianus. We study the morphology, abundance, density and distribution of the antennal sensilla and size of antennomeres and compare them between sexes in C. rhodesianus and with those of congeneric C. chinensis and C. maculatus studied by Hu et al. (2009).

Materials and methods

Insects

Adults of Callosobruchus rhodesianus were collected from cowpeas imported from South Africa in 1977. They were reared in a clear plastic cup (9 cm diameter, 5 cm height) containing about 20 g azuki bean seeds (Vigna angularis) in the laboratory, in the incubator maintained at 24 ± 1 °C, 65 ± 5 % RH, and with a 16-h light:8-h dark cycle. These beetles develop into the adult from an egg in about a month at 25 °C (Giga and Smith 1983).

Observation and measurement of antennal sensilla

We fixed newly emerging adults, killed with 50 °C heat for 24 h, to the carbon paper with water-soluble glue. We recorded the image of the scape, pedicel and each of nine flagellomeres, especially flagellomere 7, of 20 normal individuals (N = 10 for each sex; N: number of individuals examined) (Fig. 1) using the scanning electron microscope (Miniscope TM3000, Hitachi High Technologies, Tokyo). Flagellomere 7 (Fig. 1) was chosen to measure the length and width of sensilla and the width of the antennomere at 400× magnification because it is the largest segment among all antennomeres (Watanabe and Sugimoto 1988). We counted the number of sensilla trichodea 1, sensilla trichodea 2 and sensilla cavitae in the 66 μm × 100-μm area around the center of the flat surface of flagellomere 7 (N = 10 for each sex) (Fig. 2). We also recorded images of all antennomeres of each sex to check the distribution of antennal sensilla on each antennal segment and measured the length and diameter of different types of sensilla (N = 2–3 for each sex, 2–3 sensilla per individual for each type of sensilla). For classifying sensilla, we applied the terminology of Hu et al. (2009) and Zacharuk (1985).

Fig. 1
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Antennomeres on the dorsal side in Callosobruchus rhodesianus (male with 11 antennomeres). Sc scape, Pe pedicel and Fl flagellomere

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Fig. 2
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Antennal sensilla (sensilla trichodea types 1 and 2, ST1, ST2) and sensilla cavitae (SCa) on flagellomere 7 on the dorsal side of a female and b male C. rhodesianus. The rectangles indicate the 66 μm × 100 μm area for counting the sensilla to derive their density

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We also counted the total number of each type of antennal sensilla on the dorsal and ventral sides as follows. We first anesthetized freshly emerged adult beetles in the freezer (−20 °C) for 10 min and excised a pair of antennae from heads with forceps. We fixed them in 2.5 % glutaraldehyde (Nacalai Tesque, Kyoto, Japan) and kept them overnight at 4 °C. Then, they were rinsed in phosphate buffer (0.1 M, pH 7.2, Nacalai Tesque, Kyoto, Japan) three times for 15 min each. Finally, the antennae were dehydrated in a graded ethanol series of 30, 50, 70, 80, 90 and 100 % each for 15 min. Either the dorsal or ventral side of one of a pair of antenna of 2–4 individuals for each sex was fixed onto the carbon paper with double-sided sticky tape and sputter coated with osmium using the ion sputtering device (HPC-ISW, Vacuum Device, Ibaraki, Japan). We examined the morphology and abundance of antennal sensilla on each side of antennae at 2200–150,000× magnifications using the scanning electron microscope (SU-8000, Hitachi, Tokyo, Japan) set at 1 kV.

We measured the length of each antennal segment, length of the elytron and width of flagellomere 7 by using the microscope (VH-5500, Keyence, Osaka, Japan) to study the relation of antennomere sizes with the sex and body size (i.e., elytral length). For this measurement, we used specimens from the same laboratory population as above, preserved in ethanol.

