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Morphology and ultrastructure of Dufour’s and venom glands in the ant Camponotus japonicus Mayr (Hymenoptera: Formicidae)
Accepted Manuscript Title: Morphology and ultrastructure of Dufour’s and venom glands in the ant Camponotus japonicus Mayr (Hymenoptera: Formicidae) Authors: Ying Zhou, Changyou Li, Johan Billen, Hong He PII: DOI: Reference:
S0968-4328(17)30311-6 https://doi.org/10.1016/j.micron.2017.10.011 JMIC 2494
To appear in:
Micron
Received date: Revised date: Accepted date:
13-8-2017 29-10-2017 29-10-2017
Please cite this article as: Zhou, Ying, Li, Changyou, Billen, Johan, He, Hong, Morphology and ultrastructure of Dufour’s and venom glands in the ant Camponotus japonicus Mayr (Hymenoptera: Formicidae).Micron https://doi.org/10.1016/j.micron.2017.10.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title: Morphology and ultrastructure of Dufour’s and venom glands in the ant Camponotus japonicus Mayr (Hymenoptera: Formicidae)
Author names and affiliations: Ying Zhou a, Changyou Li a, Johan Billen b, Hong He a,*
a
College of Forestry, Northwest A&F University, Yangling, Shaanxi 712100, China
b
Zoological Institute, University of Leuven, Naamsestraat 59, 3000 Leuven, Belgium
*Corresponding author: Hong He:
[email protected]
Authors’ email: Ying Zhou:
[email protected]
Changyou Li: [email protected] Johan Billen: [email protected]
Highlights
Dufour’s gland shows different features among female castes. Dufour’s gland in major workers indicates for a more pronounced function. Dufour’s gland cells are characterized by basal invaginations and muscle fibers. The venom gland is morphologically similar in different female castes. Cells of venom gland contain numerous mitochondria and a prominent end apparatus.
Abstract
The Dufour’s and venom glands are two important exocrine glands in ants. In this
study, the morphology and fine structure of these two glands are described in the ant
Camponotus japonicus Mayr. The Dufour glands have a characteristic bilobed shape and show a difference in size and color between the female castes (minor and major workers, alate and dealate queens). The external surface of Dufour’s gland shows different features among the female castes. It appears more hypertrophied in major workers than in the other castes, indicating for a more pronounced function. The cells of the glandular epithelium in Dufour’s gland are characterized by abundant mitochondria, basal invaginations and developed muscle fibres. The venom gland is morphologically similar in the different female castes, and consists of a venom reservoir, a convoluted gland and a bifurcated free secretory filament, with the convoluted gland appearing as a large cap lying on top of the reservoir. Cells of the convoluted gland and the free secretory filaments contain numerous mitochondria and a prominent end apparatus. The results will contribute to understanding the functional morphologyof these glands among the different castes in Camponotus ants.
Keywords: Camponotus japonicus; Dufour gland; venom gland; morphology; ultrastructure
1. Introduction The Dufour gland and venom gland are the two common abdominal exocrine glands connected to the female reproductive system of ants, and serve a variety of functions in different phylogenetic groups (Hölldobler & Wilson, 1990; Billen, 2009). The Dufour gland among others can produce trail, recruitment and sex pheromones (Mitra, 2013). The venom gland
commonly releases offensive allomones and alarm pheromones (Schoeters & Billen, 1998). The morphology of both glands has been widely investigated in many species of various ant subfamilies (Billen, 1986a, 1986b; Billen, 1990; Billen & Taylor, 1993; Schoeters & Billen, 1995; Grasso et al., 2005; Ortiz & Camargo-Matthias, 2006; Billen et al., 2009; Fox et al., 2010; Billen et al., 2015), showing that gland morphology is linked with subfamily classification. The two glands not only display a different shape and size among the ant species, but can also change among castes and even individuals of the same species, especially, the variation of gland morphology and ultrastructure among castes may suggest a close link between gland structure and its behavioral roles (Schoeters & Billen, 1998; Abdalla & Cruz-Landim, 2001; Grasso et al., 2005). As one of the largest and highly evolved genera in Formicidae, Camponotus ants are characterized by an obvious polymorphism and division of labor (Hansen & Klotz, 2005). The morphology and ultrastructure of Dufour’s and venom glands have been investigated in several species (Billen, 1986a; Hermann & Blum, 1968), but still remain unstudied in Chinese ants. Camponotus japonicus Mayr is one of the most common and widespread ant species in China, and is also a natural enemy for many forest pests and one of the useful medical insects (Wu & Wang, 1995). In the present paper, we investigated the morphology of the Dufour and venom gland of C. japonicus, and documented their difference between the female castes (minor and major workers, alate and dealate queens) and the gland ultrastructure of workers. The results will
contribute to understanding the functional morphology of these glands among
different castes in Camponotus ants.
