Fat body of Prorhinotermes simplex (Isoptera: Rhinotermitidae): Ultrastructure, inter-caste differences and lipid composition

Micron 37 (2006) 648–656 www.elsevier.com/locate/micron

Fat body of Prorhinotermes simplex (Isoptera: Rhinotermitidae): Ultrastructure, inter-caste differences and lipid composition Jan Sˇobotnı´k a, Frantisˇek Weyda b, Robert Hanus a,c,*, Josef Cvacˇka a, Jana Nebesa´rˇova´ c,d a

Institute of Organic Chemistry and Biochemistry, Flemingovo na´m. 2, Praha 6, CZ-166 10, Czech Republic b ˇ eske´ Budeˇjovice, CZ-370 05, Czech Republic Institute of Entomology, Branisˇovska´ 31, C c Faculty of Science at Charles’ University, Albertov 6, Praha 2, CZ-128 43, Czech Republic d Institute of Parasitology, Czech Academy of Sciences, Branisˇovska´ 31, Cˇeske´ Budeˇjovice, 370 05, Czech Republic Received 9 December 2005; received in revised form 26 January 2006; accepted 26 January 2006

Abstract Ultrastructure of the fat body was studied in following castes and developmental stages of Prorhinotermes simplex: larvae of the first and the second instar, pseudergates, presoldiers, soldiers, nymphs, imagoes and mature ergatoid neotenic reproductives of both sexes. Fat body always consists of two principal cell types: adipocytes and urocytes. Adipocytes are characterized by a presence of large amounts of storage substances, namely lipid droplets, glycogen rosettes and proteins in the form of either biocrystals or vacuoles. Proportion of these components strongly varies during ontogeny. Adipocytes are equipped by a large central vacuole in which lipid droplets may resolve. Cytoplasm of urocytes contain glycogen rosettes and spherical or irregular concretions, other organelles are rare. Only adipocytes change their inner structure in the course of ontogeny: amount of glycogen decreases during the postembryonic development, it is the major kind of inclusion in the larvae but lacks in nymphs and imagoes; opposite trend is performed by lipids. The changes in protein content are less obvious but are explained and discussed. The total amount of triacylglycerols (TAGs) was found to be roughly 100 mg in a pseudergate, 250 mg in a nymph, and 30 mg in a soldier. The most abundant fatty acids in TAGs are oleic (O), stearic (S), palmitic (P) and linoleic (L) acid. TAGs form a complex mixture with OOO, OPO, OLO and OOS being the most abundant isomers. Only negligible differences exist among the castes. # 2006 Elsevier Ltd. All rights reserved. Keywords: Adipocyte; Biocrystal; Glycogen; Lipid droplets; Proteins; Termite; Triacylglycerols; Ultrastructure; Urocyte

1. Introduction The fat body of insects is a very important organ concerned especially with storage of nutrients and intermediary metabolism (Keeley, 1985; Haunerland and Shirk, 1995; Chapman, 1998). For those reasons, it has been studied in many insect groups (for a review see Dean et al., 1985; Keeley, 1985; Locke, 1998). The structure of the fat body remains relatively uniform and many similar features occur in non-relative species. Generally, the fat body is a ribbon-like tissue formed by adipocytes, but other cell types (urocytes, mycetocytes, oenocytes belong to more frequent ones) may occur as well (Dean et al., 1985; Haunerland and Shirk, 1995; Chapman, 1998). Adipocytes are characterized by a presence of lipid

* Corresponding author. Tel.: +420 220183505; fax: +420220183582. E-mail address: [email protected] (R. Hanus). 0968-4328/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2006.01.012

droplets (droplets of triacylglycerols), glycogen rosettes and protein granules (Dean et al., 1985; Haunerland and Shirk, 1995; Chapman, 1998; Locke, 1998; Canavoso et al., 2001). Urocytes contain predominantly spherical concretions made by uric acid (Haunerland and Shirk, 1995; Chapman, 1998). Termite fat body consists of adipocytes and urocytes (Grasse´, 1982; Han and Bordereau, 1982a,b). Other cell types (mycetocytes, oenocytes) were observed rarely, only in a single species (Grasse´, 1982; Sacchi et al., 2000). The postembryonic development of termites comprise several castes with clear specialization to fill particular tasks: soldiers are intended for colony defence, reproductives for breeding, workers (temporary workers = pseudergates in the genus Prorhinotermes) for food processing and taking care for the dependent castes. Therefore important differences in intermediary metabolism are expected among particular castes and variation in the fat body structure is expected to occur due to these differences. The aim of this paper is to compare the fat body structure in the fundamental castes and developmental

