Ultrastructure and distribution of sensilla on the

Received: 20 June 2017

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Revised: 4 December 2017

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Accepted: 7 December 2017

DOI: 10.1002/jmor.20791

RESEARCH ARTICLE

Ultrastructure and distribution of sensilla on the maxillary and labial palps of Chlorophorus caragana (Coleoptera: Cerambycidae) Yan-Ru Zhang1,2

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Li-Li Ren1 | Lu Zhang1 | Rong Wang3 | Yang Yu4 |

Peng-Fei Lu1 | You-Qing Luo1 1

The Key Laboratory for Silviculture and Conservation of the Ministry of Education, School of Forestry, Beijing Forestry University, Beijing 100083, People’s Republic of China

2

School of Forestry Inner Mongolia Agricultural University, Hohhot 010019, People’s Republic of China

3

School of Forestry Fujian Agriculture and Forestry University, Fuzhou 350002, People’s Republic of China

4

Institute of Plant Quarantine, Chinese Academy of Inspection and Quarantine, Beijing 100176, People’s Republic of China

Correspondence Peng-fei Lu, The Key Laboratory for Silviculture and Conservation of the Ministry of Education, School of Forestry, Beijing Forestry University, Beijing 100083. Email: [email protected] and You-qing Luo, The Key Laboratory for Silviculture and Conservation of the Ministry of Education, School of Forestry, Beijing Forestry University, Beijing 100083. Email: [email protected]

Abstract Chlorophorus caragana is a species of long-horned beetle that damages Caragana davazamcii Sancz. (Fabales: Papilionaceae) bushes in desert areas in China. The beetles cause substantial damage to local forestry plantations and the environment. Sensilla on the maxillary and labial palps of coleopterans a allow the insects to recognize their host plants. We used scanning and transmission electron microscopy to study the ultrastructure, distribution, and abundance of various sensilla on the maxillary and labial palps of C. caragana. We found four types of sensilla including ten sub€ hm’s bristles, three of sensilla chaetica, one of digitiform sensilla, and five of types: one of Bo sensilla twig basiconica. The types and distribution of the sensilla on the maxillary and labial palps were highly similar between males and females. Finally, this article discusses the functions of the

Funding information National Natural Science Foundation of China; Grant/Award Numbers: 31570643, 81774015; “Twelfth Five-year” National Science and Technology Support Project of China; Grant/Award Number: 2012BAD19B071

sensilla of related species in recognizing hosts and the significance of gustation studies in the context of the control of C. caragana.

KEYWORDS

labial palps, long-horned beetle, maxillary palps, scanning electron microscopy, transmission electron microscopy

1 | INTRODUCTION

After the insects settle on the plants, they primarily identify suitable hosts through perception from their gustatory organs, thus allowing

Chlorophorus caragana Xie & Wang, 2012 (Coleoptera: Cerambycidae)

the insects to select nutrients and avoid toxic substances (Bernays &

is a recently described species that occurs in the arid and semiarid

Chapman, 2001; Chapman, 2003; Dethier, 1976; Schoonhoven & Van

regions of northwest China and causes damage to Caragana davazamcii

Loon, 2002). It is well-known that the maxillary and labial palps of

Sancz. (Fabales: Papilionaceae; Zong, Xie, Wang, Luo, & Cao, 2012;

coleopterans play an important role in host recognition (Zhang &

Zhang, Ren, Zhang, & Luo, 2015). The larvae feed on the trunk of the

Mitchell, 1997). For example, the gustatory sensilla on the galea and

plant, and the damage from extensive boring can cause the trees to be

palps of Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae)

blown over by strong winds (Zong et al., 2013). Up to 70% of all C.

enable these beetles to respond more strongly to three types of host

davazamcii bushes are damaged in most regions of Ningxia, China,

plant sap than to non-host plant sap (Mitchell, 1994). In our earlier

directly affecting the local environment and economy. We previously

study, we found that after the beetles arrive at their oviposition sites

studied the basic bioecological relationships and plant attractants of C.

using the olfactory sensilla distributed on their antennae, C. caragana

caragana (Zong et al., 2012, 2013).

always used their maxillary and labial palps to interact with the bark

Journal of Morphology. 2018;1–15.

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C 2018 Wiley Periodicals, Inc. V

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and the sap to determine whether or not the plant was suitable for fur-

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2.3 | Transmission electron microscopy

ther oviposition. In this study, we hypothesize that the gustatory sensilla on the maxillary and labial palps of C. caragana also play a crucial

Adult C. caragana were removed from the preservation liquid. With the aid of an anatomical lens, the maxillary and labial palps were removed and

role in determining the ultimate host selection. An ultrastructural study of the gustatory sensilla provided unequivocal evidence for the function of gustatory organs and established the relationships between their structure and function (Altner & Prillinger, 1980). Although detailed ultrastructural and electrophysiological studies have been performed on antennal sensilla in many coleopteran insect species (Chen, Zhang, Wang, & Kong, 2010; Daly & Ryan, 1979; Kim & Yamasaki, 1996; Urbanek, Łuszczek, & Kapusta, 2016; Merivee, Ploomi, Luik, Rahi, & Sammelselg, 2001; Ploomi, Milius, Luik, & Heidemaa, 2005; Ploomi, Rahi, Luik, & Sammelselg, 2000; Merivee et al., rez-Gonzalez 2002; Must, Merivee, Mänd, Luik, & Heidemaa, 2006; Pe & Zaballos, 2013; Riolo et al., 2016), gustatory sensilla on the maxillary and labial palps in beetles have not been studied as frequently (Giglio, Ferrero, Perrotta, Talarico, & Zetto Brandmayr, 2010; Mitchell, 1994;

the samples washed in PBST for 3 hr, post-fixed in 1% (wt/vol) osmium tetroxide in PBS at 258C, and then carefully washed in PBS. The samples were dehydrated through a graded series of ethanolic solutions and embedded in Spurr’s resin. After solidification, each resin block was placed under a fluorescence microscope (LEICA MD2500) and photographed under blue light. The fluorescent light source of the microscope was moved to irradiate the sample from above, thus enabling the sensilla in the resin block to be clearly observed and photographed. The distances to target the sensilla were measured (Zhang, Ren, Luo, & Zong, 2013). Ultrathin sections (60–100 nm) were sliced using a Leica UC6 ultramicrotome. Fluorescence microscopy was used to accurately position the target sensilla. The sections were mounted on Formvar-coated 100-mesh copper grids, double-stained with uranyl acetate and lead citrate and observed via TEM (Hitachi model H-7500) using an operative voltage of 80 kV.