2. Material and Methods 2.1 Ants and gland dissection Three mature C. japonicus nests were excavated in Yangling, Shaanxi in China in May 2014 and 2015. All castes including alate and dealate queens and workers were studied, but workers were further distinguished into minor workers (head width 2.16 ± 0.26 mm) and major workers (head width 3.52 ± 0.03 mm). Nearly twenty individuals of minor workers, major workers, alates and six dealate queens (some queens were collected from other nests) were dissected to get glands for light microscopy as well as SEM and TEM analyses. The ants were pinned with the dorsal side up on a raised wax-based dissecting tray, and their abdomen was immersed in 0.9% NaCl. The abdominal cuticle was carefully torn off with forceps and dissected to expose the Dufour and venom glands under a stereomicroscope (Motic PXS5-T). Pictures were taken under a light microscope (Nikon SMZ 1500).
2.2 SEM method The Dufour and venom glands were respectively dissected from 5 individuals of minor workers, major workers, alates and 2 of dealate queens in 2.5% glutaraldehyde. Glands were fixed in 2.5% glutaraldehyde for 24 h at 4°C. The samples were washed 3–4 times with 0.1 M phosphate buffered saline (PBS, pH 7.2) during 15 min for each time. They were then dehydrated in a graded ethanol series (30%, 50%, 70%, 80%, 90% and 100%) and transferred to isoamyl acetate. Critical point drying was done in liquid carbon dioxide under 95 bar pressure, and was followed by gold-coatingg by cathodic spraying. Finally the samples were observed
and photographed under a JEOL JSM-6360LV SEM operated at 15 kV.
2.3 TEM method The Dufour and venom glands from 10 workers (5 major and 5 minor workers) were used for TEM observation. The glands were first fixed in 2.5% glutaraldehyde for 12 h at 4°C, rinsed four times (15 min each) in 0.1 M PBS (pH 7.2), and then post-fixed in 1% osmium tetroxide for 1.5 h. After rinsing four times (15 min each) in 0.1 M PBS (pH 7.2), glands were dehydrated in a graded ethanol series, then impregnated with a mixture of ethanol and LR-white (3:1 (v:v) for 2 h, 1:1 (v:v) for 4 h, 1:3 (v:v) for 12 h, respectively), and finally embedded in pure LR-white for 48 h. The samples were sectioned with a Leica-Ultracut ultramicrotome. Double-stained 80 nm thin sections were examined with a JEM-1230 transmission electron microscope at 80 kV.
3. Results 3.1 General anatomy The Dufour gland is a bilobed sac in all female castes of C. japonicus (Fig. 1 A–D). It is larger in the minor worker, major worker and alate female, with each lateral lobe having a length of 0.82 ± 0.02 mm (Fig. 1 A–C), and appears especially hypertrophied in major workers (Fig. 1 B). It is slightly smaller in dealate queens, each lateral lobe having a length of 0.65 ± 0.03 mm (Fig. 1 D)... In addition, the gland shows a different color in the four castes: it appears milky in minor workers, yellowish in major workers, white in alate queens, and light brown in dealate queens (Fig. 1 A–D).
The venom gland is much larger than Dufour’s gland, and consists of a venom reservoir, a convoluted gland and a bifurcating free secretory filament (Fig. 1). The convoluted gland appears as a large cap lying on top of the reservoir. It has a similar shape and size in major workers, alate and dealate queens (approximately 3.30 ± 0.20 mm length and 1.35 ± 0.10 mm width), but it is smaller in minor workers with a length of approximately 2.50 ± 0.10 mm and a width of 0.80 ± 0.15 mm. The venom gland appears light yellow in dealate queens and is more whitish in the other castes. The two free secretory filaments have a milky appearance. They have a diameter of 90 ± 2.0 m, and are approximately as long as the convoluted gland.
3.2 Ultrastructure under SEM Under SEM, the external surface of Dufour’s gland shows differences between workers and reproductive females. It is similar in major and minor workers (Fig. 2 A, C), displaying obvious protuberances surrounded by a thin reticulated layer of muscle fibres (Fig. 2 B, D). In the alate queens, however, the external surface is relatively flat without obvious protuberances; the reticulated layer of muscle fibres is very thin and fine (Fig. 3 A, B). In the dealate queens, the protuberances are relatively apparent and wrapped by an arrangement of bundles of a well-developed layer of muscle fibres (Fig. 3 C, D). Under SEM, the convoluted gland has a smooth surface (Fig. 4 A). The venom reservoir is an elongated sac with a conspicuous muscular supply at its ventrolateral sides, whereas the central part has a smooth appearance. The muscle fibres show a parallel organization in a direction perpendicular to the length of the reservoir. At each ventrolateral side, a thin longitudinal strand can be seen, that represents a nerve fibre (Fig. 4 B, C). When the reservoir is
torn open, its inner surface can be seen as a folded sheet structure (Fig. 4 D).
