Arthropod Structure & Development 40 (2011) 420e428

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Arthropod Structure & Development

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Anatomical organization of antennal-lobe glomeruli in males and females

of the scarab beetle Holotrichia diomphalia (Coleoptera: Melolonthidae)
Ji-Hua Hu, Zhi-Ying Wang, Fan Sun*
School of Forestry, Northeast Forestry University, Harbin 150040, PR China

a r t i c l e i n f o a b s t r a c t

Article history: The glomerular organization of the primary olfactory brain center, the antennal lobe, was studied in
Received 16 July 2010 males and females of Holotrichia diomphalia adults using serial histological sections labeled by the
Received in revised form reduced silver-stain technique. The results revealed an apparent sexual dimorphism. Whereas an
13 March 2011
enlarged cap-shaped glomerulus was found at the antennal nerve entrance into the antennal lobe in
Accepted 16 March 2011
males, no such unit was present in females. Also the size of the antennal lobe differed between the sexes,
the antennal lobe of males being larger than that of females. We estimated the total number of glomeruli
Keywords:
at approximately 60 units in the female antennal lobe. In males, we could discriminate only those
Olfaction
Glomeruli
glomeruli that were located in the anterior area of the antennal lobe.
Antennal lobe Ó 2011 Elsevier Ltd. All rights reserved.
Scarab beetle
Dimorphism
Macroglomerulus

1. Introduction example, allows for the identification of individual glomeruli

The olfactory sense is essential for insects, as it plays a para-
mount role in locating food sources, oviposition sites, and
conspecific individuals, mates included (Hartlieb and Anderson,
1999). The primary olfactory brain center of insects, the antennal
lobe (AL), constitutes the first synaptic relay station of the antennal
afferent pathways, as it receives input from antennal olfactory
sensory neurons and sends the output to higher brain centers
(Rospars, 1988; Hildebrand and Shepherd, 1997; Anton and
Homberg, 1999). The building blocks of the AL found in most
insect orders are the olfactory glomeruli, in which the interactions
between antennal and deutocerebral neurons take place
(Schachtner et al., 2005). Thus, the glomerular array is thought to
constitute a chemotopic map, which ultimately leads to olfactory
coding (Christensen and White, 2000; Galizia and Menzel, 2000,
2001; Ignell and Hansson, 2005).
Due to their functional significance glomeruli have been
anatomically mapped in several insect species. The size, number,
and organization of glomeruli are shown to be conserved within
individuals of one species, meaning that insects seem to have
a species-specific glomerular arrangement. Their relatively small
number of glomeruli, as compared to those of mammals for
* Corresponding author.
E-mail address: sunf-cf@nefu.edu.cn (F. Sun).
within a species (Rospars, 1988; Anton and Homberg, 1999;
Schachtner et al., 2005). The numbers of glomeruli were ranging
from 43 in fruit fly (Stocker, 1979, 1994; Stocker et al., 1990) to 442
in ants (workers) (Kuebler et al., 2010). In some species, as
Drosophila melanogaster (Stocker, 1979, 1994; Stocker et al., 1990)
and Pieris brassicae (Rospars, 1983), the glomeruli of males and
females are of approximately equal size and shape. However,
sexually dimorphic AL glomerular organization has also been
reported in a number of Hymenoptera, Lepidoptera, and Blattaria
species (Rospars, 1988; Anton and Homberg, 1999; Schachtner
et al., 2005). In particular, an arrangement of enlarged glomeruli
situated at the antennal nerve entrance, a so-called macro-
glomerulus (MG) or macroglomerular complex (MGC), has been
described in males of several species. This male-specific structure is
responsible for processing sex pheromone information underlying
reproductive behavior (Rospars, 1983; Boeckh and Selsam, 1984;
Arnold et al., 1985; Rospars and Hildebrand, 1992; Hansson, 1997).
Thus far, no study has been conducted on elucidating the
complete antennal-lobe glomerular organization of Coleoptera
species (beetles). A few publications have reported about individ-
ually recognizable glomeruli in beetles (reviewed by Schachtner
et al., 2005). Recently, Dreyer et al. (2010) described the structure
and arrangement of a part of the antennal-lobe glomeruli of the red
flour beetle, Tribolium castaneum. Regarding the peripheral system,
the fact that semiochemicals were identified in some economically
important plant-feeding scarabs (Leal, 1998) promoted studies on

