Ultrastructural ontogeny of the labial gland apparatus in termite Prorhinotermes simplex (Isoptera, Rhinotermitidae)

Arthropod Structure & Development 31 (2003) 255–270 www.elsevier.com/locate/asd

Ultrastructural ontogeny of the labial gland apparatus in termite Prorhinotermes simplex (Isoptera, Rhinotermitidae) J. Sˇobotnı´ka,*, F. Weydab a

Institute of Organic Chemistry and Biochemistry, Flemingovo na´m. 2, Praha 6, CZ-166 10, Czech Republic b Institute of Entomology, Branisˇovska´ 31, C˘eske´ Budeˇjovice, CZ-137 05, Czech Republic Received 7 October 2002; accepted 21 December 2002

Abstract The labial glands in Prorhinotermes simplex consist of secretory cells organized into acini, water sacs and the ducts connecting the gland parts to the basis of the labium. Acini are composed of central and parietal cells. Central cells type I contain predominantly lucent vacuoles and are involved probably in hydroquinone production. They are lacking in soldiers. Type II central cells produce vacuoles of proteinaceous content which are of the same electron density (type IIa) or present in more shades (type IIb). Type IIa cells are present in all older individuals, whereas type IIb are lacking in soldiers and neotenics. Type III cells represent a specific stage of type I cells development, but they are definite functional secretory cells in soldiers. Acini of first instar larvae contain undifferentiated cells which differentiate into type I cells during the second instar. Specific larval central cells start to change into type II cells during first instar. The central cells of presoldiers show a transition from the pseudergate into the soldier situation. The parietal cells keep a uniform structure throughout the whole ontogeny. Only one type of cells form the water sacs in all castes. The cells are very flat with scarce organelles. The water sac cells produce lipid-like secretion, small lucent vacuoles and bunches of angulated vacuoles. The water sac probably functions as water storage organ only. Ontogenetical changes in water sac development are small. The acinar ducts originate inside the acinus where they are formed by flat cells with rare organelles. At the acinus border, cells equipped with mitochondria, microvilli and basal invaginations appear. The water sac ducts are formed by flat cells with rare organelles. Acinar ducts outside the acinus and water sac ducts are equipped with taenidiuam. q 2003 Elsevier Science Ltd. All rights reserved. Keywords: Acini; Central cells; Parietal cells; Acinar duct; Water sac duct; Taenidium

1. Introduction Paired labial (salivary) glands in termites consist of series of lobes (the acini), connected by a branching salivary canal (acinar duct). Each labial gland possesses a thinwalled reservoir (water sac) arising from the salivary canal (Noirot, 1969; Kaib and Ziesmann, 1992). Labial glands of the same general structure were observed also in the other primitive insect orders, e.g. Blattodea or ‘Orthoptera’ (Noirot, 1969; Berridge and Oschman, 1972; Dailey and Crang, 1977). As far as the function is concerned, the labial glands in termites belong to the most complex glands since their products perform diverse roles: undoubtedly digestion (Noirot, 1969; Veivers et al., 1991), communication (Kaib and Ziesmann, 1992; Reinhard and Kaib, 1995; Reinhard * Corresponding author. Tel.: þ 42-2-20-1833-60; fax: þ 42-2-24310177. E-mail address: [email protected] (J. Sˇobotnı´k).

et al., 1997) and defense (Deligne et al., 1981; Prestwich, 1984); but they were also reported to provide food for dependent castes, material for building activities (Noirot, 1969; Grasse´, 1982) or mycostatic substances (Grasse´, 1982). The reservoirs serve for water retention for regulation of microclimatic conditions in nests or moistening of material during building activities (Grube and Rudolph, 1999a,b). Contradictory data occur about the way of water intake into the reservoirs. An ultrastructural study of the labial gland acini in all older termite castes was made by Billen et al. (1989) in Macrotermes bellicosus. Only workers were studied in other species (Mastotermes darwiniensis – Czolij and Slaytor, 1988; Heterotermes tenuis –Costa-Leonardo and Soares, 1996; Serritermes serrifer – Costa-Leonardo, 1997; Grigiotermes bequarerti – Costa-Leonardo and da Cruz-Landim, 1991). The acini are composed of one type of secretory cells in the soldiers of M. bellicosus (Billen et al., 1989), but they

1467-8039/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1467-8039(03)00002-1

256

J. Sˇobotnı´k, F. Weyda / Arthropod Structure & Development 31 (2003) 255–270

are composed of three (workers) or two types of secretory cells (other castes) and of cells forming the canals in other species (Noirot, 1969; Czolij and Slaytor, 1988; CostaLeonardo and da Cruz-Landim, 1991; Costa-Leonardo and Soares, 1996; Costa-Leonardo, 1997). The only paper concentrated on water sac ultrastructure was published by Grube et al. (1997) and is based on observation in Reticulitermes santonensis workers. Ultrastructural data on water sacs are included in some other papers (Noirot, 1969; Grasse´, 1982; Billen et al., 1989; Kaib and Ziesmann, 1992). The ontogeny of glands (including labial) was studied in Prorhinotermes simplex at the histological level (Sˇobotnı´k and Hubert, 2003). The genus Prorhinotermes reveals characteristics unique not only in Rhinotermitidae but in all termites (Sˇtys and Sˇobotnı´k, 1999). Moreover, an uncertain phylogenetic position of the genus Prorhinotermes makes it an important study object. Our study represents the first attempt to describe the fine structure of the labial gland in Prorhinotermes. Our aim is also to describe the structure of the gland in usually omitted stages (larvae, presoldiers, neotenics) and ontogenetical changes during postembryonic development.

2. Material and methods The ontogeny of the genus Prorhinotermes is summarized in Fig. 1. The following castes and developmental stages were used to study the labial gland apparatus: the first larva, the second larva, pseudergate, presoldier, soldier, nymph, imago (both sexes), neotenic reproductive (both sexes). For simplification, we use the term pseudergate for all apterous non-soldier, non-reproductive individuals, at least in the fifth instar. All individuals of P. simplex (Hagen, 1858) originated from a colony collected by Dr J. Kr˘ec˘ek in Soroa (Pinar del Rio, Cuba) in December 1964, and since then kept in the laboratory at 26 ^ 1 8C. The investigated individuals were repeatedly extracted from the colony from December 1998, to November 2001. According to changing position of the labial gland (Sˇobotnı´k and Hubert, 2003), acini were studied in the mesothorax in larvae and in the metathorax in older stages. The water sacs were studied in the metathoraces of selected individuals. The acinar ducts were studied in the mesothorax in larvae and in the metathorax in older stages (ducts inside the acini), the common acinar and the water sac ducts in the prothorax and in the head. A simple freeze-cracking method has been used for SEM study. Whole insects were placed on a metal block cooled to very low temperature with liquid nitrogen. Then, the frozen termite body was cracked under a binocular loupe with a frozen razor blade, also cooled to very low temperature. Pieces of the body were then placed into ethanol, dried by the critical point method, glued onto an aluminium holder, sputter-coated with gold and observed in a SEM Jeol 6300.