Zacharuk, Albert, & Bellamy, 1977). In the study, we explored the types of sensilla of C. caragana, their ultrastructure and their distribution on the maxillary and labial palps

2.4 | Data analysis

using scanning and transmission electron microscopy. Understanding

Sensilla identification was based on their morphology and size using

the microscopic morphological characteristics of the sensilla of C. cara-

the nomenclature of Schneider (1964), Zacharuk (1985), Zacharuk et al.

gana might help to aid in pest control.

(1977) and Zhang et al. (2013). Sensillar ultrastructure was recorded using the descriptors of Tarumingkeng, Zacharuk and Shanbhag € ller, & Steinbrecht, 1999; Tarumingkeng, Coppel, & Mat(Shanbhag, Mu

2 | MATERIALS AND METHODS

sumura, 1976; Zacharuk, 1980). The number of sensilla on each segment (sensillar density) was

2.1 | Insects

counted directly on the computer screen. The length, basal diameter,

Adult Chlorophorus caragana, Xie and Wang, 2012, were lured into field

and the size of each type of sensillum were measured using Image J 1.X

traps placed in Ling-wu County (38.05 N, 106.30 E), Ningxia Hui

software (Schneider et al., 2012). At least 30 sensilla of each type were

Autonomous Region, P. R. China, during July and August 2012 using

examined, and 12 (6 dorsal and 6 ventral) samples were measured for

plant attractants. The bodies of the adult C. caragana were cleaned and

each sex. Anterior views were routinely photographed to avoid any

preserved in 72% (wt/vol) disodium hydrogen phosphate, 28% (wt/vol)

adverse impact of the shooting angle on the results. The numbers and

monobasic sodium phosphate, 2.5% (wt/vol) glutaraldehyde, and

morphologies of the various types of male and female sensilla were

0.06% (vol/vol) Tween 20.

recorded, and independent sample t tests were used to identify signifi-

0

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cant differences between the sexes (SPSS 16.0; SPSS Inc., Chicago, IL). Scanning electron microscopy was used to count the sensilla and trace

2.2 | Scanning electron microscopy Adult C. caragana were removed from the preservation liquid and rinsed in carbon tetrachloride. With the aid of an anatomical lens, the maxillary and labial palps were removed and cleaned ultrasonically in phosphate-buffered saline (0.1 mol l21, pH 7.2) with 0.06% (vol/vol) Tween 20 (PBST) for 380 s and then transferred to 40% (vol/vol) ethyl

their distribution. The same specimens were used to obtain sensillar maps (Shanbhag et al., 1999). The list of abbreviations of types of sensilla Full names

Abbreviations Full names

Abbreviations

€ hm’s bristles Bo

BB.

Type 1 sensilla twig S.tb.1 basiconica

Type 1 sensilla chaetica

Ch.1



Ch.2



Ch.3


Digitiform sensilla Dig.


alcohol for 380 s. The samples were subsequently successively dehydrated using 20 min of treatment in 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, and 100% (all vol/vol) ethanol. After air drying, the samples were separately fixed onto stubs using carbon adhesive tape (SPI Supplies, Division of Structure Probes, Inc.) and sputter-coated with gold three times over a 45-s period while rotating the stub using an E-1010 sputter ion instrument (Hitachi Koki Co. Ltd., Tokyo, Japan). An Hitachi S-3400N SEM was used to observe the samples, and the SEM was operated at 10–25 kV.

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Chlorophorus caragana viewed by scanning electron microscopy of the maxillary and labial palps. From left to right: female maxillary palp, female labial palp, male labial palp, and male maxillary palp. Abbreviations: mp, maxillary palp; lp, labial palp; 1st, first segment; 2nd, second segment; 3rd, third segment; 4th, fourth segment

FIGURE 1

3 | RESULTS

(Figure 3g). The average length of the sensilla and the average basal diameter are shown in Tables 1 and 2.

3.1 | General structures of the maxillary and labial palps

3.2.2 | Type 1 sensilla chaetica Type 1 sensilla chaetica on the maxillary palps had sharp tips and longi-

The maxillary palp of C. caragana contained four segments. The average

tudinal grooves, were located inside open cuticular sockets, and were

length of a maxillary palp was 615.1 6 15.9 lm and 580.5 6 18.2 lm

closely inclined toward the palps (Figure 2g). On the labial palps, Ch.1

for females and males, respectively. The labial palp contained three seg-

were somewhat shorter than those on the maxillary palps, but were

ments. The average length of the labial palp was 403.3 6 10.8 lm and

similar morphologically (Figure 3f).

388.6 6 10.0 lm for females and males, respectively. Sunken areas were evident on the distal regions of the last segments of both the

3.2.3 | Type 2 sensilla chaetica

maxillary and labial palps. We found no significant difference in the

Type 1 and type 2 sensilla chaetica of the maxillary palps were morpho-

general structure of the maxillary and labial palps between males and

logically similar. The Ch.2 were longer than the Ch.1 (Figure 2i). The

females (p > .05; Figure 1).

length was adequate to allow the sensilla to reach the next segment.