Statistical analysis

We tested sensilla density, length and diameter with general linear mixed models (GLMMs) using sex as an explanatory variable and individual identity as a random variable. We tested lengths of all antennomeres, total length and the width of flagellomere 7 with general linear models (GLM) using sex and elytral length as explanatory variables. Familywise errors associated with multiple comparisons (among different antennomeres, between the length and width of sensilla, and densities among different sensilla types) were controlled with Holm’s method or Holm-Bonferroni sequential correction method (Holm 1979). All the statistical analyses except for Holm’s method, in which we manually corrected the p values by the number of comparisons, were performed using JMP10.0.

Results

General description of antennae of C. rhodesianus

Both females and males of C. rhodesianus carry serrated antennae (Fig. 1). The antenna of this species consists of the scape, pedicel and nine flagellomeres (Fig. 1). The antennomeres vary in size (Table 1). The antennomeres of males are significantly longer than those of females, except for flagellomeres 7, 8 and 9 (Table 1). The width of flagellomere 7 is larger in males than in females (Table 1). The total length of the male antenna is significantly longer than that of the female antenna (Table 1). Lengths of flagellomeres 2, 6 and 8 and total length of antennae are correlated with the elytral length (Table 1). The sexual differences remain significant after p values have been corrected with Holm’s method, but all the correlations of antennomere sizes with body size are lost with Holm’s method (Table 1). Total length of the antenna is significantly correlated with body size (Table 1).

Table 1 Lengths of antennomeres and width of flagellomere 7 of female (N = 9) and male (N = 12) Callosobruchus rhodesianus (N: number of individuals examined) (mm, mean ± SE)
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Types and distribution/abundance of sensilla

Two types of sensilla trichodea (ST1, ST2), one each of type sensilla cavitae (SCa), sensilla chaetica (SC), three types of sensilla basiconica (SB1, SB2, SB3) and Böhm bristles (BB) are differentiated according to their size and shape on the antennae of both females and males. Their distributions and abundance among antennomeres are summarized in Table 2. Overall, there is no difference in the total number of sensilla between the dorsal and ventral sides, except that there are more BBs on the ventral side than on the dorsal side of the pedicel in both sexes (Table 2).

Table 2 Abundance (mean ± SE) (N) (N: number of individuals examined) and distribution of eight sensilla types: ST1: sensilla trichodea type 1, ST2: sensilla trichodea type 2, SCa: sensilla cavitae, SC: sensilla chaetica, SB1: sensilla basiconica type 1, SB2: sensilla basiconica type 2, SB3: sensilla basiconica type 3, BB: Böhm bristles, among antennomeres of C. rhodesianus; female (F) and male (M)
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Sensilla trichodea type 1 (ST1)

The ST1s are long and have the most abundant hairs (Tables 2, 3) found on all antennomeres (Table 2). They have sharp tips with a pore (Fig. 4a, b). They have a side wall with longitudinal grooves and are nearly straight or slightly curved (Figs. 3, 4a, b). They are inserted into wide sockets (Figs. 3, 4a). The lengths of the ST1 in females and males are similar, but the diameter is shorter in females than males, even after correction with Holm’s method (Table 3). Males have more ST1s on the antenna in total than do females and on flagellomeres 3 and 5 (Table 2). More ST1s are found on the ventral than dorsal side of flagellomere 5 although the statistical significance is lost after Holm’s correction. Density of the ST1 on flagellomere 7 is significantly higher in females than in males, even after correction with Holm’s method (Table 4).

Table 3 Sizes of antennal sensilla of females and males in C. rhodesianus
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Fig. 3
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Antennal sensilla (sensilla trichodea types 1 and 2, ST1, ST2) and sensilla cavitae (SCa) of a male C. rhodesianus (on flagellomere 2)

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Fig. 4
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Antennal sensilla (sensilla trichodea type 1, ST1) of a male C. rhodesianus, a whole view, b tip

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Table 4 Density of area around the center of flat surface on flagellomere 7 in C. rhodesianus (N = 10 for each sex)
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Sensilla trichodea type 2 (ST2)

The ST2s are blunt-tipped straight hairs and have pores on their surface (Figs. 3, 5a, b). They are inserted tightly into wide sockets (Figs. 3, 5a). The ST2s are present on flagellomeres 3–9 (Table 2). The length and diameter of the ST2 in females and males are similar (Table 3). Total abundance of the ST2 on antennae is not different between sexes (Table 2). Densities of ST2 on flagellomere 7 in females and males are similar (Table 4).