Fig. 1. Developmental pathways of Prorhinotermes inopinatus (Roisin, 1988). Dotted arrows indicate situation in incipient colonies; thick arrows represent the most common developmental pathways. Abbreviations: A, alate imago; e, egg; L (1-4), larvae of first to fourth instar; Ny, nymph (wing-padded individual); Neo, neotenic reproductive; P, pseudergate; Ps, presoldier; R, regressive moult; S, soldier.

For TEM study, tissues were fixed in a mixture of 2% glutaraldehyde and 2.5% formaldehyde (Polysciences, EM Grade) in 0.1 M phosphate buffer at laboratory temperature for 1 –3 days. After washing in pure 0.1 M phosphate buffer, tissues were postfixed in 2% osmium tetroxide in 0.1 M phosphate buffer for 2 h, washed in bidistilled water and dehydrated in 50, 75% and absolute ethanol and embedded into Spurr resin (standard mixture). Ultrathin sections (silver to gold) were made with a Reichert Ultracut ultramicrotome and stained with uranyl acetate and lead citrate (standard recipe). Then they were studied in a Jeol 1010 transmission electron microscope.

3. Results 3.1. Acini The secretory cells forming the acini are of two principal types. (i) Larger central cells (up to 50 mm in the largest dimension) are apically differentiated into cell lumen bearing scarce microvilli (Figs. 2 and 3b,c). They contain many vacuoles. Within the central cells several subtypes

Fig. 2. Schematic drawing of an acinus in pseudergate. I, type I central cell; IIa, type IIa central cell; IIb, type IIb central cell; III, type III central cell; Pc, parietal cell; A, type A duct cell; B, type B duct cell; c, cuticle; cl, cell lumen; m, mitochondria; mf, myelin figure; n, nucleus.

258

J. Sˇobotnı´k, F. Weyda / Arthropod Structure & Development 31 (2003) 255–270

J. Sˇobotnı´k, F. Weyda / Arthropod Structure & Development 31 (2003) 255–270

exist (see later). (ii) Parietal cells are smaller (usually smaller than 15 mm), and appear usually (but not always, see Fig. 3a) in couples at the acinus periphery. Schema of an acinus is in Fig. 2. The apical parts of the central and the parietal cells are localized on the acinus periphery. Small duct cells are frequently inserted between the microvilli of the central and the parietal cells, but the cuticle of the duct is continuous except for the terminal opening (Fig. 3b). Such organization allows a direct transfer of the secretory cell products into the duct lumen (without passage through the cuticle). The whole acinus is covered by a basement membrane (about 50 nm thick in older stages), which is continuous with the basement membrane of the ducts. It is formed by a single lamina of diffuse structure without visible filaments. Nerves without sheath cells were frequently observed within the basement membrane of the acini. All nerves are equipped with numerous microtubules, while in some of them granules of electron dense neurosecretions (about 100 nm in diameter) are present. Rarely, nerves were also found inside an acinus. Microtubules and electron dense neurosecretion are also common within these cells but they may contain numerous small lucent vacuoles (about 40 nm in diameter). Mitochondria were observed in all nerves. Trachea were observed on the surface of acini but never under the basement membrane. 3.1.1. Parietal cells The general structure of the parietal cells remains unchanged throughout the whole ontogeny of P. simplex. They are conical in shape with a deep apical lumen filled with numerous long and dense microvilli (Figs. 3a and 4e). The plasma membrane forms deep basal invaginations, which may reach the bases of the microvilli (Fig. 3a). Nuclei are regular and oval shaped or slightly flat. Their size varies from 5 to 7 mm. Chromatin is in general dispersed, but sometimes few smaller condensations occur. The cytoplasm contains electron dense crystalloids (Fig. 3a), often disintegrated by myelin figures. Mitochondria are very abundant, elongated (up to 2 mm) and often irregular. Vacuoles are absent in parietal cells. A long septate junction connects neighbouring parietal cells. The parietal and the duct cells are connected by a desmosome and a septate junction. 3.1.2. Central cells The central cells form the major part of the acinus’ volume. Their nuclei are irregular, usually about 8 mm in

259

diameter; only if highly irregular or flat they may reach up to 12 mm in the largest dimension. Numerous small condensations occur within the chromatin. The apical part of the central cell forms a cell lumen equipped with microvilli. The basal parts of central cells are in some places connected to the basement membrane by hemidesmosomes (Fig. 3d). A general feature of two directly adjacent central cells is a sheet-like form of alternating basal invaginations. The membranes of secretory cells are locally coupled by extracellular material, probably of glycoproteinaceous nature. There are no true junctions between central cells, but they are interconnected with duct cells by a septate junction. Type I central cells (TI) possess an electron dense cytoplasm reduced in volume (Fig. 3b). Almost the whole space of the cells is filled with lucent vacuoles (0.5 – 10 mm in diameter). The vacuoles are very crowded and therefore highly irregular. They commonly fuse together and remnants of their membranes were frequently observed within them. The vacuoles contain very fine electron dense particles (smaller than 20 nm). The cytoplasm contains rough endoplasmic reticulum (RER), free ribosomes, some mitochondria and sometimes also Golgi apparatuses. The cell lumen is small or lacking in TI; microvilli may lead directly to the duct. TI is the most common type of central cells in all older non-soldier individuals. Type II central cells (TII) are rich in RER and Golgi apparatuses situated mainly in the basal regions. The vacuoles in TII are electron dense, from 1 to 2.5 mm in diameter (Fig. 3b and c). They are present in considerably lower numbers in comparison to TI and appear predominantly in the apical parts of the cells. Mitochondria are present predominantly in the basal and the apical regions. There are two types of TII. Type IIa cells (TIIa) (Fig. 3b and c) contain relatively light vacuoles with very similar electron density, while the vacuoles present in type IIb cells (TIIb) (Fig. 3c) are much darker and appear in more shades. Type III central cells (TIII) (Figs. 3d and 4e) contain a large proportion of RER and lucent vacuoles (from 1 to 4 mm in diameter) with fine irregular electron dense particles (from 50 to 200 nm in size) and/or the remnants of membranes (fusing of vacuoles is a very common process). Both structures (RER and vacuoles) are dispersed in all cell parts. Mitochondria are most numerous within TIII in comparison to other central cells. Another common organelle is Golgi apparatus. Basal invaginations are well

