- Email: [email protected]
Proboscis sensilla of the black cutworm Agrotis ypsilon (Rottemberg) (Lepidoptera: Noctuidae)
Journal of Asia-Pacific Entomology 17 (2014) 295–301
Contents lists available at ScienceDirect
Journal of Asia-Pacific Entomology journal homepage: www.elsevier.com/locate/jape
Proboscis sensilla of the black cutworm Agrotis ypsilon (Rottemberg) (Lepidoptera: Noctuidae) Shuang Xue, Bao-zhen Hua ⁎ State Key Laboratory of Crop Stress Biology for Arid Areas, Key Laboratory of Plant Protection Resources and Pest Management of the Education Ministry, Entomological Museum, Northwest A&F University, Yangling, Shaanxi 712100, China
a r t i c l e
i n f o
Article history: Received 17 August 2013 Revised 16 January 2014 Accepted 28 January 2014 Available online 5 February 2014 Keywords: Glossata Morphology Moth Mouthparts Sensilla styloconica
a b s t r a c t Proboscis sensilla are important for feeding biology in Lepidoptera, and are also valuable characters for species recognition and phylogenetic analysis. However, proboscis has not been satisfactorily explored in many groups in Lepidoptera so far. Here we examined the proboscis sensilla of the black cutworm Agrotis ypsilon (Rottemberg), a cosmopolitan agricultural pest of great economic significance, using scanning electron microscopy. Three types of sensilla were found on the proboscis: sensilla chaetica, sensilla basiconica, and sensilla styloconica. Sensilla chaetica occur only on the external surface of the proboscis and become shorter and more scattered toward the tip. Sensilla basiconica are arranged in longitudinal rows on the external proboscis and one longitudinal row in the inner food canal. Sensilla styloconica are the most characteristic sensilla on the proboscis, consisting of a single sensory cone inserted at the top of a stylus with six or seven longitudinal ribs, and are concentrated on the tip region, and are much longer and more numerous in females than in males. The role of proboscis sensilla in the feeding habit prediction is briefly discussed. © 2014 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved.
Introduction The proboscis of higher Lepidoptera is a complex fluidic system consisting of two extremely elongated galeae and functions like a drinking straw through which floral nectar and various kinds of liquids from rotten fruits, sweat, tear, dung, carrion, and urine are sucked from the tip into the head and the digestive tract (Scoble, 1992; Kingsolver and Daniel, 1995; Krenn, 2010; Monaenkova et al., 2012). The external surface of the proboscis has a sharp boundary separating a hydrophilic drinking region and a hydrophobic non-drinking region, and the hydrophilic region is at the tip region (Lehnert et al., 2013). The tip region is characterized by rows of intake slits leading into the food canal (Krenn, 1998; Lehnert et al., 2013). Each galea bears cuticular processes and various types of sensilla, which play important roles in feeding activities in the Lepidoptera (Krenn et al., 2005; Krenn, 2010). Previous investigations on proboscis sensilla in Lepidoptera are mainly concentrated on butterflies (Papilionoidea) and moths in families Noctuidae, Geometridae, Erebidae, Tortricidae, Neopseustidae, Saturniidae, and Pyralidae (Sellier, 1975; Faucheux, 1991; Paulus and Krenn, 1996; Krenn, 1998; Krenn and Penz, 1998; Walters et al., 1998; Krenn et al., 2001; Zaspel et al., 2011). Within Noctuidae, fruit-
⁎ Corresponding author. Tel./fax: +86 29 87091342. E-mail address: [email protected] (B. Hua).
piercing, nectar-feeding, skin-piercing, blood-sucking, tear-feeding, and sweat-feeding moths are frequently studied for their proboscis and sensillar morphology (Bänziger, 1975, 1982, 1988, 1992, 2007; Büttiker et al., 1996; Zaspel et al., 2007; Zenker et al., 2011). To our knowledge, however, the proboscis sensilla of the black cutworm Agrotis ypsilon (Rottemberg), a cosmopolitan agricultural pest of great economic significance, have not been investigated so far. Agrotis ypsilon (Rottemberg) is a polyphagous herbivorous moth, with the larvae severing the basal stems of seedlings of corn (Zea mays), cottons (Gossypium hirsutum, G. barbedense), tobacco (Nicotiana tobacum), wheat (Triticum aestivum), various vegetables, and turf grasses, and frequently causing serious damage to underground roots and tubers (Rings et al., 1975; Showers, 1997). This moth completes 1–7 generations per year in different areas of the world, depending on weather conditions. The larvae go through six or seven instars. The life span of the imago lasts 12–33 days depending on air humidity, temperature and feeding conditions of the larvae. The black cutworm is known as a migratory pest with extraordinary flexibility in the timing and spacing of reproduction and population growth (Showers, 1997). Although the distribution, life history, and host ranges have been well documented in A. ypsilon, its feeding behavior of the adults has not been satisfactorily investigated, especially the morphology of the proboscis sensilla. In this paper, we compared the external morphology of the proboscis sensilla of female with male A. ypsilon using scanning electron microscopy, in an attempt to predict its feeding behavior of the adults.
http://dx.doi.org/10.1016/j.aspen.2014.01.014 1226-8615/© 2014 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved.
296
S. Xue, B. Hua / Journal of Asia-Pacific Entomology 17 (2014) 295–301
Terminology for proboscis sensilla mainly follows Shields (2008) and Faucheux (2013).
Results Gross morphology of the mouthparts
Materials and methods Insect collection and preparation Adults of A. ypsilon were obtained with light traps at the Zhuque National Forest Park and the campus of the Northwest A&F University, Shaanxi Province of the central China from July to August in 2012. The heads were severed from the ether anesthetic alive insects and then fixed in Carnoy's fixative solution (95% ethanol:glacial acetic acid = 3:1, v/v) for 24 h before being stored in 75% ethanol.
Scanning electron microscopy For scanning electron microscopy (SEM), the mouthparts were cleaned with an ultrasonic cleaner for 30 s. After dehydration in ethanol series, the samples were submerged in tertiary butanol for freeze drying. They were then mounted onto SEM stubs using a double graphite adhesive tape, coated with gold in a sputter coater, and examined in a Hitachi S-3400N scanning electron microscope (Hitachi, Tokyo, Japan) at 15 kV.
Measurements Proboscis length and sensilla were measured for 20 individuals (10 females and 10 males). Sensillar length and diameter were measured using a digitizing tablet and the Imaris 7.2.3. The length and diameter of sensilla chaetica were measured in four regions: the proximal, bend, distal, and tip regions. The proboscis has a bend region approximately one-third of the length from the base (Krenn, 1998). The tip region is characterized by rows of intake slits leading into the food canal (Krenn, 1998). From the base to the bend region is the proximal region. The distal region is located between the bend and tip regions. The cuticular processes are hair-like in the proximal and bend regions, spine-like toward the distal and tip regions.
Statistical analysis Proboscis length and the number, length, and diameter of different sensilla were analyzed using the Predictive Analytics Software Statistics 18.0. For these data, the mean number and standard error of mean were calculated. To evaluate and compare differences between the sexes, Student's t-test at α = 0.05 was used.