1467-8039/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.asd.2011.03.003
 J.-H. Hu et al. / Arthropod Structure & Development 40 (2011) 420e428 421

the function of olfactory receptor neurons of beetles. Thus, antennal
sensilla housing receptor neurons specifically tuned to female-
produced pheromones and plant volatiles have been identified in
several scarab beetles (Renou et al., 1998; Hansson et al., 1999;
Larsson et al., 2001). This information represents a foundation for
investigating the structure and function of scarab beetles’ central
nerve system. As severe pests during the larval stage, these insects
cause substantial damage to agricultural crops and forestry
productions worldwide (Lee, 2003). The significance of beetles is
difficult to ignore from several points of view, the economical one
not being the least.
Some species of scarab beetles show an obvious sexual dimor-
phism of peripheral olfactory organs. This is expressed by enlarged
antennal lamellae and thereby a considerably higher number of
olfactory sensilla in males as compared to females (Meinecke, 1975;
Ågren, 1985). For other species, the size of lamellae and number of
sensilla may be similar between sexes, but the type of olfactory
sensilla and/or the sensitivity of olfactory receptor cells (ORC) are
significantly different (Kim and Leal, 2000; Larsson et al., 2001;
Nikonov et al., 2001; Stensmyr et al., 2001). Generally, sexual
dimorphism of antennal sensilla often correlates with dimorphism
of the AL (Rospars, 1988). In a previous study, using scanning and
transmission electron microscopes, the types and ultrastructure of
antennal olfactory sensilla of Holotrichia diomphalia Bates, an
important soil pest in the Northeastern region of China, was char-
acterized. The results revealed that sensilla basciconica and sensilla
placodea were two predominant types of olfactory sensilla in both
sexes and that the number of sensilla was significantly higher in
males than that in females (Sun et al., 2007).
In this study, the anatomical organization of antennal-lobe
glomeruli is described in males and females of H. diomphalia.
The results, obtained by investigating serial antennal-lobe sections
labeled via the reduced silver-stain technique, demonstrate a
clear sexual dimorphism expressed by an enlarged male-specific
macroglomerulus situated at the entennal nerve entrance. The
data provide a sound foundation for functional investigations of the
insect’s central nervous system.
2. Material and methods
2.1. Insects
H. diomphalia eclose in the soil at the end of July and over-
winter in the original place. Adults come out of overwintering in
early June the following year and the adult stage lasts about 30
days. Wild adults of H. diomphalia were collected in Sun Island Park,
Harbin, Heilongjiang province on June 15th, 2008, between 18:00
and 20:00.
China), and finally mounted using Canada balsam (Shanghai
molding plant specimens. Shanghai, China). Observations and
photos were made by using a light microscope equipped with
a 20
/0.50 Ph2 dry objective lens (Olympus BX51X) and a digital
camera (Olympus DP71).
2.3. Image processing and statistical analysis
The stained sections of at least 20 scarab beetle were compared.
Although the AL of both hemispheres were stained in some prep-
arations, we only chose a single AL from each specimen for further
analysis, disregarding possible differences between left and right
hemispheres. Four brains of each sex stained with sliver-labeling
technique were chosen for statistical analysis. The images were
processed using Photoshop 10.0. Sections containing glomeruli
were photographed in the same manner (direction and magnifi-
cation) in both males and females. Adjacent sections that contain
same glomeruli were matched by overlapping images from
consecutive cuts, comparing shapes and locations on each image,
etc. The 3-dimensional measurements of the ALs were measured
(two perpendicular lengths in mm) on the section of a given
glomerulus where it appears with the largest area. In the direction
perpendicular to the sectioning plane, we multiplied the number of
sections on which the glomerulus was visible by the section
thickness (9 mm), giving a third number (depth in mm). Methods of
measurements of the MG size were the same as AL. Here, the
maximum length of AL and MG was measured using the DP
controller 3.1.1.267 Software. All the data were analyzed with one-
way ANOVA (SPSS 13.0).
2.4. Glomerular nomenclature
The glomerular nomenclature used in the present study is based
on the general position (Boyan et al., 1993; Nishino et al., 2005;
Ghaninia et al., 2007) of single glomeruli in the AL, where two of
the following capital letters denote the position; A (anterior),
P (posterior), V (ventral), D (dorsal), L (lateral), M (medial), and
C (central) (Figs. 1, 3e5). Positions are given according to the neu-
roaxis and not to the coordinates of the body. According to the
sectional series from anterior to posterior, the first 9 sections of
female ALs were designated as region A and the rest were