3.2 | Characteristics of the sensilla on the maxillary and labial palps; SEM data

3.2.4 | Type 3 sensilla chaetica Type 3 sensilla chaetica of the labial palps were long and thin, and were located in open cuticular sockets. The Ch.3 had sharp tips and

€hm’s bristles were present on Sensilla chaetica type 1 and 2, and Bo

longitudinal grooves (Figure 3f).

both the maxillary and labial palps of C. caragana, and large numbers of the sensilla twig basiconica were noted on the distal region of the last

3.2.5 | Digitiform sensilla

segment. The sensillar types were primarily S.tb.1, S.tb.2, S.tb.3, S.tb.4,

The digitiform sensilla formed strips in the cuticular strip sockets on

and S.tb.5 (Figures 2a and 3a). In addition, the Dig. were distributed

the maxillary palps. Approximately 39 Dig. were leaf-shaped (Figure

only on the maxillary palps, and the Ch.3 sensilla were located only on

2g). One end pointed to the position of the “leaf midrib” and was read-

the labial palps. We found no difference in the sensillar types of maxil-

ily visualized (Figure 2h).

lary and labial palps between the males and females.

3.2.6 | Type 1 sensilla twig basiconica € hm’s bristles 3.2.1 | Bo

Type 1 sensilla twig basiconica on the maxillary palps were observed to

€hm’s bristles on the maxillary palps had smooth cuticular walls The Bo

be cylindrical pegs. The sensilla bore irregular longitudinal dimples of

and sharp tips, and extended perpendicularly from sunken sockets

various lengths on the peg surfaces but only a single longitudinal dim-

found on the cuticles of the first segments of the maxillary palps (Fig-

ple passed through the S.tb.1 cuticular surface (Figure 1–21). The tip of

€ hm’s bristles of the labial palps were slightly shorter ure 2i). The Bo

each Stb.1 featured 6–13 finger-shaped protrusions that were either

than those of the maxillary palps but were similar in morphology

close together or scattered. The average height of protrusions was

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ET AL.

Chlorophorus caragana viewed by scanning electron microscopy of sensilla on the maxillary palp. (a) The distal region of a maxillary palp. (b) S.tb.1, shows finger-shaped protrusions and a longitudinal dimple in the cuticular wall of the peg, and the (not-so-obvious) socket. B1 shows a tip pore and a long dimple traversing the cuticular wall. (c) S.tb.2, shows the smooth cuticular wall, the tip pore, and the elevated socket. (d) S.tb.3, shows the tip pore surrounded by protrusions, and the elevated socket. (e) S.tb.4, shows the tip pore in the center of the nipple, and the longitudinal dimple (arrow) in E1. (f) S.tb.5, shows the tip pore and the socket. (g) The ventral side of the fourth segment of the maxillary palp, showing Ch.1 and Dig.; Dig. forms a leaf (in a circle). (h) Magnified Dig., with arrows showing grooves on the cuticular wall. (i) The first segment of the maxillary palp, showing BB. in the basal region and Ch.2 in the distal region. Abbreviations: FSP, fingershaped protrusions; SO, socket; TP, tip pore

FIGURE 2

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Chlorophorus caragana viewed by scanning electron microscopy of sensilla on the labial palps. (a) The distal region of the labial palp. (b) S.tb.1, S.tb.2, and S.tb.4. (c) S.tb.1, S.tb.2, S.tb.3, and S.tb.4. (d) S.tb.2, shows the tip pore and the elevated socket. (e) S.tb.5, shows the socket. (f) The labial palp, showing Ch.1, Ch.2, and Ch.3. (g) BB. On the base of the first segment. Abbreviations: SO, socket; TP, tip pore

FIGURE 3

0.9 6 0.02 lm for both sexes. A dorsal view revealed a small tip-

3.2.7 | Type 2 sensilla twig basiconica

located pore surrounded by small protrusions (Figure 2b).

Type 2 sensilla twig basiconica on the maxillary palps were cylindrical

Type 1 sensilla twig basiconica of the labial palps were similar in

pegs with smooth walls. Each S.tb.2 stood on an elevated round cuticu-

morphology to those of the maxillary palps, although they were shorter

lar socket and had an obvious pore located at the tip. The average

than the maxillary palps. The finger-shaped protrusions averaged 0.9 6

height and average basal diameter of sockets in both sexes were 1.3 6

0.04 lm in length for both sexes (Figures 3b and 3c).

0.1 lm and 3.8 6 0.1 lm, respectively (Figure 2c).

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T AB LE 1

ET AL.

Morphological characteristics of maxillary palp sensilla of C. caragana Length (lm)

Diameter at base (lm)

Morphological characteristics of sensilla

Type

Femalea

Malea

Meanb

Femalea

Malea

Meanb

Wall

Tip

Socket

Cuticular pores

BB.

5.9 6 1.5a

5.2 6 1.6a

5.6 6 1.5

1.8 6 0.6a

2.0 6 0.7a

1.9 6 0.6

Smooth

Sharp

Wide

No

Ch.1

42.2 6 7.7b

38.2 6 6.6a

39.8 6 7.2

3.0 6 0.7a

3.1 6 0.7a

3.1 6 0.7

Grooved

Sharp

Wide

No

Ch.2

78.0 6 24.9a

71.6 6 14.5a

74.6 6 17.4

3.7 6 0.9a

3.9 6 0.6a

3.8 6 0.8

Grooved

Sharp

Wide

No

Dig.