Fig. 3. Basic structures observed in acini of older stages of P. simplex. (a) Single parietal cell in pseudergate showing deep basal invaginations (arrowheads) and crystalloids (asterisks). (bar represents 5 mm) (b) The apical parts of type I and IIa central cells and A type duct cells in nymph. Note the borders of the duct intima (arrowheads). (bar represents 5 mm) (c) Type IIb central cell in pseudergate. (bar represents 5 mm) (d) Basal part of type III central cell in nymph. Note the attachment of secretory cell to basement membrane by hemidesmosomes (arrowheads). (bar represents 1 mm) (e) Degenerative structures within acinus of female imago. (bar represents 5 mm) (Abbreviations used: I, type I central cell; IIa, type IIa central cell; IIb, type IIb central cell; III, type III central cell; A, type A duct cell; bi, basal invaginations; cl, cell lumen; dl, duct lumen; GA, Golgi apparatus; L, lysosome; m, mitochondria; mf, myelin figure; mv, microvilli; n, nucleus; RER, rough endoplasmic reticulum).

260

J. Sˇobotnı´k, F. Weyda / Arthropod Structure & Development 31 (2003) 255–270

J. Sˇobotnı´k, F. Weyda / Arthropod Structure & Development 31 (2003) 255–270

developed; their inner space is apparently enlarged (Fig. 3d). TIII are relatively rare. TIII represent a stage preceding TI (but not in soldiers, see later). The sequence of rise of TI begins in TIII with a high proportion of cytoplasm containing RER and Golgi apparatuses and a low proportion of vacuoles. Such cells possess a very large cell lumen (up to 20 mm). The amount of vacuoles increases while the amount of cytoplasm decreases. Simultaneously, the amount of electron dense particles within the vacuoles decreases and the cell changes into TI. TII and TIII may contain electron dense vesicles (probably proteinaceous), which are degraded into myelin figures. Such vesicles or myelin figures were never observed within TI. Degenerative structures were rarely found in the central cells of older stages, most commonly in presoldiers (Fig. 4d). Degenerative structures appear as lysosomes—vacuoles containing electron dense particles (Fig. 3e) and sometimes also as myelin figures (Fig. 4d). The destructive process may eliminate the whole cell (Fig. 3e). 3.1.3. Acinius structure in particular castes and developmental stages All acini of the first instar larvae contain numerous undifferentiated cells (Fig. 4a). These are small (about 10 mm in the largest dimension), with a relatively large ovoid nucleus. The cytoplasm is reduced in volume and contains only a few organelles (RER, some mitochondria) and a large amount of glycogen particles (Fig. 4a). The undifferentiated cells have no direct contact to the lumen of the ducts. The central cell structure in first instar is highly variable. Younger larvae (with the cuticle attached to the epidermis) possess a single type of central cells (L-cells) (Fig. 4b). Lcells include only a few relatively large lucent vacuoles (up to 4 mm in diameter) that contain fine particles. These particles are released from an electron dense mass attached to the internal surface of the vacuole (Fig. 4b). The dense mass originates as a granule in the cytoplasm and penetrates into the vacuole. The apical parts of L-cells are equipped with short microvilli. Other organelles comprise some mitochondria, free ribosomes, and RER dispersed in a relatively voluminous cytoplasm. The acini of older first instars (with the cuticle separated from the epidermis or those producing a new cuticle) include L-cells and transitional stages between L-cells and TIIa or TIIb. Such cells contain a variable proportion of lucent (frequently with

261

electron dense mass) and electron dense vacuoles (in more shades in the case of TIIb), other organelles are RER, Golgi apparatuses and glycogen rosettes. No TIII cells were observed. The parietal cells are fully differentiated and seem to be fully active from the beginning of the first instar on. They slightly differ from those of older stages in the presence of a smaller number of mitochondria, in less developed basal invaginations and in the presence of higher proportions of RER and glycogen. The basement membrane ranges in thickness from 15 to 25 nm. The central cells in second instar larvae show the highest variability in relation to their age. In younger individuals (the cuticle attached to the epidermis), there are many undifferentiated cells and no cells with definite structure. Typical L-cells are lacking, but various transition stages of central cells are very common. Transitional stages of TII cells are similar to those described in the first instar but they contain a higher proportion of dense vacuoles (Fig. 4c). The undifferentiated cells change into TIII. In such cells, the proportion of cytoplasm, lucent vacuoles (from the beginning on with fine electron dense particles) and RER increases while the amount of glycogen decreases. In older second instar larvae (the cuticle separated from the epidermis), the undifferentiated cells are absent, the majority of central cells reveal various transitional phases and cells with definite structure are present as well, but are rare. Parietal cells have their typical structure, but contain lower numbers of mitochondria, higher amounts of glycogen and sometimes small amounts of RER. The thickness of the basement membrane ranges from 25 to 30 nm. For details of development of acini in older stages see Table 1. 3.2. Water sac The cells forming the water sacs are usually very flat (Fig. 5b), sometimes only about 200 nm. Organelles are rare, mitochondria or small amounts of RER were observed more often. Nuclei are irregular, often extraordinarily flat. Their size is usually about 9 mm, but sections of extremely flat nuclei may be as long as 14 mm. Chromatin is condensed; the degree of condensation is the highest for all labial gland cells. Smaller amounts of glycogen rosettes are present in some cells, but they are completely lacking in others. Cell borders are of complicated structure with very deep infoldings that result in a parallel position with the epithelial surface. The water sac cells contain numerous

Fig. 4. Structure of secretory cells of acini in larvae, presoldier and soldier of P. simplex. (a) Undifferentiated cell in acinus of the first instar larva (bar represents 5 mm). (b) L-cell in the first instar (bar represents 5 mm). Note the electron dense mass attached to the internal surface of vacuole (arrowhead). (c) Cytoplasm of L-cell changing into type IIb cell in the second instar larva (bar represents 2 mm). (d) Central cell in presoldier showing transition from type IIb to type III (bar represents 5 mm). (e) Type III central cell and parietal cell in soldier (bar represents 5 mm). (Abbreviations used: A, type A duct cell; dl, duct lumen; g, glycogen; m, mitochondria; mf, myelin figure; mv, microvilli; n, nucleus; Pc, parietal cell; RER, rough endoplasmic reticulum; VL, vacuole originating in L-cell; VIIb, vacuole of type IIb cell; VIII, vacuole of type III central cell).