No distinct difference was found in mouthpart morphology between the female and male A. ypsilon. The proboscis consists of two extremely elongated galeae (Fig. 1A), which connected together to form a sucking tube. The labrum becomes a small plate over the basal proboscis joint, where bristles of pilifers contact the basal galeal joint. The mandibles are reduced and nonfunctional. The basal maxillary sclerites form a tubular component from which the coilable galeae and the maxillary palp emerge. The lacinia is vestigial. The labial palps are threesegmented, extending from the prementum sclerite and forming prominent structures in front of the head, and are equipped with a great number of scales and bristles (Fig. 1B). In the resting position, the proboscis is coiled in a tight spiral beneath the head between the setose labial palps. The coilable galeae are held together by rows of interlocking cuticular processes, which exhibit different shapes on the dorsal and ventral sides of the food canal, which is interlocked by the dorsal and ventral galeal linkages. The dorsal linkage consists of horizontally extending lancet-shaped flat plates and forms a roof over the food tube, while the ventral galeal linkage is composed of a row of hooks and provides firm interlocking structures for the proboscis. The concave median surface of the food tube is composed of smooth plates that are vertically fluted. The proboscis length is 11.86 ± 4.03 mm (9.73–13.48 mm, n = 20). The proboscis can be divided into the proximal, bend, distal, and tip regions. When uncoiled, the proximal region makes up 38% of the total length; the distance from the proximal region to the bend region comprises 10% of the total length; the distal region constitutes about 39%; and the tip region, where fluids are taken up into the food canal, comprises 13% of the total length. The outer surface of the proboscis is fluted with cuticular processes and a great number of sensilla. The cuticular processes have different shapes in different regions of the proboscis. They are hair-like in the proximal and bend regions (Fig. 2A), shorter and more spine-like toward the distal and tip regions (Figs. 2B and C), and interlocked between the consecutive coils in the resting position. Both the female and male A. ypsilon possess three types of sensilla on their proboscis: bristle-shaped sensilla chaetica, uniporous sensilla basiconica, and prominent sensilla styloconica. Different types of sensilla are located on different regions of the proboscis and assume a specific distribution pattern along the proboscis. Sensilla chaetica are the most abundant type of sensilla. Sensilla basiconica are present on the external surface of the proboscis and are the only sensilla that also exist on the internal wall of the food canal. Sensilla styloconica are confined to the tip region of the proboscis.
Fig. 1. Head of male A. ypsilon. (A) The proboscis splits into two galeae due to the preparation procedure. (B) Outer view of a galea. CE, compound eye; Ga, galea; LP, labial palp; TR, tip region. Scale bars: (A) = 500 μm; (B) = 200 μm.
S. Xue, B. Hua / Journal of Asia-Pacific Entomology 17 (2014) 295–301
297
Fig. 2. Sensilla chaetica on the proboscis of male A. ypsilon. (A)–(C) Sensilla chaetica and cuticular processes on the bend, distal and tip regions. (D) Sensilla chaetica on the bend region, showing the sensilla chaetica on the ventral side are longer than those on the dorsal side. Scale bars: (A) and (D) = 50 μm; (B) = 15 μm; (C) = 5 μm.
In A. ypsilon, the tip region of the proboscis makes up 11–15% of the total proboscis length. The tip length of the females is 1588 ± 0.74 μm (n = 10), significantly longer than that in the males (1333 ± 1.25 μm, n = 10). All the three types of sensilla are present in this region, of which sensilla chaetica and sensilla basiconica are rare, scattered, and the shortest. Sensilla chaetica Sensilla chaetica are the most abundant type of sensilla on the proboscis of A. ypsilon, with a total number ranging from 190 to 248 (228 ± 13.67, n = 40) per galea, more frequent in the proximal region
than in the bend, distal, and tip regions. They occur only on the external surface of the proboscis and become more scattered toward the tip. The sensilla chaetica each have an aporous sensory bristle extending from a collared socket and have a grooved surface (Fig. 2B). Each bristle is inserted into a distinct sunken socket, with a basal pore of approximately 3.66 ± 1.88 μm in diameter (n = 7) and protruding at an angle of 30°–45° from the surface on the proximal and bend regions of the proboscis. Then they tend to be upright toward the tip region with some slender basal diameter (Figs. 2B and C; Table 1). The sensilla chaetica become shorter gradually from the proximal to the tip region in both the females and males (Table 1) and are significantly longer on the ventral side of the proboscis than on the dorsal
Table 1 Length, diameter and number of various proboscis sensilla in A. ypsilon. Sensilla Sensilla chaetica
Female Proximal Bend Distal Tip
Length (μm) Length (μm) Diameter (μm) Length (μm) Length (μm) Diameter (μm)
Number per galea External sensilla basiconica
Internal sensilla basiconica
Sensilla styloconica
Data are presented as mean ± SE (n). NS = not significant; * p b 0.05, ** p b
Length (μm) Diameter (μm) Number per galea Length (μm) Diameter (μm) Number per galea Length (μm) Diameter (μm) Number per galea 0.01 in the independent samples t-test.
110.88 65.5 3.67 31.35 5.85 1.44 230 13.61 2.67 59 7.25 2.95 25 58.85 14.09 86
Male ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.39 (32) 1.52 (28) 0.13 (4) 0.81 (16) 0.19 (4) 0.08 (4) 4.98 (10) 0.95 (18) 1.13 (18) 0.96 (10) 0.83 (12) 1.14 (12) 0.24 (20) 4.26 (30) 0.24 (30) 0.98 (10)
108.68 60.78 3.61 28.55 5.49 1.43 224 12.43 2.51 56 7.48 2.47 23 44.69 12.00 78
t-test ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
6.28 (28) 2.44 (28) 0.24 (3) 0.96 (10) 0.20 (5) 0.15 (5) 6.48 (10) 1.19 (17) 1.86 (13) 1.08 (10) 0.25 (8) 0.39 (8) 0.32 (20) 1.37 (28) 0.42 (28) 1.14 (10)
NS * NS NS NS NS * NS NS NS NS NS NS ** * **
298
S. Xue, B. Hua / Journal of Asia-Pacific Entomology 17 (2014) 295–301
Table 2 Length difference of sensilla chaetica between the ventral and dorsal sides in A. ypsilon. Sensilla chaetica Female
Male
Proximal region Bend region Distal region Tip region Proximal region Bend region Distal region Tip region
Length on ventral side (μm)
Length on dorsal side (μm)
t-test
126.76 86.33 44.70 5.85 118.43 82.15 39.59 5.49
103.04 ± 2.11 (18) 56.37 ± 2.16 (10) 19.44 ± 3.59 (6) – 96.88 ± 1.35 (15) 49.87 ± 3.47 (10) 14.41 ± 3.56 (4) –
⁎⁎ ⁎⁎ ⁎⁎
± ± ± ± ± ± ± ±
0.58 (14) 1.24 (18) 1.23 (10) 0.19 (4) 0.17 (13) 2.65 (18) 0.36 (4) 0.20 (5)
– ⁎⁎ ⁎⁎ ⁎⁎ –
Data are presented as mean ± SE (n). –: not calculated. ⁎⁎ p b 0.01 in the independent samples t-test.
side (Fig. 2D; Table 2). On the same region, sensilla chaetica are significantly longer in females than in males (Table 3). Sensilla basiconica Sensilla basiconica each have a short blunt-tipped sensory cone that bears a single terminal pore and a short dome-shaped socket, and exist both on the external and internal surfaces of the galea (Figs. 3A and B). External sensilla basiconica are arranged in irregular rows throughout the dorsal and lateral sides of the proboscis (Fig. 3C). The mean number of the sensilla on each galea is similar between the sexes (Table 1). Internal sensilla basiconica form a single row in the food canal on each galea (Fig. 3D), and are shorter than the external sensilla basiconica, numbering 24 ± 0.83 (n = 40) on each galea. They are elongated sensory cones inserted into dome-shaped sockets. Sensilla styloconica Sensilla styloconica are the most characteristic sensilla on the proboscis, confined to the tip region (Fig. 4A), 82 ± 1.62 in number (75–88, n = 10) on each galea. From the basal to the middle part of the tip region, the sensilla styloconica are arranged in three rows, and are slender on the ventral side of the galea than on the dorsal side (Fig. 4A). Then they become scattered toward the apex. On the dorsolateral side of the tip region, the sensilla styloconica are widely spaced. Sensilla styloconica each consist of a shorter terminal uniporous sensory cone inserted into a larger stylus with longitudinal ribs, originating from an area of thin cuticle surrounded by spine-like cuticular processes (Figs. 4B–E). The gap between adjacent cuticular processes is directly in the face of the rib of the stylus. The middle part of the stylus is usually wider than the proximal part (Figs. 4B and C). Their styli possess six or seven smooth-edged longitudinal ribs (Figs. 4E and F). The sensilla styloconica with six longitudinal ribs are more abundant than those with seven. The sensory cone is fully surrounded by the longitudinal ribs on the basal and middle parts of the tip region, but extends beyond the ribs on the apex of the proboscis (Figs. 4B–E).
On the basis of t-test, a very significant difference in the total length of sensilla styloconica exists between the females and males (p b 0.01, Table 1). The sensilla styloconica are larger and more numerous in the females than in the males (Table 1). Nonetheless, no significant difference exists in the length of the sensory cone between the sexes, both being approximately 10 ± 0.46 μm (n = 28). They become gradually shorter from the base to the apex of the tip region in both the sexes.