26.5 6 2.0a

23.5 6 3.0a

24.8 6 3.0

1.1 6 0.3a

1.3 6 0.3a

1.2 6 0.3

Smooth

Blunt

Wide

No

S.tb.1

6.6 6 0.9a

7.3 6 0.9b

6.9 6 1.0

2.5 6 0.3a

2.6 6 0.4a

2.5 6 0.3

Grooved

With protrusions

Raised and tight

Tip pore

S.tb.2

5.7 6 1.4a

6.5 6 0.8b

6.0 6 1.2

2.1 6 0.3a

2.4 6 0.3b

2.3 6 0.3

Smooth

Blunt

Raised and tight

Tip pore

S.tb.3

7.4 6 1.1a

6.8 6 1.1a

7.1 6 1.1

2.1 6 0.4a

2.0 6 0.2a

2.0 6 0.3

Smooth

With protrusions

Raised and tight

Tip pore

S.tb.4

2.5 6 0.7b

1.9 6 0.4a

2.3 6 0.7

3.2 6 0.4a

3.3 6 0.4a

3.2 6 0.4

Grooved

With protrusions

Raised

Tip pore

S.tb.5

1.3 6 0.3a

1.1 6 0.4a

1.2 6 0.4

1.1 6 0.2a

1.0 6 0.2a

1.1 6 0.2

Smooth

Blunt

Raised and wide

Tip pore

a Values are the means (6 standard deviations) of the lengths and diameters of at least 15 sensilla of each type from females and males. If the means shown in the rows describing each sensillar type are followed by the same letter, the values did not differ significantly upon independent-samples t testing (p > .05). b Values are the means (6 standard deviations) of the lengths and diameters of at least 30 sensilla of each type from both sexes.

Type 2 sensilla twig basiconica of the labial palps were very similar

0.4 6 0.02 lm, and thus were shorter than those of the S.tb.1. When

to those of the maxillary palps. The average height and average basal

viewed from the dorsal side, the pores were clearly visible and sur-

diameter of sockets in both sexes were 1.2 6 0.0 lm and 2.9 6 0.1 lm,

rounded by the finger-shaped protrusions. In addition, each base bore

respectively (Figure 3b–d).

an obvious elevated socket. The average height and average diameter of sockets for both sexes were 1.0 6 0.1 lm and 3.8 6 0.1 lm,


respectively. The entire peg was embedded in the middle of the

Type 3 sensilla twig basiconica on the maxillary palps were more

socket (Figure 2d).

complex in structure than were S.tb.1 and S.tb.2. Slight longitudinal

Type 3 sensilla twig basiconica of the labial palps were morphologi-

dimples that did not traverse the entire peg were clearly visible. Oth-

cally similar to those of the maxillary palps. The average length of the

erwise, the peg surface was smooth. The pegs bore short finger-

finger-shaped protrusions was 0.3 6 0.03 lm in both sexes. The aver-

shaped protrusions that grouped together at the tip to form an open-

age height and average diameter of sockets for both sexes were 0.8 6

tip pore. The average length of S.tb.3 protrusions for both sexes was

0.1 lm and 3.8 6 0.1 lm, respectively (Figure 3c).

T AB LE 2

Morphological characteristics of labial palp sensilla of C. caragana Length (lm)

Diameter at base (lm)

Morphological characteristics of sensilla

Type

Femalea

Malea

Meanb

Femalea

Malea

Meanb

Wall

Tip

Socket

Cuticular pores

BB.

5.0 6 0.1a

4.7 6 1.2a

4.7 6 1.0

1.6 6 0.3a

1.5 6 0.4a

1.5 6 0.3

Smooth

Sharp

Wide

No

Ch.1

36.6 6 11.4a

39.0 6 9.3a

37.4 6 9.2

3.3 6 1.2a

3.2 6 0.8a

3.2 6 1.0

Grooved

Sharp

Wide

No

Ch.2

75.0 6 21.9a

100.6 6 21.0b

88.5 6 18.8

3.7 6 0.5a

3.8 6 1.2a

3.7 6 1.0

Grooved

Sharp

Wide

No

Ch.3

286.5 6 44.9a

277.4 6 29.7a

282.1 6 22.6

5.8 6 0.6a

6.5 6 0.9a

6.1 6 0.7

Grooved

Sharp

Wide

No

S.tb.1

6.5 6 1.0a

5.6 6 1.0a

6.1 6 1.1

2.1 6 0.2a

2.1 6 0.2a

2.1 6 0.2

Grooved

With protrusions

Raised and tight

Tip pore

S.tb.2

6.1 6 0.4b

5.4 6 0.6a

5.8 6 0.6

2.4 6 0.2b

2.0 6 0.2a

2.2 6 0.3

Smooth

Blunt

Raised and tight

Tip pore

S.tb.3

7.1 6 0.8b

6.0 6 0.6a

6.5 6 0.9

1.9 6 0.4a

1.8 6 0.3a

1.9 6 0.4

Smooth

With protrusions

Raised and tight

Tip pore

S.tb.4

2.4 6 0.4a

2.0 6 0.6a

2.2 6 0.5

2.7 6 0.4a

2.3 6 0.6a

2.5 6 0.5

Grooved

With protrusions

Raised

Tip pore

S.tb.5

1.1 6 0.2a

1.0 6 0.2a

1.1 6 0.2

1.0 6 0.2a

1.0 6 0.1a

1.0 6 0.2

Smooth

Blunt

Raised and wide

Tip pore

a Values are the means (6 standard deviations) of the lengths and diameters of at least 15 sensilla of each type from both females and males, with the exception of Ch.3 (n 5 12). If the means shown in the rows describing each sensillar type are followed by the same letter, the values did not differ significantly upon independent sample t testing (p > .05). b Values are the means (6 standard deviations) of the lengths and diameters of at least 30 sensilla of each type from both sexes, with the exception of Ch.3 (n 5 24).

