The wax glands and wax secretion of Matsucoccus matsumurae at different development stages

The wax glands and wax secretion of Matsucoccus matsumurae at different development stages

Arthropod Structure & Development 43 (2014) 193e204 Contents lists available at ScienceDirect Arthropod Structure & Development journal homepage: ww...

9MB Sizes 2 Downloads 14 Views

Arthropod Structure & Development 43 (2014) 193e204

Contents lists available at ScienceDirect

Arthropod Structure & Development journal homepage: www.elsevier.com/locate/asd

The wax glands and wax secretion of Matsucoccus matsumurae at different development stages Yingping Xie a, *, Fen Tian a, Weimin Liu a, Yanfeng Zhang a, Jiaoliang Xue a, Youyou Zhao b, Jun Wu c a b c

School of Life Science, Shanxi University, Taiyuan, Shanxi 030006, China Houma Entry-Exit Inspection and Quarantine Burean, Houma, Shanxi 043000, China Jinhua Forest Pest Quarantine Station, Jinhua, Zhejiang 321017, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 September 2013 Received in revised form 8 January 2014 Accepted 14 January 2014

In this paper, the wax secretions and wax glands of Matsucoccus matsumurae (Kuwana) at different instars were investigated using light microscopy, scanning electron microscopy and transmission electron microscopy. The first and second instar nymphs were found to secrete wax filaments via the wax glands located in the atrium of the abdominal spiracles, which have a center open and a series of outer ring pores. The wax gland of the abdominal spiracle possesses a large central wax reservoir and several waxsecreting cells. Third-instar male nymphs secreted long and translucent wax filaments from monolocular, biolocular, trilocular and quadrilocular pores to form twine into cocoons. The adult male secreted long and straight wax filaments in bundles from a group of 18e19 wax-secreting tubular ducts on the abdominal segment VII. Each tube duct contained five or six wax pores. The adult female has dorsal cicatrices distributed in rows, many biolocular tubular ducts and multilocular disc pores with 8e12 loculi secreting wax filaments that form the egg sac, and a rare type wax pores with 10 loculi secreting 10 straight, hollow wax filaments. The ultrastructure and cytological characteristics of the wax glands include wax-secreting cells with a large nucleus, multiple mitochondria and several rough endoplasmic reticulum. The functions of the wax glands and wax secretions are discussed. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Matsucoccidae Matsucoccus matsumurae Wax gland Wax secretion Ultrastructure Cytology

1. Introduction The pine bast scales form a group of ancient insects that parasitize on pine trees (Pinus spp., Pinaceae) and at present, include the oldest known scale insect fossils. They have a co-evolutionary relationship with their pine tree hosts (Koteja, 1984). In an earlier classification system, pine bast scales were classified as a tribe or subfamily within the family Margarodidae in the Coccoidea (Morrison, 1927; Beardsley, 1968); however, a subsequent study raised them to family status, the Matsucoccidae (Koteja, 1974). This family contains one extant genus, Matsucoccus Cockerell 1909, with the Japanese pine bast scale Matsucoccus matsumurae (Kuwana) as its the type species. Approximately 33 extant and six fossil species of this genus have been recorded worldwide; of these, Matsucoccus pini Green, Matsucoccus mugo Siewniaky, Matsucoccus josephi Bodenheimer et Harpaz, Matsucoccus boratynskii Bodenheimer et

* Corresponding author. Tel.: þ86 351 7018092. E-mail addresses: [email protected], xie[email protected] (Y. Xie). 1467-8039/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.asd.2014.01.001

Neumark, Matsucoccus feytaudi Ducasse and M. matsumurae (Kuwana) are significant pests of pine forests (Foldi, 2004). M. matsumurae (Kuwana) is a notable example. This species was first recorded in 1905 by Kuwana in Tokyo, Japan, and its original host is the Japanese black pine Pinus thunbergii Parlatore. It subsequently spread through Japan and to the Korean Peninsula and China. In the mid-1940s, M. matsumurae was first discovered at Lushun, a port city in Liaoning Province in northeastern China, and at Yantai, another port city in Shandong Province. By the 1970s, M. matsumurae had spread throughout Liaoning, Qingdao, Yantai, Shanghai, Jiangsu, Zhenjiang, and had infested the local pine forests. In northeastern area and Shandong Province, it largely parasitized Pinus tabulaeformis Carr., Pinus densiflora Sieb. et Zucc. and P. thunbergii Parlatore, whereas in southern China, it primarily infested Pinus massoniana Lamb. This pine bast scale species pierces the host bark with its piercingesucking mouthparts to draw up serum. The damaged trees showed bark dehiscence, needle defoliation, twig wilt, treetop droop. Chemical insecticides were widely applied to control the insect infestation, but the effect was limited. The species continued to spread rapidly, and its increasing population resulted in the death of forests over large areas. To control the