2.2. Preparations of brain tissues for histological observation

Heads of males and females were fixed in Bouin’s fixation

solution (Kermel Chemical Reagent Company, Tianjin, China) for 2
days. The brains were dissected under a stereo microscope and pre-
stained by placing them in 1% silver nitrate solution (Beijing
Chemical Plant, Beijing, China) in darkness (24 h). The pre-stained
brains were then embedded in paraffin and sectioned at 9 mm
thickness from anterior (A) to posterior (P) or from ventral (V) to
dorsal (D) with a Leica RM 2015 microtome. The serial sections
were dried on histological slides before being rehydrated and
stained according to Rowell’s method (Rowell, 1963). The stained
sections were then dehydrated in ascending concentrations of
Fig. 1. Photomicrograph of the dissected brain of a female H. diomphalia Bates. AL:
ethanol (Restoration Technology Development Co., Ltd. Tianjin, antennal lobe; AN: antennal nerve; CPC: circumoesophageal connective; OL: optic
China) (30%, 50%, 70%, 90%, 95%, 100%
3, 10 min each time), lobe; PT: protocerebrum; TB: tritocerebrum; A: anterior; D: dorsal; L: lateral; M:
diaphanized in xylene (Kermel Chemical Reagent Company, Tianjin, medial; P: posterior; V: ventral. Scale bar ¼ 0.5 mm.
422 J.-H. Hu et al. / Arthropod Structure & Development 40 (2011) 420e428
Fig. 2. One section of a H. diomphalia Bates brain. AL: antennal lobe; CB: central body;
MI: medulla interna; PB: protocerebral bridge.
designated as region P as glomeruli were present in 18 consecutive
sectional planes of female ALs. Similarly, the first 12 sections of
male ALs were designated as region A and the rest of 12 sections
were designated as region P as glomeruli were present in 24
consecutive sectional planes of male ALs. Two circles were drawn
with a common center located at the middle point of the maximum
diameter of the AL (the large circle diameter was the maximum
diameter of AL and the small circle diameter was half of the large
circle diameter). The small circle represented C region. The area
between the large and small circle was divided into 4 equal parts
and designed as sub-regions D, L, V, and M (Fig. 3). The number in
each glomerular name represents the sequence when it appeared
in that specific region, e.g. AD8 means that this glomerulus
appeared in the AD region in the 8th sectional plane. Glomeruli
with lower number in region A are located more anteriorly in the
antennal lobe whereas high numbers in the P region indicates
glomeruli positioned posteriorly (Fig. 2).
Glomeruli were categorized into 3 classes as described by
Ghaninia et al. (2007). Briefly, class 1 glomeruli are fairly constant
in shape, size, position, brightness, and distinctiveness. Class 2
glomeruli generally comply with Class 1 criteria and are identified
Fig. 3. The naming of glomeruli in different regions. The larger/outside circle repre-
sents the maximum diameter of the antennal lobe. The smaller/inside circle, drawn at
a diameter that is half of the larger circle diameter, defines the central region (region
C). The area between the large and small circle is divided into 4 equal parts and
designed as sub-regions D, L, V, and M.
by their relative position to Class 1 glomeruli. Class 3 glomeruli
are poorly demarcated ones with variation in more than one
criterion.
3. Results
3.1. The antennal lobe
The ALs of H. diomphalia are located at the anterior part of the
deutocerebrum in a left and right symmetric manner. The chemo-
sensory axons which constitute the antennal nerve, project to the
ipsilateral AL (Fig. 1). The stainings show, in both sexes, cell bodies
distributed at the periphery of the AL and an array of glomeruli
arranged in one to two layers around a central core (Fig. 4EeG and
Fig. 5FeG). Individual glomeruli are separated by glia and form
irregular spherical units (Figs. 4 and 5).
3.2. Sexual dimorphism
There is a pronounced sexual dimorphism concerning glomer-
ular arrangement. The males have, in addition to an assembly of
ordinary glomeruli, one cap-shaped MG occupying the entire AM
region and accounting for about 1/3 of the AL(Fig. 5BeG). Females,
on the other hand, have an assembly of similarly sized glomeruli
only(Fig. 4). In the females, these ordinary units are arranged
around a central core devoid of glomeruli whereas the corre-
sponding glomeruli of the males partly occupy this region due to
the presence of an enlarged MG. Additionally, the glomeruli at the
posterior part of male ALs were more densely packed and indi-
vidual glomeruli were difficult to delineate when compared with
females.
Furthermore, male ALs were relatively larger than those of
females as indicated by the significant difference (P ¼ 0.004) in
length at the AeP direction. The lengths along directions of MeL
and DeV, respectively, were not significantly different (P ¼ 0.13 and
0.11 respectively; Table 1).
3.3. Description and discrimination of antennal glomeruli
From analyzing the silver-stained sections of each individual,
a total number of approximately 60 antennal-lobe glomeruli were
estimated in females of H. diomphalia. Thus, the 4 stained section
series of ALs comprised 59, 59, 61, and 62 glomeruli, respectively.
The glomeruli in the specimen containing 61 units, units with
names given according to the standard previously described,
resulted in 36 Class 1 glomeruli, 18 Class 2 glomeruli, and 8 Class 3
glomeruli (Table 2, Fig. 4). Glomeruli of Class 3 are divided into 4
groups, AD3 and AD5(Fig. 4C), AD6 and AC6(Fig. 4C), PD2 and
PD3(Fig. 4H),PC5 and PC6(Fig. 4I). The two glomeruli of each group
were arranged closely to each other and their boundaries were
blurred.
Regarding the male preparations, the cap-shaped MG were
observed in all of the 4 stained section serials respectively. The 3-D
dimensions of the MG at their maximum were 131  4.68 mm
(AeV), 81.6  3.61 mm (LeM),126  5.87 (AeP) (Mean  MS,n ¼ 4
specimens). But the glomeruli at the posterior part of male ALs
were more densely packed and individual glomeruli were difficult
to delineate because of their unclear boundary(Fig. 5HeJ). The
glomeruli at the anteriorly part units, were distinguishable and
given names according to the standard previously described,
resulted in 23 Class 1 glomeruli, 14 Class 2 glomeruli (Table 2,
Fig. 5BeG).
 J.-H. Hu et al. / Arthropod Structure & Development 40 (2011) 420e428 423