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stages throughout the whole ontogeny of the model species, Prorhinotermes simplex, and to resume the changes in content of major storage inclusions. 2. Material and methods 2.1. Termites All individuals of Prorhinotermes simplex (Hagen, 1858) originated from a colony collected by Dr. J. Krˇecˇek in Soroa (Pin˜ar del Rio, Cuba) in 1964 and since that time kept in laboratory at 26  1 8C. Studied specimens were removed from the colony and promptly fixed in the period from December, 1998, to November, 2001. For ontogenetical pathways occurring in the genus Prorhinotermes see Roisin (1988). 2.2. TEM sample preparation We used larvae in the stage of the first and the second instar, pseudergates, presoldiers, soldiers, nymphs, alate imagoes of both sexes and mature neotenic reproductives (ergatoids) of both sexes. We have studied the structure of the fat body in the head, thorax and abdomen; at least two samples of each body part were observed from each category of termites. The used fixative consists of a mixture of 2% glutaraldehyde and 2.5% formaldehyde (E.M. Grade) in 0.1 M phosphate buffer. Living termites were submerged into a drop of fixative and cut into parts (head, thorax, abdomen; the latter cut into dorsal and ventral part in neotenics). Fixation last from 1–2 days at laboratory temperature. The tissues were postfixed for 2 h in 2% osmium tetroxide and consequently dehydrated through the series of ethanols (50%, 75%, 100%). Tissues were then embedded into standard Spurr resin. Silver and gold ultrathin sections (50–80 nm) cut on Ultracut Reichert-Jung were stained with uranyl acetate and lead citrate (standard recipe; 30 min in uranyl acetate followed by 20 min in lead citrate after Reynolds, 1963) and observed in Jeol 1010 transmission electron microscope. 2.3. Image analysis The TEM photographs were submitted to a quantitative analysis in order to measure the relative proportion of lipids, glycogen rosettes, and proteins in the adipocytes. The analysis was based on manual evaluation of each subcellular structure with respect to the general characteristic of proteins (heavy electron dense granules), fat (moderately electron dense droplets), and glycogen rosettes. Scion Image Beta 3D software was used to count up the selected surfaces. Areas of nuclei and central vacuoles were not included into measurements. Cumulative areas were expressed as percents of the total adipocyte section (except the nucleus and central vacuole). Between 800 and 1200 mm2 (corresponding to a median section of several adipocytes) of extranuclear cytoplasm were analysed in each of the studied castes and developmental stages.