3.3 Ultrastructure under TEM 3.3.1 Dufour’s gland ultrastructure The epithelium of Dufour’s gland is formed by a single layer of cells, of which the luminal surface is covered by a thick and uniform cuticle (Fig. 5 A). The nucleus is usually ovoid, and is often located in the central or lower part of the cell (Fig. 5 A–D). There are abundant oval to elongated mitochondria in the cytoplasm, with a conspicuous subcuticular layer (Fig. 5 A, B). The cytoplasm also contains strands of smooth endoplasmic reticulum and a clear Golgi apparatus (Fig. 5 B), as well as small lipid droplets with a diameter of up to approximately 0.8 μm and abundant irregularly shaped electron-dense secretory droplets (Fig. 5 C, D). The basement membrane is about 0.3 – 0.4 µm thick and shows numerous deep invaginations, and separates the epithelium from underlying tracheoles and well-developed muscle fibres (Fig. 5 A, C, D).
3.3.2 Venom gland ultrastructure
The convoluted gland is formed by a dense mass of tubular windings, of which the cuticular inner wall consists of a very thin cuticular layer with a thickness of nearly 1.4–1.5 μm, in which the electron-dense epicuticle is extremely thin with a thickness of about 0.13–0.14 μm and the thick electron-lucid endocuticle measures about 1.27–1.36 μm (Fig. 6 A, B). The cells of the convoluted gland have an ovoid to slightly polymorphic nucleus (Fig.6 A–C). Their cytoplasm contains numerous mitochondria and a prominent end apparatus with long and
slender microvilli (Fig. 6 B, D). Muscle fibres were never observed in relationsto the convoluted gland. The free filaments are made up by polygonal glandular cells with a large, rounded nucleus that surround the central cuticular filament lumen (Fig. 7 A). The cells have a large rounded nucleus and a cytoplasm with a distinct end apparatus, numerous mitochondria and free ribosomes (Fig. 7 B, C). Duct cells make the connection between the secretory cells and the filament lumen, and are characterized by tortuous intercellular membranes near the filament lumen (Fig. 7 D). The cuticular canal of the duct cells has a continuous electron-dense epicuticular central lining and a diameter around 0.5 µm. It continues into the end apparatus, where its inner epicuticular lining becomes considerably thinner and discontinuous (Fig. 7 E).
4. Discussion The venom and Dufour glands originally were the accessory glands of the female reproductive system. Billen (1986a) systematically described and compared the morphology and ultrastructure of Dufour’s gland from 60 ant species belonging to 8 subfamilies, and showed that it has a different shape among the subfamilies. For example, it is pyriform in Aenictus (Dorylinae) (Billen & Gotwald, 1988), elongated tubiform in Myrmecia (Mymeciinae) (Billen, 1990) and Meranoplus diversus (Myrmicinae) (Billen, 2009), bulbous in Protanilla wallacei (Leptanillinae) (Billen et al., 2013), bilobed in C. pennsylvanicus, Formica rufibarbis, F. polyctena and Polyergus rufescens (Formicinae) (Hermann and Blum, 1968; Billen et al., 2001; Grasso et al., 2005). The present study shows that the Dufour gland of C. japonicus is typically bilobed, similar to other Formicinae species.
The shape and size of Dufour's gland in C. japonicus is similar, but presents a slightly different color and external ultrastructure among the female castes. In workers, especially in major workers, it appears more hypertrophied and presents obvious protuberances at its basal side (Fig. 2). In dealate queens, however, Dufour’s gland looks reduced and apparently lacks vigor (Fig. 1D), while its external surface does not display any obvious protuberances (Fig. 3). This result indicates that there exists an apparent link between the morphological features of Dufour's gland and its function in the different female castes of C. japonicus. C. japonicus is a monogamous species that nests underground (Wang and Wu, 1992). The single dealate queen after colony foundation stays inside the nest for her entire life. Workers, including majors and minors, are in charge of nest maintainance and foraging outside. A mass of alate females and males are produced in autumn and stay inside the nest throughout winter until the nuptial mating occurs in May of the following year. Dufour's gland therefore maybe more involved in foraging and defence activities of workers to act as trail, recruitment or alarm-defense pheromone as in other ants (Mitra, 2013). Alate queens can possibly use Dufour's gland compounds to act as sex pheromones and attract males for nuptial mating (Ayasse et al., 2001), but in dealate queens, Dufour's gland will gradually degrade with age and lose its secretory capacity after colony foundation. A similar result was found in Formica polyctena in which Dufour's gland was relatively large in virgin females compared with its size in dealate queens (Löfqvist and Bergström, 1980). In the slave-making ant Polyergus rufescens, however, Grasso et al. (2005) found that Dufour's gland was small in workers, somehow of intermediate size in newly-mated queens, and hypertrophied in adult queens, whereas the gland of adult queens is no more active