Discussion To our knowledge, this paper may represent the first attempt to investigate the proboscis sensilla of A. ypsilon. No differences were found in the types of proboscis sensilla between the two sexes, suggesting that females and males have similar feeding habits in A. ypsilon. The morphology of the proboscis tip region can be used to predict the feeding habits of noctuid moths (Zenker et al., 2011). The proboscises of non-flower-visiting noctuid moths are typically armed with rasping spines, tearing hooks, and erectible barbs, and bear numerous sensilla at the tip region (Bänziger, 1982, 1988; Zenker et al., 2011). Based on our research on A. ypsilon, the tip region of the proboscis is simple with sensilla present alone, and no characteristics of non-flowervisiting moths have been found, confirming that it is a typical flowervisiting moth. In terms of proboscis sensilla, morphological differences were found mainly in the length of sensilla chaetica and the shape and number of sensilla styloconica. The terms sensilla chaetica and sensilla trichodea are often used interchangeably in literature. The longest sensillum chaeticum is 111.96 μm in flower-visiting species, but is up to 214.00 μm in nonflower-visiting species in Nymphalidae (Krenn et al., 2001). In the proboscis of A. ypsilon, the lengths of sensilla chaetica decrease greatly from the proximal region to the tip region. This is likely the adaption for nectar-feeding behavior. The longest sensillum chaeticum occurs in the proximal proboscis region in A. ypsilon, but beyond the mid-proboscis in many Heliconiini, some Limenitidinae, some Brassolinae, Morphinae, and Satyrinae (Krenn and Penz, 1998; Krenn et al., 2001). In some species of Nymphalinae (e.g. Vanessa atalanta, Nymphalis antiopa), remarkably
Table 3 Length difference of sensilla chaetica between the sexes in A. ypsilon. Sensilla chaetica Length on ventral side (μm)
Length on dorsal side (μm)
Proximal region Bend region Distal region Tip region Proximal region Bend region Distal region Tip region
Data are presented as mean ± SE (n). –: not calculated. NS = not significant; * p b 0.05, ** p b 0.01 in the independent samples t-test.
Female
Male
t-test
126.76 ± 0.58 (14) 86.33 ± 1.24 (18) 44.70 ± 1.23 (10) 5.85 ± 0.19 (4) 103.04 ± 2.11 (18) 56.37 ± 2.16 (10) 19.44 ± 3.59 (6) –
118.43 ± 0.17 (13) 82.15 ± 2.65 (18) 39.59 ± 0.36 (4) 5.49 ± 0.20 (5) 96.88 ± 1.35 (15) 49.87 ± 3.47 (10) 14.41 ± 3.56 (4) –
** * * NS ** ** ** –
S. Xue, B. Hua / Journal of Asia-Pacific Entomology 17 (2014) 295–301
299
Fig. 3. Sensilla basiconica on the proboscis of male A. ypsilon. (A) Magnification of an external sensillum basiconicum (ESB); inset shows the terminal pore (P) of a sensillum basiconicum. (B) An internal sensillum basiconicum (ISB) in the food canal. (C) Outer surface of the proboscis, showing external sensilla basiconica arranged in irregular rows. (D) Proboscis in medial view, showing the food canal (FC) interlocked by the dorsal (DGL) and ventral galeal linkages (VGL), internal sensilla basiconica (ISB) and sensilla styloconica (SS). Scale bars: (A) and (B) = 10 μm; (C) = 40 μm; (D) = 80 μm.
long sensilla chaetica are found in the proximal third of the proboscis (Krenn et al., 2001). Sensilla styloconica are the most remarkable sensillar type on the proboscis, composed of a variously shaped stylus and a shorter terminal sensory cone (Krenn, 1998; Petr and Stewart, 2004), and are regarded to be derived from sensilla basiconica (Faucheux, 2013). Their styli are either smooth and ellipsoid or longitudinally ribbed and stellate (Faucheux, 1991; Krenn, 1998; Krenn and Penz, 1998; Zaspel et al., 2011). In some species the sensilla styloconica bear terminal spines around the sensory cone, and the ribs are serrate or very short and practically absent in some other species. The styli of A. ypsilon bear longitudinal ribs, similar to those of Choristoneura fumifernana (Walters et al., 1998), Noctua pronuba, Hypocala rostrata, Eublemma pyrochroa (Büttiker et al., 1996), Helicoverpa armigera, and Helicoverpa assulta (Wang et al., 2012). However, the number of longitudinal ribs is varied among different species. In C. fumifernana, each sensillum styloconicum consists of a single sensory peg inserted at the top of a stylus that bears 6–9 ribs extending from the base of the stylus to the sensory peg socket (Walters et al., 1998). In N. pronuba and H. rostrata, their styli are ridged with 6–8 ribs. In E. pyrochroa, the sensilla styloconica are arranged in two rows and their styli are compressed, bearing 4 or 5 longitudinal ribs with apical points surrounding the sensory cone (Büttiker et al., 1996). In Helicoverpa armigera and H. assulta, each sensillum styloconicum has a stylus bearing six longitudinal ribs (Wang et al., 2012). While in A. ypsilon, the styli have 6 or 7 longitudinal rids. In general, the sensory cone is uniporous and inserted centrally at the top of the stylus. In Heliconiini, however, the cone is at the periphery of the stylus (Krenn et al., 2001). The sensory cone in an arctiid moth is with additional wall pores on the sensory cone (Altner and Altner, 1986). In A. ypsilon, this structure exhibits the common uniporous cone inserted centrally at the top of the stylus.
According to Krenn and Kristensen (2000), a stylus equipped with 4–6 longitudinal ribs was found in some species of Prodoxidae, Adelidae and all Incurvarioidea, in the Palaephatoidea, and in many Ditrysia (non-apoditrysians; Tortricidae; Arctiidae; Pyralidae; Noctuidae; Papilionoidea). Therefore, this type of stylus was regarded as the plesiomorphy for sensilla styloconica on the lepidopteran proboscis (Krenn and Kristensen, 2000). If this is the case, the sensilla styloconica of A. ypsilon proboscis may represent an apomorphy because the stylus is equipped with 6 or 7 longitudinal ribs. Sensilla chaetica are regarded as mechanosensitive on the basis of ultrastructural studies. Bristle-shaped sensilla on the proboscis may provide information on the diameters of the corolla and the depth of proboscis insertion during probing (Krenn, 1998, 2010; Krenn and Penz, 1998; Krenn et al., 2005). In A. ypsilon, sensilla chaetica become shorter toward the tip so that they can measure the width and depth of the tube without blocking the entrance for the proboscis during feeding, as in V. cardui (Krenn, 1998). Sensilla basiconica occur both on the external and internal surfaces of the galea, and are regarded as chemo- and mechanoreceptive in function (Städler and Seabrook, 1975; Walters et al., 1998). In A. ypsilon, the sensilla basiconica on the external galea each possess a single terminal pore, suggesting a gustatory function. Sensilla styloconica are regarded as chemoreceptors and bimodal chemo-mechanical receptors (Städler and Seabrook, 1975; Salama et al., 1984; Altner and Altner, 1986). Like in other lepidopteran species, the sensilla styloconica of A. ypsilon have a large robust shaft with six or seven ridges, conforming to their floral nectar feeding behaviors (Altner and Altner, 1986; Krenn, 1998; Walters et al., 1998). The sensory cone is fully surrounded by the longitudinal ribs for the sensilla styloconica on the basal and middle parts of the tip region of the proboscis, likely providing a protection for the sensory cone during the feeding process. On the apex of the proboscis, the sensory cone projects beyond the ribs at
300
S. Xue, B. Hua / Journal of Asia-Pacific Entomology 17 (2014) 295–301
Fig. 4. The tip region of the proboscis of male A. ypsilon. (A) The tip region of the proboscis. (B) Sensilla styloconica (SS) and a sensillum chaeticum (SCh) on the base of the tip region. (C) Sensilla styloconica (SS) with six longitudinal ribs and external sensilla basiconica (ESB). (D) Sensilla styloconica (SS) with six and seven longitudinal ribs and external sensilla basiconica (ESB) on the apex of the tip region. (E) Sensilla styloconica, with a terminal pore on the sensory cone (SC) and six longitudinal ribs on the stylus. (F) A sensillum styloconicum with a proximal stylus of seven ribs (R) and a distal sensory cone (SC). Scale bars: (A) = 50 μm; (B) = 10 μm; (C) and (D) = 15 μm; (E) and (F) = 5 μm.
the apex of the sensilla styloconica. This situation may have an advantage to easily detect the quality of the nectar in flowers. Apart from their role in feeding behavior prediction, proboscis sensilla exhibit remarkable differences in morphology and distribution in Noctuidae as well as other families in Lepidoptera, indicating their value in phylogenetic analysis. For this purpose, current studies on proboscis sensilla are still far from enough in Noctuidae (Bänziger, 1975, 1982, 1988, 1992, 2007; Büttiker et al., 1996; Zaspel et al., 2007; Zenker et al., 2011), and more comprehensive investigations are desperately needed. Acknowledgments We thank Junxia Zhang and Guowei Zhang for assistance in specimen collection, Qionghua Gao for assistance in the SEM, and Na Ma for help in the figure treatment. This research was supported by the National Natural Science Foundation of China (grant no. 31372186).