194

Y. Xie et al. / Arthropod Structure & Development 43 (2014) 193e204

Y. Xie et al. / Arthropod Structure & Development 43 (2014) 193e204

195

Fig. 2. Photographs of the second-instar nymph of Matsucoccus matsumurae taken under the stereomicroscope. A: Second-instar nymphs gathered beneath the bark. B: A female second-instar nymph showing the insect body resembling a reddish-brown pearl and the long wax filaments (wf) secreted and protruding outward from abdominal spiracle.

infestation, much of the damaged pine forest must be cut down (Zhao et al., 1990). The State Forestry Administration Department of China promulgated M. matsumurae as one of the worst destructive exotic pests. However, this pest continues to damage over 70,000 km2 of pine forest each year. It is difficult to control M. matsumurae via chemical insecticides, in part because this type of scale insect undergoes complex morphological changes during development and exudes wax secretions on its body surface. During development, M. matsumurae changes its morphology and the wax glands in its integument. These wax glands secrete wax substances covering onto the body surface to protect the insect from chemical insecticides and other external factors. It is important to study the characteristics of the wax glands and wax secretions at different developmental stages of the pine bast scale to understand the mechanism of self-defense through wax secretion and to develop control measures. However, studies investigating how wax glands change in form, number, and distribution in the pine bast scale between different instars are scarce. Only two have been conducted to date. Young and Yao (1986) studied the ultrastructure of the epidermal glands of female M. matsumurae; they observed two wax glands (biolocular tubular duct & multilocular disc pore) and one gland that secretes no wax (dorsal cicatrices). Foldi (2004) described the external morphology at all stages of development in both male and female M. feytaudi, and in adult females of M. matsumurae, M. pini and M. josephi. In this paper, the wax pores, wax glands and wax secretions of M. matsumurae at different stages were investigated using light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). 2. Materials and methods 2.1. Insects Specimens of M. matsumurae were collected from the P. massoniana Lamb pine forest at Jinhua (N 28 320 , E 119 140 , altitude 500 m), Zhejiang Province, China, where substantial infestation has occurred in recent years. Between March, 2011 and April, 2012, the pine bast scale at different stages was collected throughout the

study period by cutting parasitized twigs. In the laboratory, scale insect specimens were observed and photographed under an Olympus stereo-microscope. Insect samples included the 1st instar nymph, 2nd instar nymph, 3rd instar male nymph, male pupae, male adult, and female adult. A subset of samples were directly stored at 80  C in an ultra-low temperature freezer for subsequent scanning electron microscopy. Other samples were fixed in 2.5% glutaraldehyde (v/v) in 0.2 M phosphate buffer (PBS) at pH 7.2 and at 4  C for paraffin sections and transmission electron microscopy observation. The sample number for each instar was at least 100 individuals. 2.2. Scanning electron microscopy (SEM) The external morphological characteristics of the various wax glands and associated wax secretions of M. matsumurae were observed with SEM. After removal from 80  C, the samples were placed on precooled stage (below 70  C), and dried under vacuum (with a vacuum degree of 10e101 Pa) for 24 h immediately. Then the samples were mounted on microscope slides (2.5 cm  2.5 cm) and sputter-coated with gold to a thickness of approximately 20 nm. Finally, the samples were observed under a JSM-840 scanning electron microscope (JEOL Ltd. Japan) at an operating voltage of 15 kV, and micrographs were taken using a Canon EOS 350D digital camera. 2.3. Light microscopy (LM) The internal structure and cytological characters of the wax glands were observed using LM in combination with TEM following Liu et al. (2009, 2011). To prepare the paraffin sections, the insect samples were first washed with 0.2 M PBS for three times, dehydrated in a series of ethanol solutions (10 min each in 35%, 55%, 75%, 85%, 95% and 100% (v/v)), and cleared in a xylene series (10 min each in 35%,55%,75%,85%,95% and 100% (v/ v)). The samples were embedded in paraffin by immersion in a 1:1 (v/v) xylene: paraffin mixture for 12 h at 58  C and paraffin for 24 h at 58  C. The embedded specimens were then serially sectioned into thickness of 6 mm using a paraffin microtome (Reichert HistoSTAT 820) and mounted on glass slides, followed