Typical structure Rare, typical structure Absent The most common cell type, typical structure Neotenic (no sexual differences)

Typical structure

Typical structure Rare, typical structure The most common cell type, larger amount of cytoplasm, lower of vacuoles Imago (no sexual differences)

Lower proportion of vacuoles, larger cell lumen (up to 20 mm)

Typical structure Rare, larger size of particles inside the vacuoles (up to 0.5 mm) (Fig. 3d) The most common cell type, typical structure Nymph

Typical structure

The most common type (Fig. 4e) Absent Absent Soldier

Rare, typical structure

Typical structure, glycogen rossetes more numerous Absent Rarely typical; increased amount of cytoplasm and RER in others Presoldier

Both, lucent and dense vacuoles present, dense excluded or degraded by myelin figures (Fig. 4d)

Typical structure Rare, typical structure The most common cell type, typical structure Pseudergate

Typical structure

Parietal cells Type III cells Type IIb cells Type IIa cells Type I cells Caste/cell type

Table 1 Specific features of acinius development in older castes and stages of P. simplex

Typical, marked large and dense myelin figure

J. Sˇobotnı´k, F. Weyda / Arthropod Structure & Development 31 (2003) 255–270

262

microtubules. A single desmosome lays in the apical part and a long septate junction follows it. The basement membrane consists of a single lamina without apparent filaments (the thickness varies around 20 nm in older stages, about 15 nm in larvae). The cytoplasm of some water sac cells contains electron dense vesicles. These cells possess several short microvilli (about 80 nm in diameter) with a thin tubule inside (about 40 nm in diameter). Electron dense vesicles often change into myelin figures. A lipid-like substance appears in the centre of each figure and gradually replaces the figure (Fig. 5e). This substance is released from the water sac cell, always at the base of the microvilli (Fig. 5f). The lipid-like substance penetrates the cuticle by an unclear mechanism and remains in droplets after the penetration. Droplets of secretion are always attached to the luminal side of the water sac cuticle. Their size varies from 0.5 to 2 mm. The same secretion was observed inside the ducts, attached to the cuticle as well (Fig. 6d). In some parts of the water sacs, cells contain lucent vacuoles (50 – 400 nm) (Fig. 5d). Some of them are interconnected. Other membrane organelles are small vacuoles that appear united into compact bunches in the cytoplasm of some of the water sac cells (Fig. 5g). Whole bunches of these vacuoles leave the cells and occur between the cells and the cuticle. The size of a single vacuole is about 0.2 mm and the diameter of the bunch is about 0.5 mm. The vacuoles become larger after leaving the cell. The water sac is lined with a thin cuticle that is deeply and regularly folded (Fig. 5c) representing reserve for increasing the water sac volume. The cuticle is tightly pressed to the underlying epithelium in some places while it is separated from the epithelium by a space up to 1 mm elsewhere. This space contains fine particles (about 15 nm), which are not present in the lumen. The cuticle is fourlayered, always about 40 nm thick (inset in Fig. 5d). The three outer layers represent the outer epicuticle and are about half of the total thickness. Layers 1 and 3 are electron dense, while layer 2 is lucent. The innermost layer represents the inner epicuticle and is intermediary dense. Layer 2 can be divided into two equally thick sublayers by a dense but inarticulate layer. The connection of the water sac with the duct was observed only in the second instar larva, the presoldier, the soldier and the male neotenic reproductive. In the second instar larva and the presoldier, the junction between the water sac and its duct is simple, a taenidium occurs at one point of the water sac neck. On the other hand, in the soldier and the male neotenic a valve-like structure was observed; the duct is immersed into the water sac (Fig. 5b). No muscles are present here in both cases. Nerves are situated under the basement membrane locally. We suppose that they belong to receptors that control the degree of water sac filling. Tracheae are frequently present in the proximity of the water sac wall, but never under the basement membrane.

J. Sˇobotnı´k, F. Weyda / Arthropod Structure & Development 31 (2003) 255–270

The water sacs of first instar larvae are formed by thick, sometimes even columnar cells with deep infoldings penetrated by the cuticle (Fig. 5a). Mitochondria are considerably more numerous than in any other stage. Microvilli are short and scarce but more numerous than in all other stages. No secretory activity of the water sac cells was observed. The amount of glycogen is the highest among the studied castes. The situation in second instar larvae is similar, but the cells are much thinner. Production of lipidlike secretion was observed in second instar larvae but rarely. The water sac cells of pseudergates reveal the highest secretory activity (in all the three described ways) of all the castes and stages. The water sac cells in presoldier are principally the same as in other stages. They are thicker (usually more than 2 mm) and often contain glycogen particles. Nuclei are oval in shape and their chromatin is more dispersed than in other castes. In presoldiers, the water sac cells show numerous modifications related to the production of a new cuticle (a large proportion of RER, epidermal plaques, moulting vesicles). 3.2.1. Labial gland ducts The acinar ducts originate deep inside the acinus. There are several ducts inside each acinus. They are formed by simple cells (type A cells, TA) usually from 0.5 to 3 mm thick. TA are poor in organelles, microvilli are very scarce or absent (Figs. 3b and 6a). Nuclei are highly irregular, from 4 to 7 mm in the largest diameter, with numerous chromatin aggregates. Ducts formed by TA cells are equipped with a thin cuticle (from 20 to 25 nm thick) that ends opened (Fig. 3b). Except this end aperture, the cuticle of the duct is continuous. The cuticle covering a TA cell is composed of three equally thick layers of outer epicuticle (inset in Fig. 6a). The median layer is electron lucent while the inner and the outer layers are electron dense. At the acinus periphery, another cell type appears (type B cells, TB) (Fig. 6a). The apical membrane of TB forms numerous long microvilli. The nuclei of TB are irregular, 5 –7 mm in diameter and their chromatin is more dispersed in comparison to the nuclei of TA. The cytoplasm contains numerous mitochondria that are closely associated with deep infoldings of the membrane. These features show a very high transport activity of TB. The thickness of TB strongly varies but it generally increases along the duct (its ultrastructure remains unchanged). The thickness is from 1 to 7 mm near the acinus and from 2 to 15 mm in the prothorax and the head. The acinar ducts formed by TB are equipped by a thin basement membrane (20 – 30 nm thick) to which the cells are commonly attached by hemidesmosomes. TB often contain heavy electron dense vesicles. These vesicles are the results of a transformation of normal mitochondria (Fig. 6b and c). At the beginning, crystalloids appear within the mitochondria and their growth is