Fig. 4. Anterior view of the antennal lobe of a female H. diomphalia Bates, stained by the silver-labeling technique. To display all glomeruli in a limited number of images, 10 sections
at different intervals, from anterior to posterior, are selected from a total of 18 images. The depth within the AL neuropil is indicated in the upper-right corner of each image.
Anterior is up and lateral to the right. The left column displays unaltered silver-stained preparations (raw data). The middle column displays demarcated glomeruli superimposed on
gray scale inverted images. The right column displays identified glomeruli. Scale bar ¼ 100 mm.
424 J.-H. Hu et al. / Arthropod Structure & Development 40 (2011) 420e428

Fig. 4. (continued).
 J.-H. Hu et al. / Arthropod Structure & Development 40 (2011) 420e428 425

Fig. 5. Anterior view of the part of antennal lobe of a male H. diomphalia Bates, stained by the silver-labeling technique. To display all glomeruli in a limited number of images, 10
sections from anterior to posterior were selected from a total of 24 images at different intervals, where the depth within the AL neuropil is indicated in the upper-right corner of
each image, in order to display all glomeruli and to restrict the number of sections displayed. Anterior is up and lateral to the right. From A to G, left column displays unaltered
silver-stain (raw data). Middle column displays demarcated glomeruli superimposed on gray scale inverted images. Right column displays identified glomeruli. H, I and J displays
unaltered silver-stain (raw data) near the posterior end. Scale bar ¼ 100 mm. For colour, please refer to the online version of this article.
426 J.-H. Hu et al. / Arthropod Structure & Development 40 (2011) 420e428
Fig. 5. (continued).
4. Discussion
4.1. Sexual dimorphism
Sexual dimorphic features in the AL (number, location, and
arrangement of dimorphic glomeruli) differ considerably in various
insect orders, suggesting that parallel convergent evolution has
occurred toward sexual dimorphic structures in these taxa as
a consequence of the involvement of the olfactory system in sexual
communication and mate finding (Schachtner et al., 2005). The
presence of male-specific enlarged glomeruli (macroglomeruli) is
the most common form of sexual dimorphism, occurring in cock-
roaches, honey bees, ants, and moths.
The appearance of male-specific enlarged glomeruli has been
observed mainly in species with dimorphic antennae and whose
males are attracted to females by olfactory signals (Rospars, 1988).
One of the most thoroughly studied species, the male tobacco
hornworm, Manduca sexta, possesses a special MGC that correlates
with the presence of long, type-I trichoid sensilla that are found
exclusively on male antennae. These sensilla house two types of
ORCs, each of which is sensitive to one of the two key components of
the conspecific female moth’s sex pheromone (Kaissling et al., 1989;
Keil, 1989; Lee and Strausfeld, 1990). Similar to moths, many female
scarab beetle species release sex pheromones to attract mates at
long distances (Leal, 1998) and sex-specific characteristics of
antennal sensilla are quite common (Sun et al., 2007). The males of
H. diomphalia have a considerably larger number(9
)of sensilla
basiconca on their antenna as compared to females (Sun et al., 2007).
Therefore, it is plausible to hypothesize that these sensilla serve as an
organ for detection of female-produced sex pheromones. Regarding
H. diomphalia, the observation that females extend a white colored
spherical saccule at the abdominal tip, suggests a sexual behavior
including release of chemical signals to attract males for mating.
Thus, we speculate that the male-specific MG identified in the
current study receives input from pheromone-detecting receptor
neurons housed by the sensilla basiconica.
Table 1
Size comparisons of female and male antennal lobe of Holotrichia diomphalia.
Direction Female(mm) Male(mm)
MeL 163.00  10.96 A 196.00  9.25 A
DeV 181.40  9.28 A 228.50  21.59 A
AeP 160.20  10.67 A 226.75  11.49 B
Data are presented as Mean  MS (n ¼ 4 specimens).
Means within a row followed by the same letter were not significantly different at
the level of 0.05.
 J.-H. Hu et al. / Arthropod Structure & Development 40 (2011) 420e428 427