Fig. 1. First-instar nymph of Matsucoccus matsumurae. AeD: SEM photographs. A: Ventral view of the newly hatched first-instar nymph showing the head, thorax, abdomen, antenna, eye, thoracic leg and anal filament. as-abdominal segment. Scale bar ¼ 100 mm. B: Dorsal view of the nymph showing some of the abdominal spiracles (sp) on the submargin of the abdominal segment. cs-caudal setae. Scale bar ¼ 10 mm. C: Magnified view of the spiracle, showing the concave center area (ic) with granular surface and a center pore (cp) and the outer-ring containing a series of wax-secreting pores (arrow). Scale bar ¼ 1 mm. D: the abdominal spiracle, showing the wax pores (wp) surrounding the spiracle and the wax filaments (arrow). Scale bar ¼ 1 mm. E: TEM photographs. E: Wax gland under the abdominal spiracle, showing the wax reservoir (arrow) located at the center and the wax-secreting cells (*) at the side. Note: pr-procuticle. Scale bar ¼ 2 mm.

196

Y. Xie et al. / Arthropod Structure & Development 43 (2014) 193e204

Y. Xie et al. / Arthropod Structure & Development 43 (2014) 193e204

197

Fig. 4. Third-instar nymph of Matsucoccus matsumurae. AeD: Photographs taken under stereomicroscope. A: Third-instar male nymph. B: Third-instar male nymph secreting wax filaments. C: The wax filaments constructed into cocoons and covering the insect body. D: Pupae in the cocoon.

by de-waxing in xylene, washing in a series of ethanol solutions, and staining with hematoxylin (30 min). Next they were washed in water, differentiated in a hydrochloric acid and ethanol mixture for 30 s, soaked in water for 15 min and stained with eosin (2 min). Finally, the slides were dehydrated in an ethanol series, soaked in xylene twice for 1 min and enclosed with neutral balsam. The slides were observed under an OLYMPUS BX-51 light microscope (OLYMPUS Co. Ltd. Japan).

embedded in Epon 812. Then, semithin sections (1 mm) and ultrathin sections (0.07 mm) were prepared. Following 3% uranyl acetate-lead citrate double staining, the samples were examined using TEM (JSM-1200EX) operating at 80 kV.

2.4. Transmission electron microscopy (TEM)

The first-instar nymph of M. matsumurae has two live periods, a mobile period and a sedentary period. The newly hatched firstinstar nymphs (200e400 mm in length) were oblong in shape and light yellow to orange in color. They had distinct body segments and developed antennae, eyes and legs ( Fig. 1A). During this period they were crawled and did not secrete wax substances. After settling on host twigs, the nymphs began to feed and secrete white wax onto their body surfaces.

After removal from glutaraldehyde (2.5%, (v/v)), the insect samples were rinsed twice with 0.2 M PBS, fixed in 1% osmium tetroxide for 1e2 h, and twice-rinsed again with 0.2 M PBS. Next, samples were dehydrated in an acetone series (from 10% to 100% (v/ v)), impregnated (embedding agent: acetone ¼ 1:2, 3 h; embedding agent: acetone ¼ 2:1, 1.5 h; and pure embedding agent, 2 h) and

3. Results 3.1. First-instar nymph

Fig. 3. Micrographs of the second-instar nymph of Matsucoccus matsumurae. A, B, E and F: SEM photographs. C, D, G and H: TEM photographs. A: The spiracles (sp) occurring laterally along the body. Scale bar ¼ 100 mm. B: Magnified view of the wax filaments (arrow) secreted from the spiracle (*). Scale bar ¼ 10 mm. C: Wax-secreting cells (arrow) beneath a spiracle. Scale bar ¼ 2 mm. D: Magnified view of the wax-secreting cells showing many rough endoplasmic reticulum (rer) surrounding the nucleus (nu). Scale bar ¼ 500 nm. E: a layer of “wet wax” on the ventral surface. Scale bar ¼ 10 mm. F: Magnified view of the “wet wax”. Scale bar ¼ 10 mm. G: Integument showing the procuticle (*) and the epidermal cell (arrow). Scale bar ¼ 2 mm (2000). H: Magnified view of an epidermal cell (ec) showing the cell nucleus (nu). Scale bar ¼ 500 nm.