Fig. 5
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Antennal sensilla (sensilla trichodea type 2, ST2) of a male C. rhodesianus, a whole view, b tip

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Sensilla cavitae (SCa)

The diameters of the SCa in females and males are similar (Table 3; Figs. 3, 6). Males have more SCa on antennae in total and on flagellomeres 3 and 6–8 (Table 2). Densities of the SCa on flagellomere 7 in females and males are similar (Table 4). Both sexes have this type of sensilla on all antennomeres (Table 2), and we could not find hairs in the pit from outside (Fig. 6).

Fig. 6
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Antennal sensilla (sensilla cavitae, SCa) of a male C. rhodesianus

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Sensilla chaetica (SC)

The SC occurs on all antennomeres except the scape of both sexes (Table 2). This type of sensilla is the longest and thickest (Table 3) and has a grooved surface and is straight with a blunt tip (Fig. 7a, b). They are inserted into wide sockets (Fig. 7c). The length and diameter of the SC in females and males are similar (Table 3). There is no difference in the total abundance of the SC between sexes (Table 2).

Fig. 7
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Antennal sensilla (sensilla chaetica, SC) of a male C. rhodesianus, a whole view, b tip, c socket

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Sensilla basiconica type 1 (SB1)

The SB1 is a straight hair with a smooth surface with pores and blunt tip (Figs. 8, 9a, b). The length and diameter of the SB1 in females and males are similar (Table 3). The SB1s are inserted into wide sockets (Fig. 8). The SB1s are distributed on flagellomeres 3–9 in both sexes (Table 2). Most of them are located on the lateral side of the apex and front side of the above flagellomeres. There is no difference in the total abundance of the SB1 between sexes (Table 2).

Fig. 8
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Antennal sensilla (sensilla basiconica types 1, 2 and 3, SB1, SB2 and SB3) of a male C. rhodesianus (on flagellomere 9)

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Fig. 9
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Antennal sensilla (sensilla basiconica type 1, SB1) of a male C. rhodesianus, a whole view, b tip

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Sensilla basiconica type 2 (SB2)

The SB2 is a straight hair with a grooved surface and blunt tip with a pore (Figs. 8, 10). The length and diameter of the SB2 in females and males are similar (Table 3). The SB2s are inserted into wide sockets (Fig. 10). They are distributed on flagellomeres 3–9 in both sexes (Table 2). Most of them are located on the lateral side of the apex and front side of the above flagellomeres. There is no difference in total abundance of the SB2 between sexes (Table 2).

Fig. 10
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Antennal sensilla (sensilla basiconica type 2, SB2) of a male C. rhodesianus (on flagellomere 9)

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Sensilla basiconica type 3 (SB3)

The SB3 is short and has a smooth surface and blunt tip (Figs. 8, 11). The length and diameter of the SB3 in females and males are similar (Table 3). The SB3s are inserted into wide sockets (Fig. 11). They are distributed on flagellomere 9 in both sexes (Table 2) and are located a bit above the center. There is no sexual difference in total SB3 abundance on antennae.

Fig. 11
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Antennal sensilla (sensilla basiconica type 3, SB3) of a male C. rhodesianus (on flagellomere 9)

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Böhm bristles (BBs)

The BB sensillum has a triangular shape with a blunt tip and is straight with a smooth surface (Fig. 12a, b). The length and diameter of the BB in females and males are similar (Table 3). The BBs are inserted into wide sockets (Fig. 12a, b). The BB occurs on the base of the scape and pedicel at the joints between the scape and the head and between the scape and the pedicel of females and males (Table 2). There is no sexual difference in total BB abundance on antennae but there are more on the ventral side than the dorsal side of the pedicel as described above (Table 2).