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more than two outer dendritic segments (Figure 4d–f). In other sam-

Type 4 sensilla twig basiconica of the maxillary palps were circular and

ples, the outer dendritic segments were scattered in the sensillum-

elevated. They were located on the rims of sunken cuticular areas, that

lymph cavity (Figure 4g,h).

contained protuberant nipples. The average length of nipple in both

A cross-sectional view revealed that, in the pegs of the sensilla

sexes was 0.5 6 0.04 lm, and a tip-located pore was noticeable (Figure

twig basiconica (S.tb.2–S.tb.5) on the labial palps, these sensilla had no

2e). A longitudinal groove was noted on the cuticular wall that was

obvious cuticular pores in the thick sensillar walls (Figure 5a–f). Ultra-

directly connected to the base of the sensillum (Figure 2e1).

structurally, the cross-sectional views of the sensilla twig basiconica


varied. In some cases, the peg cavity was divided into outer and inner

cally similar to those of the maxillary palps. The socket protrusions

receptor lymph cavities by the dendritic sheath. The inner receptor

were 0.6 6 0.04 lm long, on average, in both sexes (Figure 3b,c).

lymph cavity contained outer dendritic segments, usually three to five, but sometimes more. The outer dendritic segments contained obvious


microtubules (Figure 5a–d). In some samples, the outer dendritic seg-

Type 5 sensilla twig basiconica on the maxillary palps were nipple-

ments exhibited a dispersed distribution inside the sensillum-lymph

shaped pegs. The sensillar tips of the sensilla were thin, and bore tip

cavity, and average diameter was 0.2 lm (Figure 5e,f).

pores. The sensilla stood inside elevated sockets and average diameter for both sexes was 2.4 6 0.1 lm (Figure 2f).

When examined by TEM, thick dendritic sheathes were visible in the cross-sections of the basal regions of the sensilla twig basiconica


(S.tb.2–S.tb.5) of the labial palps. The tubular body, surrounded by the

cally similar to those of the maxillary palps. The sockets were approxi-

dendritic sheath, was clearly separated from the outer dendritic seg-

mately 4.0 lm wide (Figure 3e).

ments (Figure 5g–i). The tubular body contained dense materials.

The average length of the S.tb.1, S.tb.2, and S.tb.4 on the maxillary palps and the Ch.2, S.tb.2, and S.tb.3 on the labial palps differed signifi-


cantly between the sexes (p < .05). The average basal diameter of the

Subsequent cross sections of the peg of the S.tb.1 on the maxillary

S.tb.2 on the labial palps of the females was also significantly greater

palps showed that the dendritic sheath surrounded the outer dendritic

than that of the males (p < .05).

segments until it reached the tip pore (Figure 6a–d). The inner receptor lymph cavity bore seven unbranched outer dendritic segments and was

3.3 | Characteristics of sensilla on the maxillary and labial palps: transmission electron microscopy data

surrounded by an outer cavity (Figure 6d). At each sensillar socket base, a dendritic sheath separated the tubular body from other outer dendritic segments. Tormogen cells

€ hm’s bristles 3.3.1 | Bo

were evident around the dendritic sheath and the cells bore microvilli.

€hm’s bristles of the maxillary palps lacked cuticular pores in their The Bo

No outer sensillum-lymph cavity was visible (Figure 6e).

thick sensillar walls. No outer dendritic segments were observed inside the sensillar lumen (Figure 4a).

In the ciliary region, eight dendrites of varying diameters were noted, thus indicating that eight bipolar neurons were present. The ciliary segment contained nine peripheral microtubule doublets. Basal

3.3.2 | Sensilla chaetica (Ch.1-Ch.2)

bodies were evident at the junction of each inner dendritic and ciliary

Type 1 sensilla chaetica of the maxillary palps had no cuticular pores in their

segment. Each basal body was embedded in an electron-dense matrix.

thick sensillar walls. The sensillum-lymph cavity was narrow and lacked

The basal bodies marked the distal ends of the inner dendritic segments.

outer dendritic segments (Figure 4b). Type 2 sensilla chaetica of the maxil-

An inner receptor lymph cavity was located between the thecogen cells

lary palps were structurally similar to those of the Ch.1 (Figure 4c).

and the dendrites, and the cavity was full of sensillar lymph. A thecogen

3.3.3 | Digitiform sensilla

cell formed the wall of the inner receptor lymph cavity. Tormogen cells and trichogen cells were located outside the thecogen cells (Figure 6f).

The sensillar cross-sections at the cracks of the maxillary palps were nearly circular. The digitiform sensilla had thick cuticular walls without cuticular pores. The sensillar lumen contained outer dendritic segments

3.4 | The distribution and abundance of the sensilla on the maxillary and labial palps

(Figure 4i). We found no difference in the sensillar distributions on the maxillary

3.3.4 | Sensilla twig basiconica (S.tb.2–S.tb.5)

and labial palps between the sexes (p > .05, Tables 3 and 4).

A cross-sectional view which revealed the pegs of the sensilla twig

Examination of the maxillary palps indicated that the BB. were dis-

basiconica (S.tb.2–S.tb.5) on the maxillary palps indicated these sensilla

tributed on the base of the first segment (Figure 7). The Ch.1 were

had no obvious cuticular pores in their thick sensillar walls (Figure 4d–

abundant on every segment, but tended to gradually increase in num-

h). Ultrastructurally, the cross-sections of the sensilla twig basiconica

ber from the first to the fourth segment. The Ch.2 were distributed dis-

varied. In some samples, the dendritic sheath divided the sensillar

tally, from the first to the third segment, and the numbers on each

lumen of the sensilla twig basiconica (S.tb.2–S.tb.5) into outer and inner

segment were similar. The Dig. were distributed on the ventral side of

receptor lymph cavities. The inner receptor lymph cavity contained

the last segment. Five types of sensilla twig basiconica were distributed

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Chlorophorus caragana viewed by transmission electron microscopy of sensilla on the maxillary palps (a) Cross-section of BB. shows the thick cuticulum and the sensillum-lymph cavity lacking dendrites. (b) Cross-section of Ch.1. (c) Crosssection of Ch.2. (d–h) Crosssections of sensilla twig basiconica (except S.tb.1). (d–f) The dendritic sheath divided the shensillar lumen of the sensilla twig basiconica into outer and inner receptor lymph cavities. (g,h) Scattered outer dendritic segments distributed in the enlarged sensillum-lymph cavity. (i) Cross-section of Dig., shows the outer dendritic segments surrounded by the dendritic sheath. Abbreviations: CW, cuticular wall; DS, dendritic sheath; iRL, inner receptor lymph cavity; oD, outer dendritic segment; oRL, outer receptor lymph cavity; SL, sensillum-lymph cavity FIGURE 4

on the distal region of the last segment. The S.tb.1 were concentrated

second segment (one on each labial palp). Five types of sensilla twig

on the last segment and were located near the lateral elevated cuticula.

basiconica were distributed on the distal region of the last segment.