263

accompanied by a gradual loss of cristae. Newly formed crystalloids reveal a regular pattern of subunit organization (Fig. 6c), while following stages are much darker and without apparent inner structure. Inside heavy electron dense vesicles, lucent gaps may appear. They enlarge and disintegrate the whole vesicle. The end product of this process is a lucent vesicle containing irregular electron dense components. Such vesicles were infrequently observed between the duct cells and the cuticle. Another way of vesicle decomposing is the change into one or several myelin figures. At the border between TA and TB, the inner epicuticle (thick about 20 nm) appears (compare insets in Fig. 6a and d). The inner epicuticle does not reveal any pattern. The supporting structure (taenidium) appears below the cuticle of the duct behind the end of the acinus. The taenidium is flat and always less electron dense than the rest of the cuticle (Fig. 6d). In older stages, the largest dimension of the taenidium cross-section increases from 150 nm to the normal size (from 400 to 600 nm). The taenidium keeps this form until the point of fusion with the water sac duct. The basic composition of the cuticle remains unchanged, but the thickness of the inner epicuticle increases anteriorly from approximately 25 nm in the mesothorax to about 70 nm in the anterior part of the head. The posterior water sac ducts are formed by cells with rare organelles. Anteriorly, the apical plasma membrane forms short microvilli and mitochondria are more numerous (Fig. 6d). The thickness of the duct wall varies from 0.5 to 3 mm along the whole length. The water sac ducts are covered by a simple basement membrane thick from 25 to 35 nm. The ducts are equipped with a thin cuticle with a marked taenidium. The cuticle of the water sac ducts (inset in Fig. 5b) is very similar to that of the acinar duct. The taenidium is oval in cross-section; the longer diameter of the taenidium varies usually from 400 to 600 nm, the shorter diameter from 250 to 400 nm. Different taenidial threads may fuse or new threads may appear in between two others (Fig. 6f). Lipid-like secretion (originated from water sac cells) is frequently stuck on the cuticle of the water sac ducts (Fig. 6d and f). The junction between neighbouring duct cells remains unchanged throughout the whole ducts of both types. Neighbouring cells are connected by an apically situated desmosome that is followed by a long septate junction. The basal parts of the cell membranes are not connected. The whole border between neighbouring cells is sinuous. The water sac ducts are similar to tracheae but can be distinguished as follows: the cuticle of the duct lacks micropapillae; in contrast to tracheae, the duct cells are always in contact with the cuticle and contain certain organelles more often; the water sac ducts often contain lipid-like secretion. The common acinar and the water sac ducts are oriented parallely from prothorax. The water sac duct is always larger in diameter but its cells are thinner. In the head, the

264

J. Sˇobotnı´k, F. Weyda / Arthropod Structure & Development 31 (2003) 255–270

J. Sˇobotnı´k, F. Weyda / Arthropod Structure & Development 31 (2003) 255–270

ducts are localized under the suboesophageal ganglion and fuse in this region. The cells forming the common duct are simple (thick from 0.8 to 2 mm) (Fig. 6e). The taenidium continues beyond the duct fusion and ends at the margin of the preoral cavity. The cuticle remains composed of the same layers after fusion. Three outer epicuticular layers retain their original thickness (together from 20 to 25 nm), but the thickness of the inner epicuticle increases up to 110 nm. Nerves without sheath cells were observed within the basement membrane of the ducts of both types. Uncommonly, nerves were observed among the acinar duct cells. Nerves enclosed within sheath cells were found under the basement membrane of the water sac duct but rarely. Tracheae are present under the basement membranes of both duct types, more frequently in the acinar ducts where they are localized within the basal invaginations. The differences in structure of the ducts among particular stages are only superficial (thickness of the duct cells, number of mitochondria, etc.). In larvae, the cells of both, the acinar and the water sac ducts contain considerably higher amounts of glycogen. Also the structure of the acinar duct cells is less developed in comparison to the following stages, e.g. basal invaginations and mitochondria are less numerous. The inner epicuticle and the taenidium are thinner in comparison to the older castes, but the thickness of the outer epicuticle is the same. All these characteristics are more apparent in first instar larvae. The basement membranes are thinner in larvae, about 15 nm for the acinar ducts and about 20 nm for the water sac ducts. In presoldiers, the basal invaginations and microvilli are rare. The duct cells reveal remarkable modifications related to the production of a new cuticle.

4. Discussion 4.1. Parietal cells The parietal cells are present and at least partially active from the first instar on. Changes consist in increasing the cell volume, increasing number of mitochondria, increasing extent of basal invagination, and, vice versa, decreasing amount of glycogen and RER. It means that the parietal cells are active since hatching but their efficiency increases during early life stages. Parietal cells are of a definite structure and size in pseudergates and subsequent changes

265

are only small. The origin of crystalloids has never been observed, even more RER is present in parietal cells only in larvae. The crystalloids may originate in mitochondria, analogously to the situation in acinar ducts. All authors agree with the fact that the main function of parietal cells is ionic transport. The differences in the shape of the apical microvillar region in comparison to some other termite species (Costa-Leonardo and da Cruz-Landim, 1991) represent probably only a minor modification in relation to their same function. The parietal cells contain no vacuoles, so there is no secretory function, similarly as in some others species (Dailey and Crang, 1977; Costa-Leonardo, 1997). On the other hand, production of certain secretion is not excluded at least for parietal cells in workers of M. darwiniensis (Czolij and Slaytor, 1988), workers of Grigiotermes bequaerti (Costa-Leonardo and da Cruz-Landim, 1991) or workers of M. bellicosus (Billen et al., 1989). The parietal cells are fully active in soldiers of P. simplex while they are lacking completely in soldiers of M. bellicosus. The only type of secretory cells in M. bellicosus soldiers is not comparable to any type of cells within the acini of other castes (Billen et al., 1989). 4.2. Ontogeny of central cells The first instar larvae possess many undifferentiated cells, which differentiate during the second instar. L-cells are probably not functional although they contain some vacuoles and are equipped with microvilli. These central cells differentiate toward TII during the first and second instar while the undifferentiated cells change into TIII only during the second instar. In the late second instars, the majority of central cells are differentiating; rarely some central cells are of definite structure. The following changes in the central cells structure are only minute. All types of central cells are fully developed in pseudergates and these cells persist through the nymphal stage into alate imagoes. The lower proportion of vacuoles in TII of imagoes is probably caused by a general deficiency of energy supply (strongly evidenced by depleted reserves in the adipocytes, personal observation). TIIb is lacking in neotenics, hence knowledge of the situation in reproductively active dealate imagoes would help us to understand better the function of these cells. In presoldiers, the development of central cells shows clear transition from the situation in pseudergates to that of soldiers. The central cells most commonly contain several

Fig. 5. Structure of water sacs in P. simplex. (a) Water sac in the first instar larva (bar represents 5 mm). (b) Valve-like connection between the water sac and its duct in soldier (bar represents 5 mm). Arrowhead marks the first taenidial thread. Inset: The cuticle of the water sac duct in presoldier under high magnification (bar represents 30 nm). (c) Cuticle of water sac in the first abdominal segment of soldier (bar represents 2 mm). Note the regular folding of cuticle. (d) Lucent vacuoles within the water sac cell in male neotenic. (bar represents 1 mm). Inset: detailed view to water sac cuticle in presoldier (bar represents 30 nm). (e) Degrading vesicles in the water sac cell of pseudergate (bar represents 2 mm). Note lipid-like droplets that rise from the centre of myelin figures (asterisk). Arrowheads mark microvilli. (f) Discharge of lipid-like secretion from the water sac cell in pseudergate (bar represents 0.5 mm). Arrowhead mark central tubule inside microvillus. (g) The water sac cell producing bunches of small vacuoles in pseudergate (bar represents 1 mm). (Abbreviations used: 1, 2, 3, three layers of outer epicuticle; 4, inner epicuticle; c, cuticle; dc, water sac duct cell; g, glycogen; m, mitochondria; n, nucleus; ws, water sac cell).