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2.4. Sample preparation and lipid analysis To get enough sample for chemical analysis we extracted whole termites instead of isolation fat body tissue from the individuals. Whole body extract gives a good picture of fat body lipids because: (i) the majority of lipids are located in fat body and (ii) termites are fed with dead sound wood only, so that contamination by food lipids is minimal. Triacylglycerols (TAGs), the main lipid class, were studied in this work. Due to the need of relatively large number of individuals for the analysis only pseudergates (20), soldiers (30) and nymphs (19) were studied. Insects were immobilized in 18 8C and homogenized with anhydrous magnesium sulphate in an agate mortar. The sample was suspended in CHCl3/CH3OH (1:1, v/v; 7 mL), sonicated for 10 min and filtered using a small column with silica gel (0.5 g). The extract was evaporated almost to dry and separated on pre-cleaned thin-layer chromatography glass plates coated with silica gel. The region containing TAGs was scraped off the plate and extracted with diethyl ether. The TAGs were reconstituted in CHCl3 and analysed by high performance liquid chromatography with atmospheric pressure chemical ionisation mass spectrometry. For GC analysis, TAGs were transesterified using a procedure described by Stra´nsky´ and Jursı´k (1996). For details about sample preparation, chromatographic analyses and data interpretation please refer to Cvacˇka et al. (2006). 3. Results 3.1. Common characters of the fat body cells Fat body of P. simplex consists of two principal cell types: adipocytes (Fig. 1) and urocytes (Fig. 5). Both types are present in all castes and developmental stages. Adipocytes and urocytes are interfused; in cross section through a single fat body ribbon, the urocyte is placed centrally and surrounded by four to six adipocytes. Adipocytes are usually about 30 mm in the largest dimension (rarely up to 40 mm, but in larvae never exceeding 25 mm). The fat body ribbons are enclosed in basement membrane formed by a single, about 30 nm thick, lamella. Basal invaginations are developed only locally at the ribbon periphery (Fig. 2). Adipocytes contain large slightly irregular nuclei (up to 10 mm in the largest diameter). Around the nucleus, there is a region of cytoplasm containing few flat cisternae of rough endoplasmic reticulum (RER), free ribosomes, numerous microtubules (Fig. 3), and sometimes Golgi apparatus as well. Mitochondria are either associated with basal invaginations or scattered through the whole cytoplasm; they are usually elongated (up to 2 mm long; see Figs. 4 and 20). Inclusions comprise glycogen rosettes, lipid droplets and proteins in the form of either biocrystals (electron dense angular granules of regular inner structures Fig. 12) or vacuoles (electron dense rounded vacuoles without apparent inner structure; see Figs. 8 and 20). Ratio between these two types of protein deposits considerably varies among studied castes (see below). Both types may be converted into myelin figures (Figs. 2 and 8) or into lamellar bodies in soldiers

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Figs. 1–7. Basic features of the fat body development in P. simplex. (1) Adipocyte in pseudergate. Arrows mark droplets of electron dense substance adhering to the inner surface of central vacuole. Scale bar represents 5 mm. (2) Basal part of adipocyte in soldier showing maximum of basal invagination development. Scale bar represents 2 mm. (3) Central part of adipocyte in soldier showing typical development of RER. Arrowheads mark microtubules in cross and longitudinal section. Scale bar represents 500 nm. (4) Cytoplasm of adipocyte in soldier with lamellar body. Scale bar represents 1 mm. (5) Urocyte in male neotenic reproductive. Scale bar represents 2 mm. (6) Detail of urate granule in presoldier. Scale bar represents 500 nm. (7) Regular and irregular urate granules in presoldier. Scale bar represents 5 mm. cv, central vacuole; g, glycogen; ld, lipid droplet; m, mitochondria; mf, myelin figure; n, nucleus; rer, rough endoplasmic reticulum; ug, urate granule.

(Fig. 4). Small droplets of lipids frequently occur inside partially decomposed protein granules (Fig. 15). The ascertained relative quantities of fat, glycogen and proteins in all studied castes and developmental stages are summarized in Fig. 21. Each adipocyte contains a large central vacuole (more vacuoles were observed rarely) into which lipid droplets may penetrate (Figs. 1 and 13) and are resolved there. Electron dense substance adheres to the inner surface of vacuole and forms droplets (usually from 0.5 to 1 mm in diameter; see Fig. 1) or rarely discontinuous sheet of central vacuole. This dense material was observed to be produced from tubular structures representing probably modified endoplasmic reticulum (Fig. 16). Central vacuole is usually lucent; it may contain

rest of lipid droplets (Fig. 13) or may be filled with glycogen particles and grains representing probably glycogen rosettes subunits (Figs. 8 and 10). The urocytes are smaller than adipocytes and their projections penetrate between neighbouring adipocytes and give them a star-like appearance. Urocytes never contact the basement membrane. The nuclei of urocytes are slightly irregular, about 8 mm in the largest diameter (Fig. 5). Urocytes are characterised by the presence of urate granules that frequently contain rod-like structures surrounded by micelles (Fig. 6). The size of urate granules varies from 2 to 5 mm. The granules may reach many different shapes, the most commonly are rounded (Figs. 5–7), but flat, irregular or even highly irregular ones were also observed (Fig. 7). The mitochondria