References Altner, H., Altner, I., 1986. Sensilla with both, terminal pore and wall pores on the proboscis of the moth, Rhodogastria bubo Walker (Lepidoptera: Arctiidae). Zool. Anz. 216, 129–150. Bänziger, H., 1975. Skin-piercing blood-sucking moths I: ecological and ethological studies on Calpe eustrigata (Lepid., Noctuidae). Acta Trop. 32, 125–144. Bänziger, H., 1982. Fruit-piercing moths (Lep., Noctuidae) in Thailand: a general survey and some new perspectives. Mitt. Schweiz. Entomol. Ges. 55, 213–240. Bänziger, H., 1988. The heaviest tear drinkers: ecology and systematics of new and unusual notodontid moths. Nat. Hist. Bull. Siam Soc. 36, 17–53. Bänziger, H., 1992. Remarkable new cases of moths drinking human tears in Thailand (Lepidoptera: Thyatiridae, Sphingidae, Notodontidae). Nat. Hist. Bull. Siam Soc. 40, 91–102. Bänziger, H., 2007. Skin-piercing blood-sucking moths VI: fruit-piercing habits in Calyptra (Noctuidae) and notes on the feeding strategies of zoophilous and frugivorous adult Lepidoptera. Mitt. Schweiz. Entomol. Ges. 80, 271–288. Büttiker, W., Krenn, H.W., Putterill, J.F., 1996. The proboscis of eye-frequenting and piercing Lepidoptera (Insecta). Zoomorphology 116, 77–83. Faucheux, M.J., 1991. Morphology and distribution of sensilla on the cephalic appendages, tarsi and ovipositor of the European sunflower moth, Homoeosoma nebulella Den. & Schiff. (Lepidoptera: Pyralidae). Int. J. Insect Morphol. Embryol. 20, 291–307.
S. Xue, B. Hua / Journal of Asia-Pacific Entomology 17 (2014) 295–301 Faucheux, M.J., 2013. Sensillum types on the proboscis of the Lepidoptera: a review. Ann. Soc. Entomol. Fr. 49, 73–90. Kingsolver, J.G., Daniel, T.L., 1995. Mechanics of food handling by fluid-feeding insects. In: Chapman, R.F., de Boer, G. (Eds.), Regulatory Mechanisms in Insect Feeding. Springer, New York, pp. 32–73. Krenn, H.W., 1998. Proboscis sensilla in Vanessa cardui (Nymphalidae, Lepidoptera): functional morphology and significance in flower-probing. Zoomorphology 118, 23–30. Krenn, H.W., 2010. Feeding mechanisms of adult Lepidoptera: structure, function, and evolution of the mouthparts. Annu. Rev. Entomol. 55, 307–327. Krenn, H.W., Kristensen, N.P., 2000. Early evolution of the proboscis of Lepidoptera (Insecta): external morphology of the galea in basal glossatan moths lineages, with remarks on the origin of the pilifers. Zool. Anz. 239, 179–196. Krenn, H.W., Penz, C.M., 1998. Mouthparts of Heliconius butterflies (Lepidoptera: Nymphalidae): a search for anatomical adaptations to pollen-feeding behavior. Int. J. Insect Morphol. Embryol. 27, 301–309. Krenn, H.W., Zulka, K.P., Gatschnegg, T., 2001. Proboscis morphology and food preferences in nymphalid butterflies (Lepidoptera: Nymphalidae). J. Zool. 254, 17–26. Krenn, H.W., Plant, J.D., Szucsich, N.U., 2005. Mouthparts of flower-visiting insects. Arthropod Struct. Dev. 34, 1–40. Lehnert, M.S., Monaenkova, D., Andrukh, T., Beard, C.E., Adler, P.H., Kornev, K.G., 2013. Hydrophobic–hydrophilic dichotomy of the butterfly proboscis. J. R. Soc. Interface. 10, 20130336. Monaenkova, D., Lehnert, M.S., Andrukh, T., Beard, C.E., Rubin, B., Tokarev, A., Lee, W.-K., Adler, P.H., Kornev, K.G., 2012. Butterfly proboscis: combining a drinking straw with a nanosponge facilitated diversification of feeding habits. J. R. Soc. Interface. 9, 720–726. Paulus, H.F., Krenn, H.W., 1996. Vergleichende Morphologie des Schmetterlingsrüssels und seiner Sensillen—Ein Beitrag zur phylogenetischen Systematik der Papilionoidea (Insecta, Lepidoptera). J. Zool. Syst. Evol. Res. 34, 203–216. Petr, D., Stewart, K.W., 2004. Comparative morphology of sensilla styloconica on the proboscis of North American Nymphalidae and other selected taxa (Lepidoptera): systematic and ecological considerations. Trans. Am. Entomol. Soc. 130, 293–409.
301
Rings, R.W., Arnold, F.J., Johnson, B.A., 1975. Host range of the black cutworm on vegetables: a bibliography. Bull. Entomol. Soc. Am. 21, 229–234. Salama, H.S., Khalifa, A., Azmy, N., Sharaby, A., 1984. Gustation in the lepidopterous moth Spodoptera littoralis (Boisd.). Zool. Jahrb. Allg. Zool. Physiol. Tiere 88, 165–178. Scoble, M.J., 1992. The Lepidoptera: Form, Function and Diversity. Oxford University Press, Oxford (404 pp.). Sellier, R., 1975. Étude ultrastructurale en microscopie électronique par balayage des organes sensoriels de la trompe des Lépidoptères Rhopalocères. Alexanor 9, 9–15. Shields, V.D.C., 2008. Ultrastructure of insect sensilla, In: Capinera, J.L. (Ed.), Encyclopedia of Entomology, 2nd ed. Springer-Verlag, Berlin, pp. 4073–4087. Showers, W.B., 1997. Migratory ecology of the black cutworm. Annu. Rev. Entomol. 42, 393–425. Städler, E., Seabrook, W.D., 1975. Chemoreceptors on the proboscis of the female eastern spruce budworm: electrophysiological study. Entomol. Exp. Appl. 18, 153–160. Walters, B.D., Albert, P.J., Zacharuk, R.Y., 1998. Morphology and ultrastructure of sensilla on the proboscis of the adult spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae). J. Zool. 76, 466–479. Wang, N., Wei, J.S., Dang, L., Wang, J.B., Hua, B.Z., 2012. Ultramorphology of sensilla on the proboscis in three noctuid moths (Lepidoptera: Noctuidae). Acta Entomol. Sin. 55, 877–884. Zaspel, J.M., Kononenko, V.S., Goldstein, P.Z., 2007. Another blood feeder? Experimental feeding of a fruit-piercing moth species on human blood in the Primorye Territory of Far Eastern Russia (Lepidoptera: Noctuidae: Calpinae). J. Insect Behav. 20, 437–451. Zaspel, J.M., Weller, S.J., Branham, M.A., 2011. A comparative survey of proboscis morphology and associated structures in fruit-piercing, tear-feeding, and blood-feeding moths in Calpinae (Lepidoptera: Erebidae). Zoomorphology 130, 203–225. Zenker, M.M., Penz, C., de Paris, M., Specht, A., 2011. Proboscis morphology and its relationship to feeding habits in noctuid moths. J. Insect Sci. 11, 42.