with an extended lumen and a thin epithelium. Combining the present results in C. japonicus, we infer that Dufour’s gland keeps active and plays more pronounced functions in workers and alated queens than it does in adult queens, and that there is a link between gland structure and the ant's behavioral roles. The Dufour gland is lined by a monolayered glandular epithelium, and its cellular organization shows considerable differences in several ant subfamilies (Billen, 1986a). In Formicinae, the epithelium has a very uniform thickness, with a characteristic subcuticular layer of mitochondria and a very thick basement membrane (Billen, 1986a). The ultrastructural features of the Dufour gland cells of C. japonicus workers are similar to those in other formicine ants such as Polyergus rufescens (Billen et al., 2001; Grasso et al., 2005), having the characteristic thick basement membrane with deep invaginations and developed muscle fibres. A large amount of electron-dense secretory droplets and mitochondria were found in the cytoplasm. We did not find clear differences in the ultrastructural features of the gland between major and minor workers. The venom gland is one of the biggest exocrine glands in ants, and to date three types of venom gland have been described (Schoeters & Billen, 1998), including the pulvinate type (with the convoluted gland on the venom sac’s dorsal side, as found uniquely in Formicinae), the ‘bourreleted’ type (with the convoluted gland inside the venom sac, in most other subfamilies), and no convoluted gland in Amblyoponinae and Leptanillinae (Schoeters et al., 1999). In C. japonicus, the venom gland is a pulvinate structure consisting of an elongated venom reservoir and a convoluted gland with a bifurcating free secretory tubule, similar to C. pennsylvanicus
(Hermann & Blum, 1968), C. vagus and Formica polyctena (Schoeters & Billen, 1998). The venom reservoir in C. japonicus is an elongated sac formed by polygonal cells and associated muscles, but these muscles do not cover the convoluted gland which is attached to the dorsal wall of the venom reservoir. Billen (1990) described the function of the convoluted gland and found it discharges the secretory compounds into the reservoir. The cytological organization of the convoluted gland and the free filaments clearly demonstrate their secretory capacity, because their glandular cells have a prominent well-developed end apparatus and numerous mitochondria. The prominent muscular supply on the ventral side of the reservoir sac, including the clear nervous provisioning is in line with the powerful spraying of formic acid by these formicine ants. We found no obvious morphological differences between four castes according to their body size under light microscopy. Our preliminary chemical analysis using gas chromatography–mass spectrometry (GC–MS), however, showed that the main chemical components in the venom gland present a clear variation between workers and queens, so more work should be done to reveal the functions of the venom gland in the female castes of C. japonicus.
Acknowledgements We are very grateful to two anonymous referees for the detailed comments and suggestions that helped us to improve the manuscript. We also express our thanks to An Vandoren (University of Leuven) for making the microscopy sections for this study and to Zhang Guoyun (Northwest A&F University) for SEM observation. The research was supported by the National
Natural Science Foundation of China (Grant No. 31070342, 31570388), Forestry Industry Research Special Funds for Public Welfare Projects (Grant No. 201404302-4) and the International Science and Technology Cooperation Project of Northwest A&F University (2016).
References
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5. Artwork with Captions
Fig.1 General morphology of Dufour’s gland and venom gland of female castes under light microscopy. A. minor worker; B. major worker; C. alate queen; D. dealate queen; DG, Dufour’s gland; VG, venom gland; ff, free filament.
Fig.2 Scanning electron micrographs of Dufour’s gland in minor and major worker. A,B. minor worker; C,D. major worker; MF, muscle fibres; pr, protuberance.
Fig.3 Scanning electron micrographs of Dufour’s gland in alate and dealate queen. A,B. alate queen; C,D. dealate queen; MF, muscle fibres; pr, protuberance.
Fig.4 Ultrastructure of the venom gland in C. japonicus workers under SEM. A. Dorsal view of convoluted gland, showing smooth surface; B,C. ventral side of reservoir, showing lateral bundles of muscle fibres (arrows indicate nerve); D. inner surface of the reservoir; cg, convoluted gland; rs, venom reservoir.
Fig.5 Ultrastructure of the Dufour’s gland in C. japonicus workers under TEM. A. General view of epithelium of minor worker. B. Detail of apical part of secretory cell, showing subcuticular layer of mitochondria (M) and Golgi apparatus (G). C,D. Detail of basal part of secretory cell of major worker, showing basal invaginations (bi) and thick basement membrane (bm). ct, cuticle; LD, lipid droplets; lu, lumen; MF, muscle fibres; N, nucleus; SD, secretory droplets; tr, trachea.
Fig.6 Ultrastructure of the convoluted gland in C. japonicus under TEM. A. the cuticular inner wall of the convoluted gland of major worker, consisting of thin epicuticle and thick endocuticle. B,C Cytoplasm contains an ovoid to slightly polymorphic nucleus and numerous mitochondria in minor worker. D. The prominent end apparatus with long and slender microvilli in minor worker. ct, cuticle; EA, end apparatus; li, lamellar inclusions; lu, lumen; M, mitochondria; Mv, microvilli; N, nucleus; nu, nucleolus.
Fig.7 Ultrastructure of the free filament in C. japonicus worker. A. Semithin section survey of free filament of minor worker with secretory cells surrounding central lumen (arrow). B,C. Cytoplasm of secretory cells in major (B) and minor worker (C). D. Survey of duct cell of major worker with folded cell membranes (cm) near filament lumen (lu). E. Junction between duct cell and end apparatus in major worker. ct, cuticle; DC, duct cell; EA, end apparatus; M, mitochondria; nu, nucleolus; SC, secretory cell.