266

J. Sˇobotnı´k, F. Weyda / Arthropod Structure & Development 31 (2003) 255–270

J. Sˇobotnı´k, F. Weyda / Arthropod Structure & Development 31 (2003) 255–270

types of vacuoles. The situation is closer to the pseudergate; typical TI were sometimes observed while TIII were never observed. The majority of central cells of soldiers belong to TIII. The absence of TI in soldiers shows that TIII are definite and functional secretory cells in this case. We suppose that TI reveal secretory cycles. TI probably releases all its vacuoles rapidly. After releasing, the cell probably repairs its cellular structures before it becomes ready to produce new vacuoles. A further stage of the cycle is probably the production of small vacuoles by cooperation of RER and Golgi apparatuses (typical TIII). The size of the vacuoles is gradually increasing by their fusing and the cell takes the form of TI. 4.3. Comparison of acini in P. simplex and in other species The central cells in P. simplex are comparable to those described for termites or other insects with acinar arrangement of secretory cells in the labial gland. Cells homologous to TI are present in all investigated termite species (Billen et al., 1989; Costa-Leonardo and da Cruz-Landim, 1991; Costa-Leonardo, 1997; Czolij and Slaytor, 1988). Workers of G. bequaerti differ in the presence of only TI (Costa-Leonardo and da Cruz-Landim, 1991). Kaib and Ziesmann (1992) described two types of vesicles within TI in the acini of workers of Schedorhinotermes lamanianus on the base of different staining in Goldner’s triple stain. In contrary to this fact, all observed TI in P. simplex reveal a similar structure. TIIa and TIIb central cells were not distinguished in the study made by Sˇobotnı´k and Hubert (2003); the differences between these cells are manifested only at the ultrastructural level. Cells similar to TII were found in the cockroach Blaberus discoidalis (Dailey and Crang, 1977). They contain dense vacuoles in more shades and are therefore more similar to TIIb. The situation in M. darwiniensis also resembles our observation, but the cells contain either homogeneously electron dense or electron lucent material or both within one vacuole (Czolij and Slaytor, 1988). Other secretory cells in various representatives frequently contain a large proportion of RER and vacuoles about 1 mm in diameter, but the latter ones are electron lucent (Billen et al., 1989; Costa-Leonardo, 1997). Costa-Leonardo and Soares (1996) describe autophagic organelles that appear probably in relation to the aging of an individual. We observe similar structures in all older castes

267

and stages (but not in all individuals), most frequently in presoldiers. This observation shows a high level of reconstruction of acini during the presoldier stage. Since our study is conceptually different, we cannot confirm if the presence of degenerative structures is a part of a reconstruction of the labial gland or a necessary component of gland development related to age polyethism. 4.4. Function of central cells There is no doubt about the production of proteinaceous secretion within TII. TIIa probably produce digestive enzymes, because they are present in all individuals older than the second instar. TIIb are present in pseudergates, nymphs and imagoes but they are completely lacking in soldiers and neotenics. We suppose that their function is the same in all cases. It is not clear if termites use the labial gland secretion for feeding the dependent castes or for building tasks. If so, TIIb probably produce a proteinaceous secretion serving for one of these functions. If not, TIIb will produce other digestive enzymes necessary for the independence of individuals. Nymphs in Prorhinotermes differ from nymphs of other termites in a high level of autonomy, they survive in pure nymph groups, for example (Hanus and Sˇobotnı´k, unpublished). Moreover, they are able to form neotenics or regress into the pseudergates (Miller, 1942; Roisin, 1988). Consequently we may suppose that they are also able to perform the same social roles as pseudergates and the similar development of acini is a piece of evidence for this fact. As neotenics appear only within groups of neuter individuals, the lack of TIIb may be the result of their redundancy in relation to the acceptance of care instead of its’ providing. The acini of soldiers contain predominantly TIII and their structure is similar to that described for the only cell type forming the acini of M. bellicosus soldiers (Billen et al., 1989). Soldiers of Termitidae: Macrotermitinae are known to produce quinones as defensive substances within their acini (Deligne et al., 1981; Prestwich, 1984). The same function could be therefore proposed for Prorhinotermes soldiers on the base of the similar ultrastructure of secretory cells. The production of a non-volatile pheromone used for marking food sources was localized into acini (Kaib and Ziesmann, 1992; Reihard and Kaib, 1995), and the signal was recently identified as hydroquinone (Reinhard et al.,

Fig. 6. Labial gland ducts in P. simplex. (a) The connection between type A (left) and type B acinar duct in female imago (bar represents 5 mm). Inset: detail of the cuticle of type A duct in nymph (bar represents 100 nm). (b) B type acinar duct in presoldier with different stages of development of vesicles from mitochondria (bar represents 2 mm). (c) Detail of mitochondria changing into vesicle corresponding to stage 1 at (b) (bar represents 0.5 mm). (d) Type B acinar duct and the water sac duct in the prothorax of female neotenic (bar represents 5 mm). Inset: Detail of the cuticle of type B acinar duct in the prothorax of female imago (bar represents 100 nm). (e) The common duct of the labial gland near the opening in male imago (bar represents 1 mm). (f) Cuticle of the water sac duct in the prothorax of pseudergate (bar represents 5 mm). White arrow marks branching of taenidium, black arrow marks appearance of a new taenidial thread in between two others. (Abbreviations used: 1, 2, 3, three layers of outer epicuticle; 4, inner epicuticle; 1s, vesicle with the last signs of origin from mitochondria; 2s, typical vesicle; 3s, partially degraded vesicle; 4s, remnants of vesicle excluded from the cell; IIb, type IIb central cell; d, desmosome; dl, duct lumen; m, mitochondria; mv, microvilli; n, nucleus; S, lipid-like secretion inside the water sac duct; sj, septate junction; T, taenidium; v, vesicle).