Contents lists available at ScienceDirect
Journal of Asia-Pacific Entomology journal homepage: www.elsevier.com/locate/jape
Proboscis sensilla of the black cutworm Agrotis ypsilon (Rottemberg) (Lepidoptera: Noctuidae) Shuang Xue, Bao-zhen Hua ⁎ State Key Laboratory of Crop Stress Biology for Arid Areas, Key Laboratory of Plant Protection Resources and Pest Management of the Education Ministry, Entomological Museum, Northwest A&F University, Yangling, Shaanxi 712100, China
a r t i c l e
i n f o
Article history: Received 17 August 2013 Revised 16 January 2014 Accepted 28 January 2014 Available online 5 February 2014 Keywords: Glossata Morphology Moth Mouthparts Sensilla styloconica
a b s t r a c t Proboscis sensilla are important for feeding biology in Lepidoptera, and are also valuable characters for species recognition and phylogenetic analysis. However, proboscis has not been satisfactorily explored in many groups in Lepidoptera so far. Here we examined the proboscis sensilla of the black cutworm Agrotis ypsilon (Rottemberg), a cosmopolitan agricultural pest of great economic significance, using scanning electron microscopy. Three types of sensilla were found on the proboscis: sensilla chaetica, sensilla basiconica, and sensilla styloconica. Sensilla chaetica occur only on the external surface of the proboscis and become shorter and more scattered toward the tip. Sensilla basiconica are arranged in longitudinal rows on the external proboscis and one longitudinal row in the inner food canal. Sensilla styloconica are the most characteristic sensilla on the proboscis, consisting of a single sensory cone inserted at the top of a stylus with six or seven longitudinal ribs, and are concentrated on the tip region, and are much longer and more numerous in females than in males. The role of proboscis sensilla in the feeding habit prediction is briefly discussed. © 2014 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved.
Introduction The proboscis of higher Lepidoptera is a complex fluidic system consisting of two extremely elongated galeae and functions like a drinking straw through which floral nectar and various kinds of liquids from rotten fruits, sweat, tear, dung, carrion, and urine are sucked from the tip into the head and the digestive tract (Scoble, 1992; Kingsolver and Daniel, 1995; Krenn, 2010; Monaenkova et al., 2012). The external surface of the proboscis has a sharp boundary separating a hydrophilic drinking region and a hydrophobic non-drinking region, and the hydrophilic region is at the tip region (Lehnert et al., 2013). The tip region is characterized by rows of intake slits leading into the food canal (Krenn, 1998; Lehnert et al., 2013). Each galea bears cuticular processes and various types of sensilla, which play important roles in feeding activities in the Lepidoptera (Krenn et al., 2005; Krenn, 2010). Previous investigations on proboscis sensilla in Lepidoptera are mainly concentrated on butterflies (Papilionoidea) and moths in families Noctuidae, Geometridae, Erebidae, Tortricidae, Neopseustidae, Saturniidae, and Pyralidae (Sellier, 1975; Faucheux, 1991; Paulus and Krenn, 1996; Krenn, 1998; Krenn and Penz, 1998; Walters et al., 1998; Krenn et al., 2001; Zaspel et al., 2011). Within Noctuidae, fruit-
⁎ Corresponding author. Tel./fax: +86 29 87091342. E-mail address: [email protected] (B. Hua).
piercing, nectar-feeding, skin-piercing, blood-sucking, tear-feeding, and sweat-feeding moths are frequently studied for their proboscis and sensillar morphology (Bänziger, 1975, 1982, 1988, 1992, 2007; Büttiker et al., 1996; Zaspel et al., 2007; Zenker et al., 2011). To our knowledge, however, the proboscis sensilla of the black cutworm Agrotis ypsilon (Rottemberg), a cosmopolitan agricultural pest of great economic significance, have not been investigated so far. Agrotis ypsilon (Rottemberg) is a polyphagous herbivorous moth, with the larvae severing the basal stems of seedlings of corn (Zea mays), cottons (Gossypium hirsutum, G. barbedense), tobacco (Nicotiana tobacum), wheat (Triticum aestivum), various vegetables, and turf grasses, and frequently causing serious damage to underground roots and tubers (Rings et al., 1975; Showers, 1997). This moth completes 1–7 generations per year in different areas of the world, depending on weather conditions. The larvae go through six or seven instars. The life span of the imago lasts 12–33 days depending on air humidity, temperature and feeding conditions of the larvae. The black cutworm is known as a migratory pest with extraordinary flexibility in the timing and spacing of reproduction and population growth (Showers, 1997). Although the distribution, life history, and host ranges have been well documented in A. ypsilon, its feeding behavior of the adults has not been satisfactorily investigated, especially the morphology of the proboscis sensilla. In this paper, we compared the external morphology of the proboscis sensilla of female with male A. ypsilon using scanning electron microscopy, in an attempt to predict its feeding behavior of the adults.
http://dx.doi.org/10.1016/j.aspen.2014.01.014 1226-8615/© 2014 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved.
296
S. Xue, B. Hua / Journal of Asia-Pacific Entomology 17 (2014) 295–301
Terminology for proboscis sensilla mainly follows Shields (2008) and Faucheux (2013).
Results Gross morphology of the mouthparts
Materials and methods Insect collection and preparation Adults of A. ypsilon were obtained with light traps at the Zhuque National Forest Park and the campus of the Northwest A&F University, Shaanxi Province of the central China from July to August in 2012. The heads were severed from the ether anesthetic alive insects and then fixed in Carnoy's fixative solution (95% ethanol:glacial acetic acid = 3:1, v/v) for 24 h before being stored in 75% ethanol.
Scanning electron microscopy For scanning electron microscopy (SEM), the mouthparts were cleaned with an ultrasonic cleaner for 30 s. After dehydration in ethanol series, the samples were submerged in tertiary butanol for freeze drying. They were then mounted onto SEM stubs using a double graphite adhesive tape, coated with gold in a sputter coater, and examined in a Hitachi S-3400N scanning electron microscope (Hitachi, Tokyo, Japan) at 15 kV.
Measurements Proboscis length and sensilla were measured for 20 individuals (10 females and 10 males). Sensillar length and diameter were measured using a digitizing tablet and the Imaris 7.2.3. The length and diameter of sensilla chaetica were measured in four regions: the proximal, bend, distal, and tip regions. The proboscis has a bend region approximately one-third of the length from the base (Krenn, 1998). The tip region is characterized by rows of intake slits leading into the food canal (Krenn, 1998). From the base to the bend region is the proximal region. The distal region is located between the bend and tip regions. The cuticular processes are hair-like in the proximal and bend regions, spine-like toward the distal and tip regions.
Statistical analysis Proboscis length and the number, length, and diameter of different sensilla were analyzed using the Predictive Analytics Software Statistics 18.0. For these data, the mean number and standard error of mean were calculated. To evaluate and compare differences between the sexes, Student's t-test at α = 0.05 was used.
No distinct difference was found in mouthpart morphology between the female and male A. ypsilon. The proboscis consists of two extremely elongated galeae (Fig. 1A), which connected together to form a sucking tube. The labrum becomes a small plate over the basal proboscis joint, where bristles of pilifers contact the basal galeal joint. The mandibles are reduced and nonfunctional. The basal maxillary sclerites form a tubular component from which the coilable galeae and the maxillary palp emerge. The lacinia is vestigial. The labial palps are threesegmented, extending from the prementum sclerite and forming prominent structures in front of the head, and are equipped with a great number of scales and bristles (Fig. 1B). In the resting position, the proboscis is coiled in a tight spiral beneath the head between the setose labial palps. The coilable galeae are held together by rows of interlocking cuticular processes, which exhibit different shapes on the dorsal and ventral sides of the food canal, which is interlocked by the dorsal and ventral galeal linkages. The dorsal linkage consists of horizontally extending lancet-shaped flat plates and forms a roof over the food tube, while the ventral galeal linkage is composed of a row of hooks and provides firm interlocking structures for the proboscis. The concave median surface of the food tube is composed of smooth plates that are vertically fluted. The proboscis length is 11.86 ± 4.03 mm (9.73–13.48 mm, n = 20). The proboscis can be divided into the proximal, bend, distal, and tip regions. When uncoiled, the proximal region makes up 38% of the total length; the distance from the proximal region to the bend region comprises 10% of the total length; the distal region constitutes about 39%; and the tip region, where fluids are taken up into the food canal, comprises 13% of the total length. The outer surface of the proboscis is fluted with cuticular processes and a great number of sensilla. The cuticular processes have different shapes in different regions of the proboscis. They are hair-like in the proximal and bend regions (Fig. 2A), shorter and more spine-like toward the distal and tip regions (Figs. 2B and C), and interlocked between the consecutive coils in the resting position. Both the female and male A. ypsilon possess three types of sensilla on their proboscis: bristle-shaped sensilla chaetica, uniporous sensilla basiconica, and prominent sensilla styloconica. Different types of sensilla are located on different regions of the proboscis and assume a specific distribution pattern along the proboscis. Sensilla chaetica are the most abundant type of sensilla. Sensilla basiconica are present on the external surface of the proboscis and are the only sensilla that also exist on the internal wall of the food canal. Sensilla styloconica are confined to the tip region of the proboscis.