The S.tb.2 were almost always confined to the distal regions of the max-

The S.tb.1 were concentrated on the elevated cuticula close to the lat-

illary palps. The S.tb.3 and S.tb.4 were distributed along the edges of

eral side; S.tb.2 had the greatest density on the distal regions of the

the distal regions of the last segments of the maxillary palps. The S.tb.5

labial palps, and the distributions of the S.tb.3, S.tb.4, and S.tb.5 were

were distributed on the outer edges of the distal regions. The S.tb.2

similar to those of the maxillary palps. The S.tb.2 were the most abun-

were the most dense of the sensilla twig basiconica types, and S.tb.1

dant, and the other four types of sensilla twig basiconica were present

was the next dense. The density of S.tb.4 was the lowest observed.

at similarly low densities.

Examination of the labial palps indicated that the BB. were distributed on the base of the first segment (Figure 7). The Ch.1 were abundant on every segment but tended to gradually increase in number

4 | DISCUSSION

from the first to the third segment. The Ch.2 were distributed on the distal regions of the first and second segments but were present in

The maxillary and labial palps are considered to be important sensory

greater numbers on the second. The Ch.3 were distributed on the

organs for herbivorous insects especially with respect to host

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9

Chlorophorus caragana viewed by transmission electron microscopy of sensilla twig basiconica (S.tb.2–S.tb.5) on the labial palps. (a–d) Cross-section of sensilla twig basiconica pegs shows the dendritic sheathes that divide the peg cavities into inner and outer receptor lymph cavities. (e–f) Scattered outer dendritic segments distributed in the enlarged sensillum-lymph cavity. (g–i) Cross-sections of the basal region of the peg, showing the thick dendritic sheath and the tubular body full of dense material. Abbreviations: Cu, cuticle; CW, cuticular wall; DS, dendritic sheath; iRL, inner receptor lymph cavity; M, microtubules; oD, outer dendritic segment; oRL, outer receptor lymph cavity; TB, tubular body FIGURE 5

recognition and feeding (Bernays & Chapman, 2001; Chapman, 2003;

This study is the first identification and analysis of several types of

Dethier, 1976; Schoonhoven & Van Loon, 2002). Most studies on the

sensilla in the maxillary and labial palps of the oligophagous pest C. car-

insect sensilla of the maxillary and labial palps focused on the Diptera,

agana, including five types of sensilla twig basiconica, three of sensilla

Lepidoptera, Orthoptera, and Coleoptera (Blaney, 1974; Eilers, Talarico,

€ hm’s bristles. When chaetica, one of digitiform sensilla and one of Bo

Hansson, Hilker, & Reinecke, 2012; Giglio et al., 2010; Lee, Selzer, &

the C. caragana sensilla are compared with those of other coleopteran

Altner, 1985; Wasserman & Itagaki, 2003). In particular, the function of

insects such as the Chrysomelidae, both similarities and differences are

the sense organs located on the palps has been extensively elucidated

apparent. Three types of sensilla twig basiconica are found on the max-

in model insect species such as Drosophila melanogaster Meigen (Dip-

illary and labial palps of Chrysolina aeruginosa Fald. (Coleoptera: Chryso-

tera: Drosophilidae) and Manduca sexta Linnaeus (Lepidoptera: Sphingi-

melidae) similar to their distribution in C. caragana. However, C.

dae) (Mitchell, Itagaki, & Rivet, 1999; Shanbhag et al., 1999; Shanbhag,

aeruginosa lacks the S.tb.4 and S.tb.5 (Zhang et al., 2013). Three types

Park, Pikielny, & Steinbrecht, 2001). We therefore assumed that the

of basiconica sensilla are located on the distal regions of the labial palps

maxillary and labial palps of C. caragana with gustatory sensilla could

of Siagona europaea Dejean (Coleoptera: Chrysomelidae) similar to the

play a role in host recognition.

S.tb.1, S.tb.2, and S.tb.3 of C. caragana. Another sensillar type termed

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Chlorophorus caragana viewed by transmission electron microscopy of sensilla type 1 twig basiconica (S.tb.1) on the maxillary palps. (a) Scanning electron micrographs of S.tb.1; the dotted line is a region close to the cross-section taken. (b–f) Transmission electron microscopy of S.tb.1. (b) Cross-section of the finger-shaped protrusions shows the scattered cuticula. (c) Cross-section of the basal regions of finger-shaped protrusions shows the inner receptor lymph cavities without outer dendritic segments. (d) Cross-section of the middle region of the peg shows the dendritic sheath dividing the sensillum-lymph cavity into an inner and an outer cavity, with seven outer dendritic segments in the inner cavity. (e) Basal region of the peg shows the tubular body surrounded by the dendritic sheath, and separated from outer dendritic segments. The outside of the dendritic sheath is formed by a tormogen cell. (f) Cross-section of the ciliary region showing eight dendrites of different diameter. Abbreviations: bb, basal body; cs, ciliary segment; Cu, cuticle; CW, cuticular wall; DS, dendritic sheath; iRL, inner receptor lymph cavity; M, Microtubule; Mi, microvilli; oD, outer dendritic segment; oRL, outer receptor lymph cavity; TB, tubular body; TH, thecogen cell; TO, tormogen cell; TR, trichogen cell

FIGURE 6

sensilla coeloconica by Giglio et al. (2010) is similar to S.tb.5 in mor-

(Bloom, Zacharuk, & Holodniuk, 1981; Zacharuk et al., 1977). Digiti-

phology and distribution and may be just synonymous. In addition, the

form sensilla are also found on the heads of coleopteran larvae, but the

sensilla twig basiconica of the palps of C. caragana are notably similar

numbers vary (Eilers et al., 2012; Giglio et al., 2003; Whitehead, 1981).

to the uniporous sensilla of the larval heads of other Coleoptera

For example, each maxillary palp of the larvae of Melolontha melolontha

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T AB LE 3

11

Numbers of sensilla in the male and female maxillary palps of C. caragana

Type

Sex

First

Second

Third

Fourth

Distal region of 4th

BB.