S0968-4328(17)30311-6 https://doi.org/10.1016/j.micron.2017.10.011 JMIC 2494
To appear in:
Micron
Received date: Revised date: Accepted date:
13-8-2017 29-10-2017 29-10-2017
Please cite this article as: Zhou, Ying, Li, Changyou, Billen, Johan, He, Hong, Morphology and ultrastructure of Dufour’s and venom glands in the ant Camponotus japonicus Mayr (Hymenoptera: Formicidae).Micron https://doi.org/10.1016/j.micron.2017.10.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title: Morphology and ultrastructure of Dufour’s and venom glands in the ant Camponotus japonicus Mayr (Hymenoptera: Formicidae)
Author names and affiliations: Ying Zhou a, Changyou Li a, Johan Billen b, Hong He a,*
a
College of Forestry, Northwest A&F University, Yangling, Shaanxi 712100, China
b
Zoological Institute, University of Leuven, Naamsestraat 59, 3000 Leuven, Belgium
*Corresponding author: Hong He:
[email protected]
Authors’ email: Ying Zhou:
[email protected]
Changyou Li: [email protected] Johan Billen: [email protected]
Highlights
Dufour’s gland shows different features among female castes. Dufour’s gland in major workers indicates for a more pronounced function. Dufour’s gland cells are characterized by basal invaginations and muscle fibers. The venom gland is morphologically similar in different female castes. Cells of venom gland contain numerous mitochondria and a prominent end apparatus.
Abstract
The Dufour’s and venom glands are two important exocrine glands in ants. In this
study, the morphology and fine structure of these two glands are described in the ant
Camponotus japonicus Mayr. The Dufour glands have a characteristic bilobed shape and show a difference in size and color between the female castes (minor and major workers, alate and dealate queens). The external surface of Dufour’s gland shows different features among the female castes. It appears more hypertrophied in major workers than in the other castes, indicating for a more pronounced function. The cells of the glandular epithelium in Dufour’s gland are characterized by abundant mitochondria, basal invaginations and developed muscle fibres. The venom gland is morphologically similar in the different female castes, and consists of a venom reservoir, a convoluted gland and a bifurcated free secretory filament, with the convoluted gland appearing as a large cap lying on top of the reservoir. Cells of the convoluted gland and the free secretory filaments contain numerous mitochondria and a prominent end apparatus. The results will contribute to understanding the functional morphologyof these glands among the different castes in Camponotus ants.
Keywords: Camponotus japonicus; Dufour gland; venom gland; morphology; ultrastructure
1. Introduction The Dufour gland and venom gland are the two common abdominal exocrine glands connected to the female reproductive system of ants, and serve a variety of functions in different phylogenetic groups (Hölldobler & Wilson, 1990; Billen, 2009). The Dufour gland among others can produce trail, recruitment and sex pheromones (Mitra, 2013). The venom gland
commonly releases offensive allomones and alarm pheromones (Schoeters & Billen, 1998). The morphology of both glands has been widely investigated in many species of various ant subfamilies (Billen, 1986a, 1986b; Billen, 1990; Billen & Taylor, 1993; Schoeters & Billen, 1995; Grasso et al., 2005; Ortiz & Camargo-Matthias, 2006; Billen et al., 2009; Fox et al., 2010; Billen et al., 2015), showing that gland morphology is linked with subfamily classification. The two glands not only display a different shape and size among the ant species, but can also change among castes and even individuals of the same species, especially, the variation of gland morphology and ultrastructure among castes may suggest a close link between gland structure and its behavioral roles (Schoeters & Billen, 1998; Abdalla & Cruz-Landim, 2001; Grasso et al., 2005). As one of the largest and highly evolved genera in Formicidae, Camponotus ants are characterized by an obvious polymorphism and division of labor (Hansen & Klotz, 2005). The morphology and ultrastructure of Dufour’s and venom glands have been investigated in several species (Billen, 1986a; Hermann & Blum, 1968), but still remain unstudied in Chinese ants. Camponotus japonicus Mayr is one of the most common and widespread ant species in China, and is also a natural enemy for many forest pests and one of the useful medical insects (Wu & Wang, 1995). In the present paper, we investigated the morphology of the Dufour and venom gland of C. japonicus, and documented their difference between the female castes (minor and major workers, alate and dealate queens) and the gland ultrastructure of workers. The results will
contribute to understanding the functional morphology of these glands among
different castes in Camponotus ants.