Fig. 1. Head of male A. ypsilon. (A) The proboscis splits into two galeae due to the preparation procedure. (B) Outer view of a galea. CE, compound eye; Ga, galea; LP, labial palp; TR, tip region. Scale bars: (A) = 500 μm; (B) = 200 μm.
S. Xue, B. Hua / Journal of Asia-Pacific Entomology 17 (2014) 295–301
297
Fig. 2. Sensilla chaetica on the proboscis of male A. ypsilon. (A)–(C) Sensilla chaetica and cuticular processes on the bend, distal and tip regions. (D) Sensilla chaetica on the bend region, showing the sensilla chaetica on the ventral side are longer than those on the dorsal side. Scale bars: (A) and (D) = 50 μm; (B) = 15 μm; (C) = 5 μm.
In A. ypsilon, the tip region of the proboscis makes up 11–15% of the total proboscis length. The tip length of the females is 1588 ± 0.74 μm (n = 10), significantly longer than that in the males (1333 ± 1.25 μm, n = 10). All the three types of sensilla are present in this region, of which sensilla chaetica and sensilla basiconica are rare, scattered, and the shortest. Sensilla chaetica Sensilla chaetica are the most abundant type of sensilla on the proboscis of A. ypsilon, with a total number ranging from 190 to 248 (228 ± 13.67, n = 40) per galea, more frequent in the proximal region
than in the bend, distal, and tip regions. They occur only on the external surface of the proboscis and become more scattered toward the tip. The sensilla chaetica each have an aporous sensory bristle extending from a collared socket and have a grooved surface (Fig. 2B). Each bristle is inserted into a distinct sunken socket, with a basal pore of approximately 3.66 ± 1.88 μm in diameter (n = 7) and protruding at an angle of 30°–45° from the surface on the proximal and bend regions of the proboscis. Then they tend to be upright toward the tip region with some slender basal diameter (Figs. 2B and C; Table 1). The sensilla chaetica become shorter gradually from the proximal to the tip region in both the females and males (Table 1) and are significantly longer on the ventral side of the proboscis than on the dorsal
Table 1 Length, diameter and number of various proboscis sensilla in A. ypsilon. Sensilla Sensilla chaetica
Female Proximal Bend Distal Tip
Length (μm) Length (μm) Diameter (μm) Length (μm) Length (μm) Diameter (μm)
Number per galea External sensilla basiconica
Internal sensilla basiconica
Sensilla styloconica
Data are presented as mean ± SE (n). NS = not significant; * p b 0.05, ** p b
Length (μm) Diameter (μm) Number per galea Length (μm) Diameter (μm) Number per galea Length (μm) Diameter (μm) Number per galea 0.01 in the independent samples t-test.
110.88 65.5 3.67 31.35 5.85 1.44 230 13.61 2.67 59 7.25 2.95 25 58.85 14.09 86
Male ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.39 (32) 1.52 (28) 0.13 (4) 0.81 (16) 0.19 (4) 0.08 (4) 4.98 (10) 0.95 (18) 1.13 (18) 0.96 (10) 0.83 (12) 1.14 (12) 0.24 (20) 4.26 (30) 0.24 (30) 0.98 (10)
108.68 60.78 3.61 28.55 5.49 1.43 224 12.43 2.51 56 7.48 2.47 23 44.69 12.00 78
t-test ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
6.28 (28) 2.44 (28) 0.24 (3) 0.96 (10) 0.20 (5) 0.15 (5) 6.48 (10) 1.19 (17) 1.86 (13) 1.08 (10) 0.25 (8) 0.39 (8) 0.32 (20) 1.37 (28) 0.42 (28) 1.14 (10)
NS * NS NS NS NS * NS NS NS NS NS NS ** * **
298
S. Xue, B. Hua / Journal of Asia-Pacific Entomology 17 (2014) 295–301
Table 2 Length difference of sensilla chaetica between the ventral and dorsal sides in A. ypsilon. Sensilla chaetica Female
Male
Proximal region Bend region Distal region Tip region Proximal region Bend region Distal region Tip region
Length on ventral side (μm)
Length on dorsal side (μm)
t-test
126.76 86.33 44.70 5.85 118.43 82.15 39.59 5.49
103.04 ± 2.11 (18) 56.37 ± 2.16 (10) 19.44 ± 3.59 (6) – 96.88 ± 1.35 (15) 49.87 ± 3.47 (10) 14.41 ± 3.56 (4) –
⁎⁎ ⁎⁎ ⁎⁎
± ± ± ± ± ± ± ±
0.58 (14) 1.24 (18) 1.23 (10) 0.19 (4) 0.17 (13) 2.65 (18) 0.36 (4) 0.20 (5)
– ⁎⁎ ⁎⁎ ⁎⁎ –
Data are presented as mean ± SE (n). –: not calculated. ⁎⁎ p b 0.01 in the independent samples t-test.
side (Fig. 2D; Table 2). On the same region, sensilla chaetica are significantly longer in females than in males (Table 3). Sensilla basiconica Sensilla basiconica each have a short blunt-tipped sensory cone that bears a single terminal pore and a short dome-shaped socket, and exist both on the external and internal surfaces of the galea (Figs. 3A and B). External sensilla basiconica are arranged in irregular rows throughout the dorsal and lateral sides of the proboscis (Fig. 3C). The mean number of the sensilla on each galea is similar between the sexes (Table 1). Internal sensilla basiconica form a single row in the food canal on each galea (Fig. 3D), and are shorter than the external sensilla basiconica, numbering 24 ± 0.83 (n = 40) on each galea. They are elongated sensory cones inserted into dome-shaped sockets. Sensilla styloconica Sensilla styloconica are the most characteristic sensilla on the proboscis, confined to the tip region (Fig. 4A), 82 ± 1.62 in number (75–88, n = 10) on each galea. From the basal to the middle part of the tip region, the sensilla styloconica are arranged in three rows, and are slender on the ventral side of the galea than on the dorsal side (Fig. 4A). Then they become scattered toward the apex. On the dorsolateral side of the tip region, the sensilla styloconica are widely spaced. Sensilla styloconica each consist of a shorter terminal uniporous sensory cone inserted into a larger stylus with longitudinal ribs, originating from an area of thin cuticle surrounded by spine-like cuticular processes (Figs. 4B–E). The gap between adjacent cuticular processes is directly in the face of the rib of the stylus. The middle part of the stylus is usually wider than the proximal part (Figs. 4B and C). Their styli possess six or seven smooth-edged longitudinal ribs (Figs. 4E and F). The sensilla styloconica with six longitudinal ribs are more abundant than those with seven. The sensory cone is fully surrounded by the longitudinal ribs on the basal and middle parts of the tip region, but extends beyond the ribs on the apex of the proboscis (Figs. 4B–E).
On the basis of t-test, a very significant difference in the total length of sensilla styloconica exists between the females and males (p b 0.01, Table 1). The sensilla styloconica are larger and more numerous in the females than in the males (Table 1). Nonetheless, no significant difference exists in the length of the sensory cone between the sexes, both being approximately 10 ± 0.46 μm (n = 28). They become gradually shorter from the base to the apex of the tip region in both the sexes.