Female

7.7 6 1.6a

0

0

0

0

Male

8.2 6 0.8a

0

0

0

0

Female

3.3 6 1.0a

12.3 6 1.6a

18.8 6 1.9a

38.5 6 4.9a

0

Male

4.2 6 1.2a

13.0 6 2.0a

20.7 6 5.6a

41.2 6 1.5a

0

Female

4.8 6 0.8a

5.2 6 0.8a

4.8 6 1.5a

0

0

Male

5.2 6 1.5a

5.0 6 0.9a

5.2 6 0.4a

0

0

Female

0

0

0

26.0 6 2.4a

0

Male

0

0

0

26.0 6 2.1a

0

Female

0

0

0

0

48.3 6 4.2a

Male

0

0

0

0

50.0 6 3.9a

Female

0

0

0

0

89.0 6 2.3a

Male

0

0

0

0

88.5 6 3.6a

Female

0

0

0

0

17.3 6 1.6a

Male

0

0

0

0

18.7 6 1.8a

Female

0

0

0

0

5.0 6 1.4a

Male

0

0

0

0

5.2 6 1.7a

Female

0

0

0

0

21.0 6 2.4a

Male

0

0

0

0

19.0 6 1.3a

Ch.1

Ch.2

Dig.

S.tb.1

S.tb.2

S.tb.3

S.tb.4

S.tb.5

First–fourth, first to fourth Segments. Values shown are the means (6 standard deviations) of the data from 12 maxillary palps from each sex. If the means shown in the rows describing each sensillar type are followed by the same letter, the values did not differ significantly upon independentsamples t testing (p > .05).

L. (Coleoptera: Scarabaeinae) has only one Dig., while that of the adult

we suggest that the Ch.1 of the maxillary and labial palps of C. caragana

C. caragana has approximately 39 (Eilers et al., 2012).

may be stimulated upon contact with solid objects and could be

Based on previous studies of palpal structure and function in other insect species, we suggest potential functions of each type of sensilla of C. caragana (Altner & Prillinger, 1980).

involved in the identification of the physical characteristics of the plant surface while selecting the oviposition site. The Ch.2 were distributed on the distal region of each segment (except for the final one) which were considered to perceive the posi-

€ hm’s bristles 4.1 | Bo The BB. are considered to be important receptors of mechanical stimuli (Schneider, 1964) that are widespread in many insect species (Merivee et al., 2000; Yang, Yan, & Liu, 2009). We suggest that the BB. on the maxillary and labial palps of C. caragana could help to identify its palpal position as C. caragana changed position.

4.2 | Sensilla chaetica

tion of the palps and control the direction of movement of each palpal segment. Keil (1997) found that the mechanoreceptive bristles are critical for the regulation of body position. For example, the mechanoreceptors in the neck region of the honeybee can monitor the relative position of the head and thorax (Lindauer & Nedel, 1959; Thurm, 1965). The sensilla chaetica on the distal region of each flagellomere of Psacothea hilaris Pascoe (Coleoptera: Cerambycidae) can help control the extent of antennal bending and perceive the movements of adjacent flagellomeres (Dai & Honda, 1990).

The sensilla chaetica of the maxillary and labial palps of C. caragana have no cuticular pores, and there are no outer dendritic segments inside the sensillar lumen that are considered to be typical mechano-

4.3 | Digitiform sensilla

sensitive sensilla (Keil, 1997; Klowden, 2013). The sensilla chaetica are

The digitiform sensilla of C. caragana had no visible cuticular pores, but

widely distributed in most Coleoptera (Bartlet, Romani, Williams, & Isi-

they did have outer dendritic segments that were inside the sensillar

doro, 1999; Chen et al., 2010; Ren, Shi, Zhang, & Luo, 2012).

lumen. These sensilla are present on the heads of most other adult or

The morphology and ultrastructure of the Ch.1 observed in this

larval insects. The fine structures of the Dig. of C. caragana were similar

study were similar to the mechanoreceptive bristles described by Keil

to those of other insects (Eilers et al., 2012; Honomichl & Guse, 1981;

(1997) that were considered to be useful for direct touch. Therefore,

Whitehead, 1981). The digitiform pegs on the labial palps of the larvae

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T AB LE 4

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The numbers of sensilla in the male and female labial palps of C. caragana

Type

Sex

First

Second

Third

Distal region of 3rd

BB.

Female

5.8 6 1.2a

0

0

0

Male

5.0 6 1.4a

0

0

0

Female

6.3 6 0.8a

18.2 6 1.0a

64.2 6 2.3a

0

Male

6.3 6 1.2a

17.7 6 1.4a

63.3 6 3.9a

0

Female

3.8 6 1.2a

11.5 6 1.6a

0

0

Male

4.3 6 1.6a

11.8 6 2.5a

0

0

Female

0

1.0 6 0.01a

0

0

Male

0

1.0 6 0.02a

0

0

Female

0

0

0

13.2 6 1.6a

Male

0

0

0

14.3 6 2.0a

Female

0

0

0

111.2 6 3.2a

Male

0

0

0

112. 7 6 3.1a

Female

0

0

0

13.2 6 1.2a

Male

0

0

0

12.8 6 2.9a

Female

0

0

0

7.5 6 1.1a

Male

0

0

0

8.0 6 1.8a

Female

0

0

0

14.8 6 1.5a

Male

0

0

0

14.8 6 1.3a

Ch.1

Ch.2

Ch.3

S.tb.1

S.tb.2

S.tb.3

S.tb.4

S.tb.5

First–Fourth, first to fourth segments. Values shown are the means (6 standard deviations) of data on 12 labial palps of each sex. If the means shown in the rows describing each sensillar type are followed by the same letter, the values did not differ significantly upon independent-samples t testing (p > .05).