198

Y. Xie et al. / Arthropod Structure & Development 43 (2014) 193e204

Fig. 5. Micrographs of the third-instar nymph of Matsucoccus matsumurae. AeG: SEM photographs. A: Monolocular pore (*). Scale bar ¼ 1 mm. B: Biolocular pore (*), Scale bar ¼ 1 mm. C: Trilocular pore (*). Scale bar ¼ 1 mm. D: Quadrilocular pore (*). Scale bar ¼ 1 mm. E: A monolocular pore secreting a wax filament. Scale bar ¼ 1 mm. F: A biolocular is secreting wax filaments (wf). Scale bar ¼ 1 mm. G: Wax filaments (wf) coagulated into a sphere at the terminal end. Scale bar ¼ 10 mm. H: TEM photograph of the wax glands showing the large and bottle-shaped central cells (arrow), lateral cells (*). Scale bar ¼ 2 mm.

Y. Xie et al. / Arthropod Structure & Development 43 (2014) 193e204

199

During SEM observation, no wax pores were observed on the body surface of the scale insect; however, seven pairs of abdominal spiracles located laterally along each abdominal segment were observed (Fig. 1B). The spiracles were circular and 2.5e3.0 mm in diameter. Each spiracle consists of an inner concave area (ic) with dense granules, a center pore (cp) and an outer ring divided into several pores functioning as wax-secreting pore (wp) (Fig. 1C). The wax filaments (wf) secreted from the pores accumulated around the spiracle (Fig. 1D). Using TEM observation, a large, developed wax gland was observed beneath the spiracle (Fig. 1E). The wax gland was a complex structure consisting of a wax reservoir (wr) at the center and several laterally surrounding wax-secreting cells (wsc). The wax reservoir was irregularly cylindrical in shape (6e 10 mm in diameter) and the wax-secreting cells were fusiform to cylindrical. 3.2. Second-instar nymph

Fig. 6. General organization of biolocular tubular duct, showing two large central cells (cc) closely ranged together and surrounded by the lateral cells (lc), which possessed very large nuclei. ep-epicuticle, pr-procuticle, epi-epidermis.

The second instar nymph stage was called the pearl-shape stage, or the cyst, because its antennae, eyes and legs disappeared with only the mouthparts remaining; and its body became spherical or slightly flattened, resembling a reddish-brown pearl. In this instar, the nymphs usually occurred in clusters underneath cracks in the bark (Fig. 2A). Under the stereomicroscope, several long wax filaments (approximately 2.5 mm long) extending out from beneath the pearl body were readily observed (Fig. 2B). Using SEM, these long wax filaments were observed to be secreted from the

Fig. 7. Adult male of Matsucoccus matsumurae. A: Adult male showing wax filaments (wf) in bundles secreted from the abdominal segment VII. B and C: SEM photographs. B: The abdominal segments (*) of the adult male showing the tubular ducts (td) on the abdominal segment VII. Scale bar ¼ 10 mm. C: Magnified view of the 18e19 tubular ducts (td), each with 5e6 small wax-secreting pores. Scale bar ¼ 10 mm. D: Light micrograph of the tubular wax glands showing the tubular duct (du) and the wax-secreting cells (wsc). Note: mumuscle, *-testis. Scale bar ¼ 50 mm.