Table 2 et al., 1998, 2005; Lee et al., 2006a). Thus, the number of macro-
Glomerular neuropils in the antennal lobe of female and male Holotrichia diomphalia glomerular sub-structures seems to be correlated with the number
by the silver-labeling technique.
of physiological male-specific receptor neuron categories. However,
Female Malea there also are exceptions, e.g. in Helicoverpa zea, ORNs that detect
Glomerulus Classb Commentc Glomerulus Classb Commentc different semiochemicals are reported to project to a single MGC
AC1 1 AC1 1 subunit (Lee et al., 2006b). Moreover, in Lobesia botrana and Cydia
AC2 2 d AC2 1 pomonella (Tortricidea), only one large glomerulus is present in the
AC3 2 d AC3 1 ALs of males, while three different components serve as phero-
AC4 1 AC4 1
mones (Masante-Roca et al., 2005; Varela et al., 2009). Also in the
AC5 1 AC5 1
AC6 3 sh, p AC6 2 sh
cockroach, Periplaneta americana, two types of pheromone receptor
AC7 1 AC7 1 neurons tuned to the female-produced attractants periplanone A
AC8 2 sz AC8 1 and periplanone B, respectively, are reported to target one male-
AD1 1 AC9 1 specific unit, the MG (Boeckh et al., 1970; Boeckh and Selsam,
AD2 1 AC10 1
1984). The results presented here, demonstrate the presence of
AD3 3 sh, sz, p AC11 2 sh
AD4 1 AC12 2 sh a male-specific antennal-lobe structure of H. diomphalia, an MG
AD5 3 sz, p AC13 1 similar to that of cockroaches. The prominence of the MG in this
AD6 3 sh, p AD1 1 Coleopteran species, both as concern size and position, indicates
AD7 2 sh AD2 1 the importance of pheromone signals for reproductive behavior.
AD8 1 AD3 1
AD9 2 sh AD4 2 sz
AD10 2 sh, d AL1 1 4.2. Antennal glumeruli of H. diomphalia: number and description
AD11 2 d AL2 1
AL1 1 AL3 1 The number of approximately 60 antennal-lobe glomeruli in
AL2 1 AL4 2 d
H. diomphalia, as determined for the females in the present study, is
AL3 1 AL5 2 d
AL4 1 AL6 1 in correspondence with previous reports from other insect species.
AL5 1 AL7 2 sh In particular, this number is similar to the finding of ca 70 glomeruli
AL6 1 AL8 1 in the red flour beetle T. castaneum (Dreyer et al., 2010). As
AL7 2 d AL9 2 d compared to mammals, which often have thousands of glomeruli in
AL8 2 d AV1 1
AM1 1 AV2 1
their primary olfactory center (Menco, 1980), insects usually
AM2 1 AV3 1 possess a considerably smaller number of typically 50e250
AM3 1 AV4 2 sh, d glomeruli (Rospars, 1988; Anton and Homberg, 1999; Schachtner
AM4 2 d AV5 2 sz, d et al., 2005). It seems that the numbers of glomeruli are similar
AM5 2 d AV6 2 sz, d
across species of the same insect group. Thus, Diptera generally
AM6 1 AV7 2 sh, d
AV1 1 AV8 2 sh, d have numbers ranging from 40 to 50, Lepidoptera 60 to 65, and
AV2 1 PD1 1 Hymenoptera more than 100 (Schachtner et al., 2005; Ignell and
AV3 1 PD2 2 sh Hansson, 2005; Ghaninia et al., 2007; Masante-Roca et al., 2005;