of Ctenicera destructor Brown (Coleoptera: Elateridae) respond electro-

4.4 | Sensilla twig basiconica

physiologically to contact and vibratory stimuli but not to the amino acids, sugars, salts, and water tested (Zacharuk et al., 1977). Other studies also concluded that the digitiform organ is hygro- and thermosensitive (Eilers et al., 2012; Guse, 1980) or engaged in the recognition of CO2 (Honomichl & Guse, 1981). In lepidopteran larvae, the digitiform organ is considered to be mechanoreceptive (Devitt & Smith, 1982). We suggest that the Dig. of C. caragana has some of the characteristics of contact chemoreceptors based on all of these studies cited on sensillum ultrastructural research.

The sensilla twig basiconica on the maxillary and labial palps of C. caragana were distributed on the distal region of the maxillary and labial palps and could contribute to the behavioral pattern of C. caragana that entails continual use of its palps to contact the plant surfaces after arriving at the plant. In each S.tb.1 with a tip pore, one of the neurons ends in a tubular body at the base of the peg, and the other outer dendritic segments were surrounded by the dendritic sheath until the tip pore that is a

F I G U R E 7 Distribution of sensilla twig basiconica in the distal region of the maxillary and labial palps of C. caragana. (a) The distal regions of the maxillary palp. (b) The distal regions of the labial palp [Color figure can be viewed at wileyonlinelibrary.com]

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13

typical characteristic of contact chemoreceptors (Slifer, 1970;

ACKNOWLE DGME NTS

Zacharuk, 1980). The sensillar channel in the S.tb.1 peg may provide a

We appreciate the generous assistance of the Beijing Vocational

hydraulic mechanism for finger opening and closing that is similar to

College of Agriculture, the Institute for the Application of Atomic

the taste sensilla of Locusta migratoria Linne (Orthoptera: Acrididae)

Energy (Chinese Academy of Agricultural Science), the Bioresearch

(Blaney, Chapman, & Cook, 1971). Pore opening or closing by similar

Center of Beijing Forestry University and Professor Shan-gan Zhang

fingers was reported to occur in the taste receptors of Colorado potato

of the Institute of Zoology, Chinese Academy of Sciences. This

beetle larvae in response to chemical stimuli (Mitchell & Schoonhoven,

research was supported by the National Natural Science Foundation

1974). There were eight neurons inside the S.tb.1 of C. caragana, one

of China (Grant No. 31570643, 31270693), “Twelfth Five-year”

of which ended in a tubular body at the base of the peg and was inter-

National Science and Technology Support Project of China (Grant

preted to be a typical mechanosensilla characteristic (Devitt & Smith,

No. 2012BAD19B071).

1982; Keil, 1997; Zacharuk, 1980). The rest of the seven are considered to be chemosensory neuron (Zacharuk, 1980). Studies of insect taste receptor cells suggest that individual gustatory neurons can sense

ORC ID Yan-Ru Zhang

http://orcid.org/0000-0002-0185-5591

a variety of simple and compound sugars, salts, amino acids, certain acidic odors (Dethier, 1976; Pollack & Balakrishnan, 1997; Shiraishi & Kuwabara, 1970), and a broad range of structurally heterogeneous alkaloids and bitter molecules (Chapman, Ascoli-Christensen, & White, 1991; Glendinning & Hills, 1997). These chemicals can elicit positive or negative responses. For example, sugars, amino acids, and host plantspecific secondary substances often have stimulating effects on the neurons, while salt, non-host secondary substances, and toxic chemicals tend to have inhibitory effects (Bernays & Chapman, 2001; Schoonhoven, Simmonds, & Blaney, 1991; Schoonhoven & Van Loon, 2002). Certain quinones are considered to be gustation inhibitors in a number of insects (Gilbert, Baker, & Norris, 1967; Gilbert & Norris, 1968; Norris, 1969). The terminal sensilla of Schistocerca gregaria Forskal (Orthoptera: Acrididae) and Locusta migratoria migratorioides Reiche and Frm. (Orthoptera: Acrididae) contain neurons specifically sensitive to secondary materials synthesized by the neem plant (Haskell & Schoonhoven, 1969). In this study, we suggest that the seven neurons of the S.tb.1 were either phagostimulatory and/or deterrent neurons that played an important role in identifying stimulators and/or inhibitors during C. caragana feeding, oviposition and host selection. The group of sensilla (S.tb.2–S.tb.5) of C. caragana all had an opening at one point in the cuticle through which chemical communication could occur between the dendrites and the external environment (Zacharuk, 1980). These sensilla (S.tb.2–S.tb.5) were structurally similar to the thick walled sensilla (Slifer & Sekhon, 1969). All of the microtubules within the dendrites are supporting elements that receive the ordering from the branching of the dendrites during molting (Ernst, 1969). In particular, the S.tb.2 and S.tb.3 are typical contact chemoreceptors with tip pores and flexible sockets (Altner & Prillinger, 1980; Zacharuk, 1980, 1985) that had already been observed in many insect species such as Chrysolina aeruginosa Faldermann (Coleoptera: Chrysomelidae), Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae), Siagona europaea Dejean (Coleoptera: Carabidae), Ips typographus Linnaeus (Coleoptera: Scolytidae), and Dendroctonus ponderosae Hopkins (Coleoptera: Scolytidae) (Giglio et al., 2010; Hallberg, 1982; Sen, 1988; Whitehead, 1981; Zhang et al., 2013). Generally, these types of sinsilla are known to be gustatory receptors that play an important role in host-plant recognition by phytophagous insects (Giglio et al., 2010).

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