200

Y. Xie et al. / Arthropod Structure & Development 43 (2014) 193e204

abdominal spiracles. There were seven pairs of abdominal spiracles arranged ventrolaterally along the insect body (Fig. 3A). Several bundles of wax filaments protruded around each spiracle, with each bundle of wax filaments measuring approximately 8 mm in diameter (Fig. 3B). The complex wax gland structure and wax-secreting cells (wsc) under the spiracle were observed using TEM (Fig. 3C). Within the wax-secreting cells, the nucleus (nu) was very large and occupied approximately 80% of the cell area. Several rough endoplasmic reticulum (RER) surrounded the nucleus (Fig. 3D). A very thin wax layer was observed on the pearl-shaped body surface of the second-instar nymphs. The SEM photographs revealed the thin wax layer arranged in a scale-like manner (Fig. 3E). Magnified 3000, the newly secreted fluid wax from the cuticle was observable (Fig. 3F). These waxy substances were “wet” upon secretion from the wax cells in the integument and subsequently coagulated and accumulated into the scale-like structure. No wax pores were identified on the body surface that were responsible for secreting this wet wax. Using TEM observation, only dense epidermal cells were observed in the integument. Each epidermal cell was 5e6 mm in length and approximately 2 mm in width (Fig. 3G). The magnified view revealed the nucleus and rough endoplasmic reticulum inside these epidermal cells (Fig. 3H). There were more of these cells in the epidermal layer on the dorsal side of the nymphs than on the ventral side. 3.3. Third-instar male nymph and pupa In late March, the overwintering generation of M. matsumurae completed its development of the pearl-shaped stage. The male scale insects molted into the 3rd instar. The nymphs were dark orange, 1.5 mm long and 0.5e1.0 mm wide (Fig. 4A). A few hours after molting, each nymph began secreting white, semitransparent wax filaments from the wax pores symmetrically distributed over its body surface (Fig. 4B). This wax secretion coated the insect with wax filaments. Finally, the wax filaments produced a cocoon 1.4e 1.7 mm long and 0.6e0.9 mm wide. Those insects with wax cocoons sheltered together in clusters within spaces beneath the bark (Fig. 4C). With the cocoon, the insect completed its prepupa and pupa stages and developed compound eyes, thoracic legs and wing buds, but no longer secreted any wax substances (Fig. 4D). The wax pores on the body surface of the 3rd instar nymphs were visible via SEM. Of the wax pores, almost 70% were biolocular pores; the remainder were monolocular, trilocular or quadrilocular pores. The characteristics of the wax pores were clearly apparent at

5000e13000 magnification. Each wax pore had a smooth protuberant rim 2.5e4.0 mm in diameter and an invaginated opening 1.5e3.0 mm in diameter. Each monolocular pore contained a single hole in the inner opening (Fig. 5A). Biolocular pores each possessed two holes arranged in a Fig. 8 configuration (Fig. 5B), trilocular pores had three holes triangularly arranged (Fig. 5C), and quadrilocular pores possessed four holes arranged in a square (Fig. 5D). As shown in Fig. 5E and F the wax pore secreted wax filaments from its opening, and each pore secreted one wax filament. In some cases, the wax coagulated into a spherical shape (Fig. 5G). The complex structure of the wax gland was observed under TEM (Fig. 5H). This wax gland contained two large central cells and several small lateral cells. The two center cells were close ranged together and surrounded by the lateral cells, which possessed very large nuclei (Fig. 6). 3.4. Adult male After emergence, the adult males possessed developed antennae, compound eyes, legs and forewings allowing them to search for female adults for copulation. Adult males secreted wax solely from the tergum of abdominal segment VII, producing a cluster of straight wax filaments 1.5e2 times the male’s body length (Fig. 7A). Using SEM, it was observed that the tergum of abdominal segment VII was lined with a group of 18e20 tubular, upwardprotruding ducts. Each tubular duct was 10e13 mm in diameter and contained five or six wax-secreting pores; through each pore, one wax filament was secreted (Fig. 7B). Longitudinal sections of the tubular ducts and wax gland cells were observed using the paraffin sections of the abdominal segment VII. The tubular ducts were complex glandular structures. They each contained a tubular duct as a top channel for wax excreting outward, a wax reservoir at the center, and a group of wax-secreting cells at the bottom, of these latter cells was 40 mm long and 10 mm wide (Fig. 7C and D). 3.5. Adult female The second-instar female nymph exuviated and developed into the adult female stage. Adult females were 2.5e3.5 mm in length and possessed developed antennae, eyes, and legs (Fig. 8A). They were similar to the 3rd instar male nymphs in appearance, but were larger with broader abdomens. After mating, adult females secreted a mass of wax filaments from the wax pores distributed over the

Fig. 8. Adult female of Matsucoccus matsumurae. A and B: Photograph taken under stereomicroscope. A: Adult female. B: The adult female was secreting wax filaments to form the ovisac.