Discussion To our knowledge, this paper may represent the first attempt to investigate the proboscis sensilla of A. ypsilon. No differences were found in the types of proboscis sensilla between the two sexes, suggesting that females and males have similar feeding habits in A. ypsilon. The morphology of the proboscis tip region can be used to predict the feeding habits of noctuid moths (Zenker et al., 2011). The proboscises of non-flower-visiting noctuid moths are typically armed with rasping spines, tearing hooks, and erectible barbs, and bear numerous sensilla at the tip region (Bänziger, 1982, 1988; Zenker et al., 2011). Based on our research on A. ypsilon, the tip region of the proboscis is simple with sensilla present alone, and no characteristics of non-flowervisiting moths have been found, confirming that it is a typical flowervisiting moth. In terms of proboscis sensilla, morphological differences were found mainly in the length of sensilla chaetica and the shape and number of sensilla styloconica. The terms sensilla chaetica and sensilla trichodea are often used interchangeably in literature. The longest sensillum chaeticum is 111.96 μm in flower-visiting species, but is up to 214.00 μm in nonflower-visiting species in Nymphalidae (Krenn et al., 2001). In the proboscis of A. ypsilon, the lengths of sensilla chaetica decrease greatly from the proximal region to the tip region. This is likely the adaption for nectar-feeding behavior. The longest sensillum chaeticum occurs in the proximal proboscis region in A. ypsilon, but beyond the mid-proboscis in many Heliconiini, some Limenitidinae, some Brassolinae, Morphinae, and Satyrinae (Krenn and Penz, 1998; Krenn et al., 2001). In some species of Nymphalinae (e.g. Vanessa atalanta, Nymphalis antiopa), remarkably
Table 3 Length difference of sensilla chaetica between the sexes in A. ypsilon. Sensilla chaetica Length on ventral side (μm)
Length on dorsal side (μm)
Proximal region Bend region Distal region Tip region Proximal region Bend region Distal region Tip region
Data are presented as mean ± SE (n). –: not calculated. NS = not significant; * p b 0.05, ** p b 0.01 in the independent samples t-test.
Female
Male
t-test
126.76 ± 0.58 (14) 86.33 ± 1.24 (18) 44.70 ± 1.23 (10) 5.85 ± 0.19 (4) 103.04 ± 2.11 (18) 56.37 ± 2.16 (10) 19.44 ± 3.59 (6) –
118.43 ± 0.17 (13) 82.15 ± 2.65 (18) 39.59 ± 0.36 (4) 5.49 ± 0.20 (5) 96.88 ± 1.35 (15) 49.87 ± 3.47 (10) 14.41 ± 3.56 (4) –
** * * NS ** ** ** –
S. Xue, B. Hua / Journal of Asia-Pacific Entomology 17 (2014) 295–301
299
Fig. 3. Sensilla basiconica on the proboscis of male A. ypsilon. (A) Magnification of an external sensillum basiconicum (ESB); inset shows the terminal pore (P) of a sensillum basiconicum. (B) An internal sensillum basiconicum (ISB) in the food canal. (C) Outer surface of the proboscis, showing external sensilla basiconica arranged in irregular rows. (D) Proboscis in medial view, showing the food canal (FC) interlocked by the dorsal (DGL) and ventral galeal linkages (VGL), internal sensilla basiconica (ISB) and sensilla styloconica (SS). Scale bars: (A) and (B) = 10 μm; (C) = 40 μm; (D) = 80 μm.
long sensilla chaetica are found in the proximal third of the proboscis (Krenn et al., 2001). Sensilla styloconica are the most remarkable sensillar type on the proboscis, composed of a variously shaped stylus and a shorter terminal sensory cone (Krenn, 1998; Petr and Stewart, 2004), and are regarded to be derived from sensilla basiconica (Faucheux, 2013). Their styli are either smooth and ellipsoid or longitudinally ribbed and stellate (Faucheux, 1991; Krenn, 1998; Krenn and Penz, 1998; Zaspel et al., 2011). In some species the sensilla styloconica bear terminal spines around the sensory cone, and the ribs are serrate or very short and practically absent in some other species. The styli of A. ypsilon bear longitudinal ribs, similar to those of Choristoneura fumifernana (Walters et al., 1998), Noctua pronuba, Hypocala rostrata, Eublemma pyrochroa (Büttiker et al., 1996), Helicoverpa armigera, and Helicoverpa assulta (Wang et al., 2012). However, the number of longitudinal ribs is varied among different species. In C. fumifernana, each sensillum styloconicum consists of a single sensory peg inserted at the top of a stylus that bears 6–9 ribs extending from the base of the stylus to the sensory peg socket (Walters et al., 1998). In N. pronuba and H. rostrata, their styli are ridged with 6–8 ribs. In E. pyrochroa, the sensilla styloconica are arranged in two rows and their styli are compressed, bearing 4 or 5 longitudinal ribs with apical points surrounding the sensory cone (Büttiker et al., 1996). In Helicoverpa armigera and H. assulta, each sensillum styloconicum has a stylus bearing six longitudinal ribs (Wang et al., 2012). While in A. ypsilon, the styli have 6 or 7 longitudinal rids. In general, the sensory cone is uniporous and inserted centrally at the top of the stylus. In Heliconiini, however, the cone is at the periphery of the stylus (Krenn et al., 2001). The sensory cone in an arctiid moth is with additional wall pores on the sensory cone (Altner and Altner, 1986). In A. ypsilon, this structure exhibits the common uniporous cone inserted centrally at the top of the stylus.
According to Krenn and Kristensen (2000), a stylus equipped with 4–6 longitudinal ribs was found in some species of Prodoxidae, Adelidae and all Incurvarioidea, in the Palaephatoidea, and in many Ditrysia (non-apoditrysians; Tortricidae; Arctiidae; Pyralidae; Noctuidae; Papilionoidea). Therefore, this type of stylus was regarded as the plesiomorphy for sensilla styloconica on the lepidopteran proboscis (Krenn and Kristensen, 2000). If this is the case, the sensilla styloconica of A. ypsilon proboscis may represent an apomorphy because the stylus is equipped with 6 or 7 longitudinal ribs. Sensilla chaetica are regarded as mechanosensitive on the basis of ultrastructural studies. Bristle-shaped sensilla on the proboscis may provide information on the diameters of the corolla and the depth of proboscis insertion during probing (Krenn, 1998, 2010; Krenn and Penz, 1998; Krenn et al., 2005). In A. ypsilon, sensilla chaetica become shorter toward the tip so that they can measure the width and depth of the tube without blocking the entrance for the proboscis during feeding, as in V. cardui (Krenn, 1998). Sensilla basiconica occur both on the external and internal surfaces of the galea, and are regarded as chemo- and mechanoreceptive in function (Städler and Seabrook, 1975; Walters et al., 1998). In A. ypsilon, the sensilla basiconica on the external galea each possess a single terminal pore, suggesting a gustatory function. Sensilla styloconica are regarded as chemoreceptors and bimodal chemo-mechanical receptors (Städler and Seabrook, 1975; Salama et al., 1984; Altner and Altner, 1986). Like in other lepidopteran species, the sensilla styloconica of A. ypsilon have a large robust shaft with six or seven ridges, conforming to their floral nectar feeding behaviors (Altner and Altner, 1986; Krenn, 1998; Walters et al., 1998). The sensory cone is fully surrounded by the longitudinal ribs for the sensilla styloconica on the basal and middle parts of the tip region of the proboscis, likely providing a protection for the sensory cone during the feeding process. On the apex of the proboscis, the sensory cone projects beyond the ribs at
300
S. Xue, B. Hua / Journal of Asia-Pacific Entomology 17 (2014) 295–301
Fig. 4. The tip region of the proboscis of male A. ypsilon. (A) The tip region of the proboscis. (B) Sensilla styloconica (SS) and a sensillum chaeticum (SCh) on the base of the tip region. (C) Sensilla styloconica (SS) with six longitudinal ribs and external sensilla basiconica (ESB). (D) Sensilla styloconica (SS) with six and seven longitudinal ribs and external sensilla basiconica (ESB) on the apex of the tip region. (E) Sensilla styloconica, with a terminal pore on the sensory cone (SC) and six longitudinal ribs on the stylus. (F) A sensillum styloconicum with a proximal stylus of seven ribs (R) and a distal sensory cone (SC). Scale bars: (A) = 50 μm; (B) = 10 μm; (C) and (D) = 15 μm; (E) and (F) = 5 μm.
the apex of the sensilla styloconica. This situation may have an advantage to easily detect the quality of the nectar in flowers. Apart from their role in feeding behavior prediction, proboscis sensilla exhibit remarkable differences in morphology and distribution in Noctuidae as well as other families in Lepidoptera, indicating their value in phylogenetic analysis. For this purpose, current studies on proboscis sensilla are still far from enough in Noctuidae (Bänziger, 1975, 1982, 1988, 1992, 2007; Büttiker et al., 1996; Zaspel et al., 2007; Zenker et al., 2011), and more comprehensive investigations are desperately needed. Acknowledgments We thank Junxia Zhang and Guowei Zhang for assistance in specimen collection, Qionghua Gao for assistance in the SEM, and Na Ma for help in the figure treatment. This research was supported by the National Natural Science Foundation of China (grant no. 31372186).