Fig. 12
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a Antennal sensilla (Böhm bristles, BBs) of a male C. rhodesianus (on the scape), b whole

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Discussion

We compared the abundance and sizes of eight types of sensilla and the densities of the ST1, ST2 and SCa between females and males in the bruchine bean beetle, C. rhodesianus, a pest of cowpeas and other stored beans. In congeneric species, the ST1 is considered to serve as an olfactory receptor (Hu et al. 2009), and this sensillum is also similar to sensilla chaetica reported in two other Callosobruchus species (type 2 in Mbata et al. 1997). The ST1 is the most abundant sensillum with grooves, and its tip has a pore (Table 2, Fig. 4a), which may indicate that this sensillum has a gustatory function in this species. The ST2 is considered to serve as a sex pheromone receptor in C. maculatus (Hu et al. 2009). The number of the ST2s was not different between sexes, and the surface is smooth with pores (Fig. 5b), which may indicate that this sensillum has an olfactory function in this species. The SCa is considered to function as a receptor for chemical substances, temperature and humidity (Zacharuk 1985). We found more ST1 and SCa on male antennae than on females’ (Table 2), which may indicate these sensilla detect female pheromone. The sexual difference in ST1 abundance was found also in C. chinensis but not in C. maculatus (Hu et al. 2009). By contrast, the sexual difference in SCa abundance was not observed in C. chinensis (Hu et al. 2009). The SCa is absent in C. maculatus (Hu et al. 2009).

The ST1 is sparser and thicker in males than in females in C. rhodesianus (Tables 3, 4; Fig. 2). The STs are also thicker in males in the peach fruit fly, Bactrocera zonata (Awad et al. 2014). This is probably because there are additional sensory cells in each ST1 hair of males to receive sex pheromone from females. By contrast, neither the abundance nor the density of the ST2 was significantly different between the sexes (Tables 2, 4). The abundances of SB1, SB2, SB3 and BB were also similar in both sexes (Table 2). There may also be no sexual difference in their functions, which contrasts with previous suggestions for congeners (Hu et al. 2009; Zacharuk 1985). The SB1 has pores on its surface (Fig. 9b), which indicates that this sensillum is a chemoreceptor. The SB2 has a grooved surface with a pore on its tip (Fig. 10), which indicates that this sensillum is also a chemoreceptor.

There are more ST1s on the ventral than dorsal side on flagellomere 5 (although the statistical significance is lost after Holm’s correction). This is probably because when beetles search for cues for food and oviposition site, they normally use the ventral side. There are also more BBs on the ventral than on the dorsal side of the pedicel (even after Holm’s correction). This sensillum, located only on intersegmental joints of the scape and pedicel, may work as a mechanoreceptor (Zacharuk 1985) and sense the position and movement of antennae. When moving their antennae downward, C. rhodesianus may perceive the antennal position more sensitively than when moving the antennae upward.

Most of antennomeres in this species are longer in males than in females (Table 1) as in C. chinensis (Hu et al. 2009). The sexual difference in the shape of antennae is smaller in this species than in C. chinensis in which males have comb-shaped antennae at their terminal ends (Nakamura 1969). Interestingly, the sexual difference in antennomere length is absent for terminal antennomeres in C. rhodesianus, while it is most pronounced in C. chinensis and C. maculatus (Table 1; Hu et al. 2009).