Y. Xie et al. / Arthropod Structure & Development 43 (2014) 193e204

thorax and abdomen (particularly at posterior end of abdomen) to form an ovisac. They then deposited eggs in the ovisac (Fig. 8B). The body surface and wax pores of adult females were observed using SEM. The cuticle surface was characterized by a compact and plump reseau, on which the dorsal cicatrices and wax pores were distributed. The dorsal cicatrices were distributed in line with the body segments. Each cicatrice consisted of a large hollow pit 9 mm in diameter (Fig. 9A). Multiple fine wax filaments were observed around the dorsal cicatrices (Fig. 9B). The wax pores were distributed along the thorax and abdomen with more along the latter. Among these wax pores, monolocular, biolocular and trilocular pores were in the minority, with the majority being multilocular pores surrounding the vulva region (Fig. 9C). Each multilocular pore contained 8e12 loculi in an outer ring (Fig. 9D). Each loculus of the multilocular pore secreted one wax filament, and the wax filaments were solid and either straight or curly. Fig. 9E and F shows the multilocular disc pores. In addition, another type of wax pore with 10 loculi was observed at the body margin. This 10-locular pore secreted 10 straight and hollow wax filaments. The 10 wax filaments assembled together into three rows, resembling a multitube cannon with 10 tubes (Fig. 9G and H). In the cross section of the abdominal terminal of the adult female, a series of wax glands were observed close together in the epidermis. There were more wax glands on the dorsal than ventral surface (Fig. 10A). However, the wax glands were dense and arranged in multiple layers in the V-shaped vulva region (Fig. 10B). The multilocular pore had a complex structure. The transverse sections of the multilocular pores containing eight loculi clearly revealed a central cell and eight surrounding lateral cells (Fig. 10C). The longitudinal sections showed a central, bottle-shaped cell at the center of the gland, several lateral cells encompassing the central cell, a wax reservoir located at the top of the central cell, and a tube duct with its bottom end connected to the center cell. The tube duct, through which the wax substances excreted led to the outer cuticle surface (Fig. 10D). A longitudinal section of a central cell of a multilocular pore was observed using TEM. The wax reservoir at the apical end of the central cell was very large (Fig. 10E) and filled with synthesized wax substances (Fig. 10F). These wax substances were secreted through the tube ducts to the outer cuticle. The number of ducts was equal to the number of pores in the multilocular pore. 4. Discussion The present study showed that the wax gland and wax secretions of M. matsumurae vary considerably across different development stages. However, within the Matsucoccus genus, the wax gland and wax secretions within the same development stage are very similar morphologically and are distinguished only by small differences, such as wax pores and cicatrices. A discussion of the differences between M. matsumurae and other species of the genus and the functions of the different wax glands at different development stages follows. In the newly hatched nymph or crawler stage, M. matsumurae lacked wax substances on its body surface; however, after settling the first-instar nymph began to secrete wax filaments around the spiracle. In the second instars, long wax filaments were also found around the abdominal spiracles. A similar morphology was also described for M. feytaudi (Foldi, 2004). In general, insect spiracles function in gas exchange. However, the abdominal spiracles of M. matsumurae had a central opening surrounded by series of pores (as observed by SEM); the central opening was used for insect respiration. Foldi (2004) described and illustrated the morphological characters of all the development stages of M. feytaudi and noted that the wax filaments around the spiracles of both the first-