References Altner, H., Altner, I., 1986. Sensilla with both, terminal pore and wall pores on the proboscis of the moth, Rhodogastria bubo Walker (Lepidoptera: Arctiidae). Zool. Anz. 216, 129–150. Bänziger, H., 1975. Skin-piercing blood-sucking moths I: ecological and ethological studies on Calpe eustrigata (Lepid., Noctuidae). Acta Trop. 32, 125–144. Bänziger, H., 1982. Fruit-piercing moths (Lep., Noctuidae) in Thailand: a general survey and some new perspectives. Mitt. Schweiz. Entomol. Ges. 55, 213–240. Bänziger, H., 1988. The heaviest tear drinkers: ecology and systematics of new and unusual notodontid moths. Nat. Hist. Bull. Siam Soc. 36, 17–53. Bänziger, H., 1992. Remarkable new cases of moths drinking human tears in Thailand (Lepidoptera: Thyatiridae, Sphingidae, Notodontidae). Nat. Hist. Bull. Siam Soc. 40, 91–102. Bänziger, H., 2007. Skin-piercing blood-sucking moths VI: fruit-piercing habits in Calyptra (Noctuidae) and notes on the feeding strategies of zoophilous and frugivorous adult Lepidoptera. Mitt. Schweiz. Entomol. Ges. 80, 271–288. Büttiker, W., Krenn, H.W., Putterill, J.F., 1996. The proboscis of eye-frequenting and piercing Lepidoptera (Insecta). Zoomorphology 116, 77–83. Faucheux, M.J., 1991. Morphology and distribution of sensilla on the cephalic appendages, tarsi and ovipositor of the European sunflower moth, Homoeosoma nebulella Den. & Schiff. (Lepidoptera: Pyralidae). Int. J. Insect Morphol. Embryol. 20, 291–307.
S. Xue, B. Hua / Journal of Asia-Pacific Entomology 17 (2014) 295–301 Faucheux, M.J., 2013. Sensillum types on the proboscis of the Lepidoptera: a review. Ann. Soc. Entomol. Fr. 49, 73–90. Kingsolver, J.G., Daniel, T.L., 1995. Mechanics of food handling by fluid-feeding insects. In: Chapman, R.F., de Boer, G. (Eds.), Regulatory Mechanisms in Insect Feeding. Springer, New York, pp. 32–73. Krenn, H.W., 1998. Proboscis sensilla in Vanessa cardui (Nymphalidae, Lepidoptera): functional morphology and significance in flower-probing. Zoomorphology 118, 23–30. Krenn, H.W., 2010. Feeding mechanisms of adult Lepidoptera: structure, function, and evolution of the mouthparts. Annu. Rev. Entomol. 55, 307–327. Krenn, H.W., Kristensen, N.P., 2000. Early evolution of the proboscis of Lepidoptera (Insecta): external morphology of the galea in basal glossatan moths lineages, with remarks on the origin of the pilifers. Zool. Anz. 239, 179–196. Krenn, H.W., Penz, C.M., 1998. Mouthparts of Heliconius butterflies (Lepidoptera: Nymphalidae): a search for anatomical adaptations to pollen-feeding behavior. Int. J. Insect Morphol. Embryol. 27, 301–309. Krenn, H.W., Zulka, K.P., Gatschnegg, T., 2001. Proboscis morphology and food preferences in nymphalid butterflies (Lepidoptera: Nymphalidae). J. Zool. 254, 17–26. Krenn, H.W., Plant, J.D., Szucsich, N.U., 2005. Mouthparts of flower-visiting insects. Arthropod Struct. Dev. 34, 1–40. Lehnert, M.S., Monaenkova, D., Andrukh, T., Beard, C.E., Adler, P.H., Kornev, K.G., 2013. Hydrophobic–hydrophilic dichotomy of the butterfly proboscis. J. R. Soc. Interface. 10, 20130336. Monaenkova, D., Lehnert, M.S., Andrukh, T., Beard, C.E., Rubin, B., Tokarev, A., Lee, W.-K., Adler, P.H., Kornev, K.G., 2012. Butterfly proboscis: combining a drinking straw with a nanosponge facilitated diversification of feeding habits. J. R. Soc. Interface. 9, 720–726. Paulus, H.F., Krenn, H.W., 1996. Vergleichende Morphologie des Schmetterlingsrüssels und seiner Sensillen—Ein Beitrag zur phylogenetischen Systematik der Papilionoidea (Insecta, Lepidoptera). J. Zool. Syst. Evol. Res. 34, 203–216. Petr, D., Stewart, K.W., 2004. Comparative morphology of sensilla styloconica on the proboscis of North American Nymphalidae and other selected taxa (Lepidoptera): systematic and ecological considerations. Trans. Am. Entomol. Soc. 130, 293–409.
301
Rings, R.W., Arnold, F.J., Johnson, B.A., 1975. Host range of the black cutworm on vegetables: a bibliography. Bull. Entomol. Soc. Am. 21, 229–234. Salama, H.S., Khalifa, A., Azmy, N., Sharaby, A., 1984. Gustation in the lepidopterous moth Spodoptera littoralis (Boisd.). Zool. Jahrb. Allg. Zool. Physiol. Tiere 88, 165–178. Scoble, M.J., 1992. The Lepidoptera: Form, Function and Diversity. Oxford University Press, Oxford (404 pp.). Sellier, R., 1975. Étude ultrastructurale en microscopie électronique par balayage des organes sensoriels de la trompe des Lépidoptères Rhopalocères. Alexanor 9, 9–15. Shields, V.D.C., 2008. Ultrastructure of insect sensilla, In: Capinera, J.L. (Ed.), Encyclopedia of Entomology, 2nd ed. Springer-Verlag, Berlin, pp. 4073–4087. Showers, W.B., 1997. Migratory ecology of the black cutworm. Annu. Rev. Entomol. 42, 393–425. Städler, E., Seabrook, W.D., 1975. Chemoreceptors on the proboscis of the female eastern spruce budworm: electrophysiological study. Entomol. Exp. Appl. 18, 153–160. Walters, B.D., Albert, P.J., Zacharuk, R.Y., 1998. Morphology and ultrastructure of sensilla on the proboscis of the adult spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae). J. Zool. 76, 466–479. Wang, N., Wei, J.S., Dang, L., Wang, J.B., Hua, B.Z., 2012. Ultramorphology of sensilla on the proboscis in three noctuid moths (Lepidoptera: Noctuidae). Acta Entomol. Sin. 55, 877–884. Zaspel, J.M., Kononenko, V.S., Goldstein, P.Z., 2007. Another blood feeder? Experimental feeding of a fruit-piercing moth species on human blood in the Primorye Territory of Far Eastern Russia (Lepidoptera: Noctuidae: Calpinae). J. Insect Behav. 20, 437–451. Zaspel, J.M., Weller, S.J., Branham, M.A., 2011. A comparative survey of proboscis morphology and associated structures in fruit-piercing, tear-feeding, and blood-feeding moths in Calpinae (Lepidoptera: Erebidae). Zoomorphology 130, 203–225. Zenker, M.M., Penz, C., de Paris, M., Specht, A., 2011. Proboscis morphology and its relationship to feeding habits in noctuid moths. J. Insect Sci. 11, 42.