The length and width of C. rhodesianus’s antennal sensilla (Table 3) are shorter than those of C. maculatus and C. chinensis (Hu et al. 2009), although the basic structure of antenna and sensilla types are similar except the SB3. The SB3 is similar to the grooved pegs (GP) in the two congeneric species (Hu et al. 2009). Since the total length of an antenna is correlated with body size (Table 1), the smaller antennomeres might result from a correlation with the smaller body of C. rhodesianus than the body of the congeneric species (Sakurai, Yanagi, Tuda unpublished), if the intraspecific relation between antennal length and body size holds for interspecies comparison.

Distributions of the ST1, SC, SB1, SB2 and BB among different antennomeres are the same as those in congeneric species (Table 5). By contrast, our observation shows that distributions of the SCa, ST2 and SB3 are different among C. rhodesianus, C. maculatus and C. chinensis (Hu et al. 2009; Table 5). The SCa is absent from all antennomeres in C. maculatus but present on antennomeres except for the scape in C. chinensis (Hu et al. 2009; Table 5). We found the SCa on all antennomeres in C. rhodesianus (Tables 2, 5). The following causes are possible for the difference in SCa distributions among the three congeneric species. First is the difference in the habitat climate. Callosobruchus maculatus inhabits tropical areas where temperature changes are small, while C. rhodesianus is distributed in a temperate/subtropical zone where there is more seasonality. Therefore, the SCa may have developed to sense and accommodate ambient conditions in C. rhodesianus. Second is the difference of sex pheromone. Two kinds of sex pheromones are known in Callosobruchus. One is volatile chemicals transmitted through the air, and the other is nonvolatile chemicals triggering copulation as males touch females (i.e., contact pheromone; Cork 1991; Nojima et al. 2007; Phillips et al. 1996; Shimomura 2007; Shimomura et al. 2010d, 2016; Tanaka et al. 1981, 1982). The SCa is found both in C. chinensis and C. rhodesianus, and males have more SCa in C. rhodesuanus. Their sex attractant pheromones are similar (homosesquiterpene aldehydes; Shimomura 2007; Shimomura et al. 2010a, b), while the contact pheromone of C. rhodesianus is quite different from that of other species (Shimomura et al. 2016). From an evolutionary point of view, C. rhodesianus is more closely related to C. maculatus than to C. chinensis based on the molecular phylogeny of the genus (Tuda et al. 2006). The SCa may be either ancestral, being lost in C. maculatus later, or obtained independently in C. chinensis and C. rhodesianus (i.e., convergence) because of a common selective force, such as the wide range of habitat climate (Giga and Smith 1983) and/or the similar sex pheromone. By contrast, we suggest that the difference between C. rhodesianus/C. maculatus and C. chinensis in the distribution of the ST2 (Table 5) is due to the effect of a common ancestor (i.e., a phylogenetic signal). The SB3 occurred on flagellomeres 3–9 in C. chinensis and C. maculatus, but only on flagellomere 9 in C. rhodesianus. The SB3 resembles type 6 (sensilla basiconica) of C. subinnotatus (Mbata et al. 1997), which may work as chemo- or thermoreceptors (Hu et al. 2009; Mbata et al. 1997; Zacharuk 1985). The distribution of SB3 on the antenna of C. rhodesianus is similar to that of C. subinnotatus (Mbata et al. 1997). The habitats for these two species overlap in part (Appleby and Credland 2001; Delobel 2012). For the phylogenetic relation of C. rhodesianus and C. subinnotatus, they share a common ancestor (Tuda et al. 2006). The interspecies similarity in the SB3 distribution might be due to independent losses of the SB3 by a common selective force, stochasticity or to a common ancestor.

Table 5 Comparison of antennal sensilla distribution in C. rhodesianus with that in other Callosobruchus species
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In this study, we compared the morphology, abundance and per area density of antennal sensilla between females and males in C. rhodesianus for the first time. We found that the abundance, density and thickness of the ST1, which is the most abundant, long sensilla type, and the abundance of the SCa differ between sexes. In comparison with Hu et al.’s (2009) study of C. maculatus and C. chinensis, we also found interspecies differences in among-antennomere distributions of the ST2, SCa and SC.