2. Material and Methods 2.1 Ants and gland dissection Three mature C. japonicus nests were excavated in Yangling, Shaanxi in China in May 2014 and 2015. All castes including alate and dealate queens and workers were studied, but workers were further distinguished into minor workers (head width 2.16 ± 0.26 mm) and major workers (head width 3.52 ± 0.03 mm). Nearly twenty individuals of minor workers, major workers, alates and six dealate queens (some queens were collected from other nests) were dissected to get glands for light microscopy as well as SEM and TEM analyses. The ants were pinned with the dorsal side up on a raised wax-based dissecting tray, and their abdomen was immersed in 0.9% NaCl. The abdominal cuticle was carefully torn off with forceps and dissected to expose the Dufour and venom glands under a stereomicroscope (Motic PXS5-T). Pictures were taken under a light microscope (Nikon SMZ 1500).
2.2 SEM method The Dufour and venom glands were respectively dissected from 5 individuals of minor workers, major workers, alates and 2 of dealate queens in 2.5% glutaraldehyde. Glands were fixed in 2.5% glutaraldehyde for 24 h at 4°C. The samples were washed 3–4 times with 0.1 M phosphate buffered saline (PBS, pH 7.2) during 15 min for each time. They were then dehydrated in a graded ethanol series (30%, 50%, 70%, 80%, 90% and 100%) and transferred to isoamyl acetate. Critical point drying was done in liquid carbon dioxide under 95 bar pressure, and was followed by gold-coatingg by cathodic spraying. Finally the samples were observed
and photographed under a JEOL JSM-6360LV SEM operated at 15 kV.
2.3 TEM method The Dufour and venom glands from 10 workers (5 major and 5 minor workers) were used for TEM observation. The glands were first fixed in 2.5% glutaraldehyde for 12 h at 4°C, rinsed four times (15 min each) in 0.1 M PBS (pH 7.2), and then post-fixed in 1% osmium tetroxide for 1.5 h. After rinsing four times (15 min each) in 0.1 M PBS (pH 7.2), glands were dehydrated in a graded ethanol series, then impregnated with a mixture of ethanol and LR-white (3:1 (v:v) for 2 h, 1:1 (v:v) for 4 h, 1:3 (v:v) for 12 h, respectively), and finally embedded in pure LR-white for 48 h. The samples were sectioned with a Leica-Ultracut ultramicrotome. Double-stained 80 nm thin sections were examined with a JEM-1230 transmission electron microscope at 80 kV.
3. Results 3.1 General anatomy The Dufour gland is a bilobed sac in all female castes of C. japonicus (Fig. 1 A–D). It is larger in the minor worker, major worker and alate female, with each lateral lobe having a length of 0.82 ± 0.02 mm (Fig. 1 A–C), and appears especially hypertrophied in major workers (Fig. 1 B). It is slightly smaller in dealate queens, each lateral lobe having a length of 0.65 ± 0.03 mm (Fig. 1 D)... In addition, the gland shows a different color in the four castes: it appears milky in minor workers, yellowish in major workers, white in alate queens, and light brown in dealate queens (Fig. 1 A–D).
The venom gland is much larger than Dufour’s gland, and consists of a venom reservoir, a convoluted gland and a bifurcating free secretory filament (Fig. 1). The convoluted gland appears as a large cap lying on top of the reservoir. It has a similar shape and size in major workers, alate and dealate queens (approximately 3.30 ± 0.20 mm length and 1.35 ± 0.10 mm width), but it is smaller in minor workers with a length of approximately 2.50 ± 0.10 mm and a width of 0.80 ± 0.15 mm. The venom gland appears light yellow in dealate queens and is more whitish in the other castes. The two free secretory filaments have a milky appearance. They have a diameter of 90 ± 2.0 m, and are approximately as long as the convoluted gland.
3.2 Ultrastructure under SEM Under SEM, the external surface of Dufour’s gland shows differences between workers and reproductive females. It is similar in major and minor workers (Fig. 2 A, C), displaying obvious protuberances surrounded by a thin reticulated layer of muscle fibres (Fig. 2 B, D). In the alate queens, however, the external surface is relatively flat without obvious protuberances; the reticulated layer of muscle fibres is very thin and fine (Fig. 3 A, B). In the dealate queens, the protuberances are relatively apparent and wrapped by an arrangement of bundles of a well-developed layer of muscle fibres (Fig. 3 C, D). Under SEM, the convoluted gland has a smooth surface (Fig. 4 A). The venom reservoir is an elongated sac with a conspicuous muscular supply at its ventrolateral sides, whereas the central part has a smooth appearance. The muscle fibres show a parallel organization in a direction perpendicular to the length of the reservoir. At each ventrolateral side, a thin longitudinal strand can be seen, that represents a nerve fibre (Fig. 4 B, C). When the reservoir is
torn open, its inner surface can be seen as a folded sheet structure (Fig. 4 D).