268

J. Sˇobotnı´k, F. Weyda / Arthropod Structure & Development 31 (2003) 255–270

2002). This signal probably originates within TI. This view is supported by the same function of hydroquinone throughout the whole order (Reinhard et al., 2002) and by the presence of TI in at least workers of all investigated species. Thus we suppose, that TI and TIII are involved in quinone production in all individuals but the quinones play different roles. At the ontogenetic level, it is clear, that the more important cells producing digestive enzymes (TII) are recognizable from the age of late first instar while the less important quinone producing cells stay as undifferentiated and are recognizable from the age of the late second instar. Reinhard et al. (2002) suppose that defensive quinones in soldiers could have evolved from food-marking quinones in workers. This hypothesis is evidenced on the ultrastructural level as an arrestment of further development of TI in the stage of TIII. 4.5. Ontogenetical changes in water sac development The water sac undergoes only minor changes during the ontogeny. In the first instar larvae, the water sacs are relatively smaller since their cells are thicker. The cells become flat in the second instar. Secretory activity of the water sac cells appears in the second instar and reaches its maximum in pseudergates. The secretory activity lacks in presoldiers and the only secretory process is connected to the production of a new cuticle. The surprising structural variability of the connection between the water sac and the duct may be related to moulting. Both castes in which a more complicated valvelike structure was observed (the soldier and the male neotenic) are terminal stages. A simple connection was observed in the second instar and the presoldier. We suppose, that this simple structure may be advantageous for moulting. 4.6. Function of water sacs The water sac cells have no characteristics typical for transporting epithelia (basal invaginations associated with mitochondria, apical microvilli, etc.; Berridge and Oschman, 1972) and serve only for isolation of water within the body. Our data basically correspond with Grube et al. (1997) and Grube and Rudolph (1999a), while they contradict Watson et al. (1971) who postulate water transfer from the gut to the water sacs through hemolymph. In spite of their main function, the water sac cells produce chemicals by at least three ways. These are lucent vacuoles (Fig. 5d), bunches of angulated vacuoles (Fig. 5g) and lipid-like secretion (Fig. 5e and f) originating in the myelin degradation of electron dense vesicles. We suppose that the droplets are of fat nature due to the characteristic undulation of sections just at the secretion droplets (typical for lipid droplets within the adipocytes). This view is supported by the evident hydrophobicity of the secretion: it is insoluble in water and always remains in droplets attached

to the luminal side of the cuticle. None of these ways of secretion were observed in water sacs of other termite species. A possible function of these secretions may be selfprotection against pathogens coming into the reservoir with sucked water or modification of the physical or chemical properties of water, e.g. the regulation of viscosity or osmolarity. Lipid droplets may protect the cuticle or provide the hydrophobicity necessary for a complete emptying of the water sac. 4.7. Labial gland ducts The labial gland ducts in P. simplex are objects of only minor changes during ontogeny. The observed general structure of the labial gland ducts is similar to descriptions in other termite species (Quennedey, 1984; Czolij and Slaytor, 1988; Billen et al., 1989). The intraacinar ducts of S. serrifer differ from TA duct of P. simplex in several characteristics, e.g. the ducts inside the acinus are discontinuous and possess short and spaced microvilli (Costa-Leonardo, 1997). 4.8. Structure of the cuticular intima of labial glands The cuticle reveals a similar arrangement during the whole ontogeny and throughout the whole labial gland. The structure of the outer epicuticle is very similar to that described for the cuticle of physogastric termitid queens (Bordereau, 1982) but strongly differs from the cuticle within labial glands of M. bellicosus (Billen et al., 1989). The cuticle of the ducts is composed of epicuticular layers and taenidium only in some parts. Except TA ducts, the inner and the outer epicuticle are present. The thickness of the inner epicuticle is constant in the water sac ducts, but it increases within the acinar duct anteriorly. The cuticle inside the acinus is continuous (except the terminal opening), but it is fenestrated in some other termite species (Billen et al., 1989; Costa-Leonardo, 1997). Some authors (Deligne et al., 1981; Quennedey, 1984; Czolij and Slaytor, 1988) describe the presence of endocuticular layers in labial glands, but such a cuticle was not observed in the ducts of P. simplex. The use of the term taenidium is well founded because its appearance is very similar to the taenidium in tracheae. The presence of the taenidium seems to be a common character of the labial gland ducts, its absence is described only for the acinar duct in R. santonensis (Grube et al., 1997). Particular authors use different terms for the taenidium: endocuticle (Czolij and Slaytor, 1988); cuticular ridges, taenidia-like circular threads (Grube et al., 1997). The specific appearance of the acinar duct, described as a crenellated pattern (Billen et al., 1989), is due to the flatness of the taenidium (Fig. 6d). The taenidium serves for the same function as in tracheae, it keeps the constant diameter of the tube. It is present from the beginning of the water sac ducts, but appears further from the acini in the acinar ducts of

J. Sˇobotnı´k, F. Weyda / Arthropod Structure & Development 31 (2003) 255–270

P. simplex unlike in M. darwiniensis, in which it reaches into the acini (Czolij and Slaytor, 1988). 4.9. Function of labial glands as a whole We would like to propose a probable function for the whole labial gland apparatus. According to the observed structure of central cells, considerable differences in the mode of action exist between TI on one hand and opposite TII and TIII on the other hand. TI are poor in mitochondria; hence the releasing of vacuoles should be a passive process, probably on the base of different osmolarities between the cell inside and the duct lumen. The composition of the duct lumen probably depends on the action of parietal cells. Stimulation by different neurons could cause both, either a change of permeability of the TI membrane or a change of transport activity of parietal cells. Change of permeability of the membrane of TII and TIII could be very important too, but the process may be not fully dependent on it because of the presence of mitochondria providing energy for active transport. These processes probably induce overpressure, which causes the movement of primary secretion along the duct. The acinar ducts contain only minute amounts of secretion in our samples. We suppose, that the secretion is released in relation to specific activities, e.g. defence in soldiers or food intake in others. TB acinar ducts reveal characteristics typical for transporting epithelia (Berridge and Oschman, 1972). These ducts serve probably for backward resorption of watery portion and formation of final secretion. In opposite, TA ducts are much simpler and serve probably predominantly for conducting the primary secretion from the acini to TB ducts. The water sac duct cells contain mitochondria more often but the main function is probably only the conduction of water to the water sac during its receiving and its conducting backwards during water regurgitation. Neither the water sacs nor their ducts are equipped with a closing apparatus, in contrary to cockroaches, which possess specific muscles for closing and/or emptying the reservoir (Sutherland and Chillseyzn, 1968). The mechanisms of water intake, retention and release are not understood. The content of the water sac probably functions as a water reserve. We suppose that it does not dilute the products of the acini because resorption of water probably takes place in the acinar ducts.