AV4 1 MG 1 Varela et al., 2009; Nishikawa et al., 2008; Zube and Rössler, 2008).
AV5 1
AV6 2 sh
Although we numbered and named all found glomeruli, one
PC1 2 sh must recognize that it is an estimation. Two factors could affect this
PC2 1 estimation. One is that smaller glomeruli may not be visible due to
PC3 1 the thickness of the sections causing a smaller estimation. The
PC4 1
other is that the unclear boundary of Class 3 glomeruli may
PC5 3 sh, sz, p
PC6 3 sh, sz, p increase the estimation. Total of 8 Class 3 glomeruli were identified
PC7 1 in females. Unclear boundaries were present between AD3 and
PC8 1 AD5, AD6 and AC6, PC5 and PC6, PD2 and PD3. Therefore, it is
PC9 1 possible that the pairs we named in the study are actual one
PC10 1
PD1 1
glomerulus. These two factors are the likely reasons for the
PD2 3 sh, sz, p discrepancy of number of glomeruli in the four samples. In males
PD3 3 sh, sz, p the glomeruli at the posterior part of male ALs were more densely
PL1 1 packed and individual glomeruli were difficult to delineate because
PL2 1
of the unclear boundary. This matter of fact may of course be due to
PM1 2 sh, d
PM2 1 technical problems concerning penetration of dye in the deeper
PM3 2 sh, d brain layers. However, the distinctiveness of glomeruli may also be
PV1 1 closely related to the composition of the neural network, i.e., the
PV2 2 sh density of neuropil. The number of afferent neurons in posteriorly
PV3 2 sh
located glomeruli may for instance be lower than that of more
PV4 1
a
anteriorly positioned glomeruli (Ghaninia et al., 2007). Regardless
Comprising only the glomeruli located at the anterior antennal lobe of male.
b of the cause, the estimates should be improved by alternative
Classes refer to the distinctness of glomeruli (for details see Section 2).
c
Deviation(s) from class distinctness is indicated under comment: sh: shape; sz: staining methods or novel molecular techniques in order to verify
size; p: position; d: demarcation. and complement the presented data.

In male moths the MGC consists of 2e4 glomeruli positioned Acknowledgments

close to the entrance of the antennal nerve. Each of these male-
specific units receive input from a population of ORNs that detect We acknowledge Dr. Bente G. Berg (Norwegian University of
one particular constituent of female-produced semiochemicals Science and Technology) for critically reading and greatly improving
(Hansson et al., 1992; Ochieng et al., 1995; Todd et al., 1995; Berg the manuscript. We also thank two anonymous reviewers for their
428 J.-H. Hu et al. / Arthropod Structure & Development 40 (2011) 420e428
helpful comments.This work was supported by National Natural
Science Foundation of China (30300276) and Natural Science
Foundation of Province Heilongjiang.
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