3.3 Ultrastructure under TEM 3.3.1 Dufour’s gland ultrastructure The epithelium of Dufour’s gland is formed by a single layer of cells, of which the luminal surface is covered by a thick and uniform cuticle (Fig. 5 A). The nucleus is usually ovoid, and is often located in the central or lower part of the cell (Fig. 5 A–D). There are abundant oval to elongated mitochondria in the cytoplasm, with a conspicuous subcuticular layer (Fig. 5 A, B). The cytoplasm also contains strands of smooth endoplasmic reticulum and a clear Golgi apparatus (Fig. 5 B), as well as small lipid droplets with a diameter of up to approximately 0.8 μm and abundant irregularly shaped electron-dense secretory droplets (Fig. 5 C, D). The basement membrane is about 0.3 – 0.4 µm thick and shows numerous deep invaginations, and separates the epithelium from underlying tracheoles and well-developed muscle fibres (Fig. 5 A, C, D).
3.3.2 Venom gland ultrastructure
The convoluted gland is formed by a dense mass of tubular windings, of which the cuticular inner wall consists of a very thin cuticular layer with a thickness of nearly 1.4–1.5 μm, in which the electron-dense epicuticle is extremely thin with a thickness of about 0.13–0.14 μm and the thick electron-lucid endocuticle measures about 1.27–1.36 μm (Fig. 6 A, B). The cells of the convoluted gland have an ovoid to slightly polymorphic nucleus (Fig.6 A–C). Their cytoplasm contains numerous mitochondria and a prominent end apparatus with long and
slender microvilli (Fig. 6 B, D). Muscle fibres were never observed in relationsto the convoluted gland. The free filaments are made up by polygonal glandular cells with a large, rounded nucleus that surround the central cuticular filament lumen (Fig. 7 A). The cells have a large rounded nucleus and a cytoplasm with a distinct end apparatus, numerous mitochondria and free ribosomes (Fig. 7 B, C). Duct cells make the connection between the secretory cells and the filament lumen, and are characterized by tortuous intercellular membranes near the filament lumen (Fig. 7 D). The cuticular canal of the duct cells has a continuous electron-dense epicuticular central lining and a diameter around 0.5 µm. It continues into the end apparatus, where its inner epicuticular lining becomes considerably thinner and discontinuous (Fig. 7 E).
4. Discussion The venom and Dufour glands originally were the accessory glands of the female reproductive system. Billen (1986a) systematically described and compared the morphology and ultrastructure of Dufour’s gland from 60 ant species belonging to 8 subfamilies, and showed that it has a different shape among the subfamilies. For example, it is pyriform in Aenictus (Dorylinae) (Billen & Gotwald, 1988), elongated tubiform in Myrmecia (Mymeciinae) (Billen, 1990) and Meranoplus diversus (Myrmicinae) (Billen, 2009), bulbous in Protanilla wallacei (Leptanillinae) (Billen et al., 2013), bilobed in C. pennsylvanicus, Formica rufibarbis, F. polyctena and Polyergus rufescens (Formicinae) (Hermann and Blum, 1968; Billen et al., 2001; Grasso et al., 2005). The present study shows that the Dufour gland of C. japonicus is typically bilobed, similar to other Formicinae species.
The shape and size of Dufour's gland in C. japonicus is similar, but presents a slightly different color and external ultrastructure among the female castes. In workers, especially in major workers, it appears more hypertrophied and presents obvious protuberances at its basal side (Fig. 2). In dealate queens, however, Dufour’s gland looks reduced and apparently lacks vigor (Fig. 1D), while its external surface does not display any obvious protuberances (Fig. 3). This result indicates that there exists an apparent link between the morphological features of Dufour's gland and its function in the different female castes of C. japonicus. C. japonicus is a monogamous species that nests underground (Wang and Wu, 1992). The single dealate queen after colony foundation stays inside the nest for her entire life. Workers, including majors and minors, are in charge of nest maintainance and foraging outside. A mass of alate females and males are produced in autumn and stay inside the nest throughout winter until the nuptial mating occurs in May of the following year. Dufour's gland therefore maybe more involved in foraging and defence activities of workers to act as trail, recruitment or alarm-defense pheromone as in other ants (Mitra, 2013). Alate queens can possibly use Dufour's gland compounds to act as sex pheromones and attract males for nuptial mating (Ayasse et al., 2001), but in dealate queens, Dufour's gland will gradually degrade with age and lose its secretory capacity after colony foundation. A similar result was found in Formica polyctena in which Dufour's gland was relatively large in virgin females compared with its size in dealate queens (Löfqvist and Bergström, 1980). In the slave-making ant Polyergus rufescens, however, Grasso et al. (2005) found that Dufour's gland was small in workers, somehow of intermediate size in newly-mated queens, and hypertrophied in adult queens, whereas the gland of adult queens is no more active