201

instar and second-instar nymphs were secreted by multilocular pores located at the base of atrium of the spiracles. In the present study, a complex wax gland structure with a central wax reservoir and several wax-secreting cells beneath the abdominal spiracle was observed by TEM. In addition, the wax-secreting cells possessed large nuclei and abundant rough endoplasmic reticulum. In biochemistry, the rough endoplasmic reticulum is often associated with lipid synthesis in the cell, and lipids are the primary constituent of the wax secretions of scale insects (Tamaki, 1997; Xie et al., 2006). The pores in the atrium of each spiracle played a role in wax synthesis and secretion. A thin layer of wet wax was observed on the body surface of the 2nd instar nymph, but no obvious wax pore was apparent. Using TEM observation, we observed dense epidermal cells in the integument and speculated that these epidermal cells likely function in the synthesis and secretion of wet-wax. In the third-instar male nymph or prepupa of M. feytaudi, the biolocular pores observed on the head, thorax and abdomen function in secreting waxy filaments for constructing the cocoon (Foldi, 2004). In the present study, the third-instar male nymph of M. matsumurae secreted multiple long, translucent wax filaments to form the cocoon. The biolocular pores were observed using SEM and represented 70% of all the wax glands. TEM revealed that this gland has a complex glandular structure with two large central cells and several small lateral cells. In addition to the biolocular pores, we observed monolocular, trilocular and quadrilocular pores that secreted wax filaments. Adult males of scale insects in the Coccidae, Pseudococcidae and Eriococcidae typically secrete two long, straight wax filaments from the abdominal terminus through multilocular pores presented on each side of the tergum. These waxy filaments are very conspicuous in living males, but their function is unclear (Giliomee, 1997). However, adult male M. matsumurae secreted very long, white wax filaments in several bundles at the abdominal terminus. We observed a group of 18e19 wax-secreting tube ducts protruding upward from the tergum of abdominal segment VII, with each tube duct having five or six smaller pores (as observed under SEM). The bundles of wax filaments appear to be secreted from the tube ducts. A similar structure was also reported by Foldi (2004) in M. feytaudi, but with 20e37 tube ducts. As the wax filaments were equal in length to or longer than the male antennas, we speculate that adult males might apply these wax filaments for balance during flying or other movement. Using SEM, we observed many dorsal cicatrices distributed in rows on the body segments of adult female M. matsumurae. The dorsal cicatrices of M. matsumurae were also observed by Young and Yao (1986), who suggested that these structures secreted no wax. However, we observed multiple fine wax filaments adhering to the margins of the dorsal cicatrices. In contrast, Foldi (2004) reported very fine secretions from the central regions of the dorsal cicatrices of M. feytaudi. It has been said that the dorsal cicatrices are a type of lenticel gland through which a sex pheromone was secreted (Tang and Hao, 1995). The adult female of M. matsumurae secreted wax filaments through multilocular disc pores to form an ovisac (Young and Yao, 1986). By using SEM, we not only observed multilocular pores (8e 12 loculi) secreting curly and solid wax filaments, but we also observed a type of wax pore containing 10 loculi that secreted 10 straight, hollow wax filaments arranged closely together in three rows. This type of multilocular pore and manner of wax secretion have rarely been observed in prior studies. As mentioned above, we found that most of the wax filaments secreted by M. matsumurae were solid (i.e., non-hollow) in structure, similar to those wax filaments secreted by Icerya purchasi Maskell and Icerya aegyptiaca (Douglas) in the family

Fig. 9. SEM photographs of the adult female of Matsucoccus matsumurae. A: the dorsal cicatrices (arrow) distributed along the body segments. Scale bar ¼ 10 mm. B: Dorsal cicatrices (arrow) and surrounding wax filaments (wf). Scale bar ¼ 10 mm. C: Multilocular disc pore (mlp) distributed around the vulva region (arrow). Scale bar ¼ 10 mm. D: Multilocular disc pore (*) with 10 loculi. Scale bar ¼ 1 mm. E: A wax gland secreting wax filaments (wf) with each pore secreting a single wax filament. Scale bar ¼ 1 mm. F: Multiple wax filaments (*), curly and solid, secreted from the multilocular pore. Scale bar ¼ 10 mm. G: A special type of wax filaments (wf). Scale bar ¼ 10 mm. H: Magnified view of the special wax filaments. Scale bar ¼ 1 mm.

Y. Xie et al. / Arthropod Structure & Development 43 (2014) 193e204

203

Fig. 10. Micrographs of the adult female of Matsucoccus matsumurae. AeD: Light micrograph. A: Transection of the abdomen showing the wax glands (arrow), which were more abundant in the dorsal surface than in the ventral surface. co-common oviduct, oc-oocyte. Scale bar ¼ 100 mm. B: Dense wax glands (arrow) arranged in multiple layers in the vulva region. Scale bar ¼ 50 mm. C: Longitudinal section of a wax gland, showing epidermal cells (ec), lateral cells (lc), a bottle-shaped central cell (cc) and a wax duct (arrow). Scale bar ¼ 30 mm. D: Transection of a multilocular pore showing a center cell (cc) and eight lateral cells (arrow). Scale bar ¼ 30 mm. E and F: TEM photographs. E: Longitudinal section of a wax gland showing lateral cells (**), a center cell (*) filled with wax substances (bounding box) and a wax duct (arrow). Scale bar ¼ 2 mm. F: Magnified view of the wax substance in the center cell. Scale bar ¼ 500 nm.