Acknowledgements We would like to thank Pavel Sˇtys (Faculty of Sciences at Charles University, Prague) and Johan Billen (Zoological Institute, Leuven, Belgium) for their critical reading of the manuscript completed with valuable remarks. We thank to Ivan Hrdu´ (Institute of Organic Chemistry and Biochemistry, Prague) for his support. We are grateful to Jitka

269

Pflegerova´ (Institute of Entomology, Czech Academy of Sciences) and Jana Nebesa´r˘ova´, Tonda Pola´k, Petra Masar˘ova´, Lad’ka Nova´kova´ (staff of Laboratory of Electron Microscopy, Institute of Parasitology, Czech Academy of Sciences) for their help in electron microscopical investigations. We thank to Martina Janousˇkova´ (Institute of Botany, Praha) for English review. This research was supported by the grant agency of Czech Republic, project No. 522/97/0126. Senior author thanks to Z4 055 905 project realized in IOCB, Prague.

References Berridge, M.J., Oschman, J.L., 1972. Transporting Epithelia, Academic Press, New York. Billen, J., Joye, L., Leuthold, R.H., 1989. Fine structure of the labial gland in Macrotermes bellicosus (Isoptera, Termitidae). Acta Zoologica (Stockholm) 70, 37–45. Bordereau, Ch., 1982. Utrastructure and formation of the physogastric termite queen cuticle. Tissue and Cell 14, 371–396. Costa-Leonardo, A.M., 1997. Secretion of salivary glands of the Brazilian termite Serritermes serrifer Hagen and Bates (Isoptera: Serritermitidae). Annales de la Societe Entomologique de France 33, 29–37. Costa-Leonardo, A.M., da Cruz-Landim, C., 1991. Morphology of the salivary gland acini in Grigiotermes bequaerti (Isoptera: Termitidae: Apicotermitinae). Entomologia Generalis 16, 13 –21. Costa-Leonardo, A.M., Soares, H.X., 1996. Degenerative structures in the salivary glands of the termite Heterotermes tenuis (Isoptera; Rhinotermitidae). Biociencias (Porto Alegre) 4, 145– 153. Czolij, R.T., Slaytor, M., 1988. Morphology of the salivary glands of Mastotermes darwiniensis Froggatt (Isoptera: Mastotermitidae). International Journal of Insect Morphology and Embryology 17, 207–220. Dailey, P.J., Crang, R.E., 1977. Ultrastructure of salivary gland of the cockroach, Blaberus discoidalis Serville (Blattaria: Blaberidae). International Journal of Insect Morphology and Embryology 62, 61– 66. Deligne, J., Quennedey, A., Blum, M.S., 1981. In: Hermann, H.R., (Ed.), The Enemies and Defense Mechanisms of Termites, Social Insects, vol. II. Academic Press, New York, pp. 1–76. Grasse´, P.-P., 1982. Termitologia, vol. I. Masson, Paris. Grube, S., Rudolph, D., 1999a. The labial gland reservoirs (water sacs) in Reticulitermes santonensis (Isoptera: Rhinotermitidae): studies of the functional aspects during microclimatic moisture regulation and individual water balance. Sociobiology 33, 307–323. Grube, S., Rudolph, D., 1999b. Water supply during building activities in the subterranean termite Reticulitermes santonensis De Feytaud (Isoptera: Rhinotermitidae). Insectes Sociaux 46, 192–193. Grube, S., Rudolph, D., Zerbst-Boroffka, I., 1997. Morphology, fine structure, and functional aspects of the labial gland reservoirs of the subterranean termite Reticulitermes santonensis de Feytaud (Isoptera: Rhinotermitidae). International Journal of Insect Morphology and Embryology 26, 49–53. Kaib, M., Ziesmann, J., 1992. The labial gland in termite Schedorhinotermes lamanianus (Isoptera: Rhinotermitidae): morphology and function during communal food exploitation. Insectes Sociaux 39, 373– 384. Miller, E.M., 1942. The problem of castes and caste differentiation in Prorhinotermes simplex (Hagen). Bulletin of University of Miami 15, 3–27. Noirot, Ch., 1969. In: Krishna, K., Weesner, F.M. (Eds.), Glands and Secretions, Biology of Termites, vol. I. Academic Press, New York, pp. 89–123. Prestwich, G.D., 1984. Defense mechanisms of termites. Annual Review of Entomology 29, 201–232.

270

J. Sˇobotnı´k, F. Weyda / Arthropod Structure & Development 31 (2003) 255–270

Quennedey, A., 1984. Morphology and ultrastructure of termite defence glands. In: Hermann, H.R., (Ed.), Defensive Mechanisms in Social Insects, Praeger Publishers, New York, pp. 151–200. Reinhard, J., Kaib, M., 1995. Interaction of pheromones during food exploitation by the termite Schedorhinotermes lamanianus. Physiological Entomology 20, 266– 272. Reinhard, J., Hertel, H., Kaib, M., 1997. Feeding stimulation signal in labial gland secretion of the subterranean termite Reticulitermes santonensis. Journal of Chemical Ecology 23, 2371–2381. Reinhard, J., Lacey, M.J., Ibarra, F., Schroeder, F.C., Kaib, M., Lenz, M., 2002. Hydroquinone: a general phagostimulating pheromone in termites. Journal of Chemical Ecology 28, 1–14. Roisin, Y., 1988. Morphology, development and evolutionary significance of the working stages in the caste system of Prorhinotermes (Insecta, Isoptera). Zoomorphology 107, 339–347. Sˇobotnı´k, J., Hubert, J., 2003. The morphology of the exocrine glands of

Prorhinotermes simplex (Isoptera: Rhinotermitidae) and their ontogenetical aspects. Acta Societatis Zoologicae Bohemicae in press. Sutherland, D.J., Chillseyzn, J.M., 1968. Function and operation of the cockroach salivary reservoir. Journal of Insect Physiology 14, 21–31. Sˇtys, P., Sˇobotnı´k, J., 1999. Comments on classifications of insect ontogenies, and occurrence of proneometabolous wing development in termite genus Prorhinotermes (Hexapoda: Isoptera). Acta Societatis Zoologicae Bohemicae 63, 483–492. Veivers, P.C., Mu¨hlemann, R., Slaytor, M., Leuthold, R.H., Bignell, D.E., 1991. Digestion, diet and polyethism in two fungus-growing termites: Macrotermes subhyalinus Rambur and M. michaelseni Sjo¨dtedt. Journal of Insect Physiology 37, 675– 682. Watson, J.A.L., Hewitt, P.H., Nel, J.J.C., 1971. The water-sacs of Hodotermes mossambicus. Journal of Insect Physiology 17, 1705–1709.