with an extended lumen and a thin epithelium. Combining the present results in C. japonicus, we infer that Dufour’s gland keeps active and plays more pronounced functions in workers and alated queens than it does in adult queens, and that there is a link between gland structure and the ant's behavioral roles. The Dufour gland is lined by a monolayered glandular epithelium, and its cellular organization shows considerable differences in several ant subfamilies (Billen, 1986a). In Formicinae, the epithelium has a very uniform thickness, with a characteristic subcuticular layer of mitochondria and a very thick basement membrane (Billen, 1986a). The ultrastructural features of the Dufour gland cells of C. japonicus workers are similar to those in other formicine ants such as Polyergus rufescens (Billen et al., 2001; Grasso et al., 2005), having the characteristic thick basement membrane with deep invaginations and developed muscle fibres. A large amount of electron-dense secretory droplets and mitochondria were found in the cytoplasm. We did not find clear differences in the ultrastructural features of the gland between major and minor workers. The venom gland is one of the biggest exocrine glands in ants, and to date three types of venom gland have been described (Schoeters & Billen, 1998), including the pulvinate type (with the convoluted gland on the venom sac’s dorsal side, as found uniquely in Formicinae), the ‘bourreleted’ type (with the convoluted gland inside the venom sac, in most other subfamilies), and no convoluted gland in Amblyoponinae and Leptanillinae (Schoeters et al., 1999). In C. japonicus, the venom gland is a pulvinate structure consisting of an elongated venom reservoir and a convoluted gland with a bifurcating free secretory tubule, similar to C. pennsylvanicus
(Hermann & Blum, 1968), C. vagus and Formica polyctena (Schoeters & Billen, 1998). The venom reservoir in C. japonicus is an elongated sac formed by polygonal cells and associated muscles, but these muscles do not cover the convoluted gland which is attached to the dorsal wall of the venom reservoir. Billen (1990) described the function of the convoluted gland and found it discharges the secretory compounds into the reservoir. The cytological organization of the convoluted gland and the free filaments clearly demonstrate their secretory capacity, because their glandular cells have a prominent well-developed end apparatus and numerous mitochondria. The prominent muscular supply on the ventral side of the reservoir sac, including the clear nervous provisioning is in line with the powerful spraying of formic acid by these formicine ants. We found no obvious morphological differences between four castes according to their body size under light microscopy. Our preliminary chemical analysis using gas chromatography–mass spectrometry (GC–MS), however, showed that the main chemical components in the venom gland present a clear variation between workers and queens, so more work should be done to reveal the functions of the venom gland in the female castes of C. japonicus.
Acknowledgements We are very grateful to two anonymous referees for the detailed comments and suggestions that helped us to improve the manuscript. We also express our thanks to An Vandoren (University of Leuven) for making the microscopy sections for this study and to Zhang Guoyun (Northwest A&F University) for SEM observation. The research was supported by the National
Natural Science Foundation of China (Grant No. 31070342, 31570388), Forestry Industry Research Special Funds for Public Welfare Projects (Grant No. 201404302-4) and the International Science and Technology Cooperation Project of Northwest A&F University (2016).
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5. Artwork with Captions
Fig.1 General morphology of Dufour’s gland and venom gland of female castes under light microscopy. A. minor worker; B. major worker; C. alate queen; D. dealate queen; DG, Dufour’s gland; VG, venom gland; ff, free filament.
Fig.2 Scanning electron micrographs of Dufour’s gland in minor and major worker. A,B. minor worker; C,D. major worker; MF, muscle fibres; pr, protuberance.
Fig.3 Scanning electron micrographs of Dufour’s gland in alate and dealate queen. A,B. alate queen; C,D. dealate queen; MF, muscle fibres; pr, protuberance.
Fig.4 Ultrastructure of the venom gland in C. japonicus workers under SEM. A. Dorsal view of convoluted gland, showing smooth surface; B,C. ventral side of reservoir, showing lateral bundles of muscle fibres (arrows indicate nerve); D. inner surface of the reservoir; cg, convoluted gland; rs, venom reservoir.
Fig.5 Ultrastructure of the Dufour’s gland in C. japonicus workers under TEM. A. General view of epithelium of minor worker. B. Detail of apical part of secretory cell, showing subcuticular layer of mitochondria (M) and Golgi apparatus (G). C,D. Detail of basal part of secretory cell of major worker, showing basal invaginations (bi) and thick basement membrane (bm). ct, cuticle; LD, lipid droplets; lu, lumen; MF, muscle fibres; N, nucleus; SD, secretory droplets; tr, trachea.
Fig.6 Ultrastructure of the convoluted gland in C. japonicus under TEM. A. the cuticular inner wall of the convoluted gland of major worker, consisting of thin epicuticle and thick endocuticle. B,C Cytoplasm contains an ovoid to slightly polymorphic nucleus and numerous mitochondria in minor worker. D. The prominent end apparatus with long and slender microvilli in minor worker. ct, cuticle; EA, end apparatus; li, lamellar inclusions; lu, lumen; M, mitochondria; Mv, microvilli; N, nucleus; nu, nucleolus.
Fig.7 Ultrastructure of the free filament in C. japonicus worker. A. Semithin section survey of free filament of minor worker with secretory cells surrounding central lumen (arrow). B,C. Cytoplasm of secretory cells in major (B) and minor worker (C). D. Survey of duct cell of major worker with folded cell membranes (cm) near filament lumen (lu). E. Junction between duct cell and end apparatus in major worker. ct, cuticle; DC, duct cell; EA, end apparatus; M, mitochondria; nu, nucleolus; SC, secretory cell.