Monophlebidae (Zhang et al., 2011). The wax filaments of these latter species likely exhibit a simple structure and ancestral characteristics because Matsucoccidae and Monophlebidae are relatively ancient groups in the Coccoidea. In contrast, the Coccidae,

Eriococcidae and Pseudococcidae are relatively evolutionary groups in the Coccoidea, and most of their wax filaments are hollow. This hollow structure increased the strength of the wax filaments but decreased the amount of material consumption, which may be a

204

Y. Xie et al. / Arthropod Structure & Development 43 (2014) 193e204

derived trait (Foldi, 1991, 1997; Xie et al., 2006; Zhang et al., 2009, 2011). In summary, our study provides comprehensive information about the wax secretions, wax-secreting pores, and wax-secreting glands of M. matsumurae at different stages of development. This study contributes greatly to our understanding of the waxsecreting diversity of M. matsumurae and may inform the development of scientific control techniques for this pest. Acknowledgments This research was supported by the National Natural Science Foundation of China (31070584) and the Specialized Research Fund for the Doctoral Program of Higher Education of China (20101401110008). References Beardsley, J.W., 1968. External morphology of the adult male of Matsucoccus bisetosus. Ann. Entomol. Soc. Am. 61, 1449e1459. Foldi, I., 1991. The wax glands in scale insects: comparative ultrastructure, secretion, function and evolution (Homoptera: Coccoidea). Ann. Soc. entomol. Fr. 27, 163e 188. Foldi, I., 1997. Ultrastructure of the integumentary gands. In: Ben-Dov, Hodgson (Eds.), Soft Scale Insects, Their Biology, Natural Enemies and Control. Elsevier Science Publ., Pays-Bas, pp. 73e90. Foldi, I., 2004. The Matsucoccidae in the Mediterranean basin with a world list of species (Hemiptera: Sternorrhyncha: Coccoidea). Ann. Soc. Entomol. Fr. 40 (2), 145e168.

Giliomee, J.H., 1997. The adult male. In: Ben-Dov, Y., Hodgson, C.J. (Eds.), Soft Scale Insects e Their Biology, Natural Enemies and Control, vol. 7A, Elsevier, Amsterdam & New York, pp. 23e30. Koteja, J., 1974. Comparative studies on the labium in the Coccinea (Homoptera). Zesz. Nauk. Akad. Rol. Krak. 89, 1e162. Koteja, J., 1984. The Baltic amber Matsucoccidae (Homoptera, Coccinea). Pol. J. Entomol. 69, 187e218. Liu, W.M., Xie, Y.P., Xue, J.L., Zhang, Y.F., Zhang, X.M., 2011. Ultrastructural and cytochemical characterization of brown soft scale Coccus hesperidum (Hemiptera: Coccidae) infected by the Lecanicillium lecanii (Ascomycota: Hypocreales). Micron 42, 71e79. Liu, W.M., Xie, Y.P., Xue, J.L., Gao, Y., Zhang, Y.F., Zhang, X.M., Tan, J.S., 2009. Histopathological changes of Ceroplastes japonicus infected by Lecanicillium lecanii. J. Invertebr. Pathol. 101, 96e105. Morrison, H., 1927. Descriptions of new genera and species belonging to the coccid family Margarodidae. Proc. Biol. Soc. Wash. 40, 99e109. Tamaki, Y., 1997. Chemistry of the test cover. In: Ben-Dov, Y., Hodgson, C.J. (Eds.), Soft Scale Insects-Their Biology, Natural Enemies and Control, vol. 7A, Elsevier, Amesterdam & New York, pp. 55e72. Tang, F.D., Hao, J.J., 1995. The Margarodidae and Others of China (Homoptera: Coccinea of Insecta). Chinese Agricultural Science Technology Press, Beijing. Xie, Y.P., Xue, J.L., Zheng, L.Y., 2006. Wax Secretions of Soft Scale Insects, Their Ultrastructure & Chemical Composition. China Forestry Publishing House, Beijing. Young, B.L., Yao, H., 1986. Studies on ultrastructure and function of epidermal glands on female Matsucoccus matsumurae (Kuwana) (Coccoidea: Margarodidae). Contrib. Shanghai Inst. Entomol. 6, 253e260. Zhang, Y.F., Xie, Y.P., Xue, J.L., Liu, W.M., 2009. Developmental variation in the dermal glands and wax secretions of the mealy bug, Phenaciccus fraxinus. J. Entomol. Sci. 44 (1), 59e68. Zhang, Y.F., Xie, Y.P., Xue, J.L., 2011. Ultrastructure of wax secretions of Icerya purchase and Icerya aegyptiaca. Sichuan J. Zool. 30 (5), 751e752. Zhao, S.F., Chang, G.B., Dang, Z.Y., 1990. The infestation and control of Matsucoccus matsumurae (Kuwana) in China. For. Sci. Technol. 12, 1e3.