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Figs. 8–12. Adipocyte ultrastructure in larvae, presoldiers, and nymphs of P. simplex. (8) Central part of adipocyte in the first instar larva. Scale bar represents 2 mm. (9) Basal parts of three neighbouring adipocytes in the second instar larva. Note the absence of intercellular junctions. Scale bar represents 2 mm. (10) Adipocyte in presoldier. Arrowheads mark typical glycogen rosettes inside the central vacuole filled with granular material. Scale bar represents 2 mm. (11) Central part of adipocyte in nymph. Scale bar represents 2 mm. (12) Detailed view on biocrystals and cytoplasm of adipocyte in nymph. Note the absence of glycogen rosettes. Arrowheads mark fine particles of high electron density. Scale bar represents 500 nm. b, biocrystal; cv, central vacuole; g, glycogen; ld, lipid droplet; n, nucleus; v, electron dense protein vacuole.

are present in low numbers, other organelles are rare. Cytoplasm is relatively voluminous and contains considerable amount of glycogen rosettes. Urocytes do not show any differences among the studied castes and developmental stages except the mentioned case (see below), but it seems that quantity of urocytes is increasing during the ontogeny. 3.2. Larvae of the 1st instar Typical characteristic of the fat body cells in the first instar larvae is a high proportion of glycogen particles and presence of lower proportion of lipid droplets (Fig. 21). RER occurs in higher amounts in comparison to the other castes (except neotenic females). Protein vacuoles occur frequently; they are sometimes changing into myelin figures (Fig. 8). 3.3. Larvae of the 2nd instar The development of adipocytes is similar to the 1st instar with exception of higher proportion of glycogen particles (see Fig. 21 or compare Figs. 8 and 9) and lower amount of RER. 3.4. Pseudergate The amount of lipid droplets and glycogen rosettes is intermediate (see Fig. 21). Biocrystals and protein vacuoles are equally frequent in the adipocytes.

3.5. Presoldier Adipocytes of presoldier contain slightly lower proportion of glycogen and slightly higher proportion of proteins (both biocrystals and vacuoles). Central vacuole is often filled with granular material (subunits of glycogen rosettes) mixed with glycogen particles (Fig. 10). RER and Golgi apparatus occur more frequently in comparison to other sterile castes. 3.6. Soldier Proportion of proteins (biocrystals, vacuoles) is relatively low in soldiers (Fig. 21). Decomposition of proteins runs frequently through lamellar bodies (Fig. 4), which were never observed in any other caste. The central vacuole often contains granular material (subunits of glycogen), glycogen rosettes and sometimes myelin figures as well. 3.7. Nymph Central vacuole absents in the adipocytes of nymphs. The proportion of lipids and protein biocrystals is the highest among studied stages (Figs. 11 and 21). Protein vacuoles and glycogen rosettes are lacking. Total volume of cytoplasm is very low. Cytoplasm contains fine particles of high electron density, which were not observed in any other caste (Fig. 12). Nuclei of adipocytes are highly irregular due to squeezing by lipid

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Figs. 13–16. Adipocyte ultrastructure in imagoes of P. simplex. (13) Adipocyte in male imago. Arrow marks remnant of dissolved lipid droplet. Scale bar represents 2 mm. (14) Central part of adipocyte in male imago showing dissolving the lipid droplets directly in the cytoplasm. Scale bar represents 1 mm. (15) Adipocyte in female imago. Note the occurrence of dense spot within the enlarged mitochondria with well-developed cristae. Asterisk marks lipids occurring within the decomposing electron dense granule. Scale bar represents 1 mm. (16) Adipocyte in female imago showing various stages of transformation of mitochondria into the dense granules. Arrow marks production of electron dense substance (adhering to the inner surface of central vacuole). Scale bar represents 2 mm. av, autophagic vacuole; cv, central vacuole; dg, electron dense granule originated in transformation of mitochondrion (for explanation see the text); ds, electron dense spots occurring within the matrix of the mitochondria; em, enlarged mitochondrion; ld, lipid droplet; m, mitochondria; mf, myelin figure; n, nucleus; ug, urate granule.

droplets and biocrystals; their largest dimension may reach up to 13 mm. 3.8. Alate imago Adipocytes of imagoes reveal considerable differences in comparison to other stages. Adipocytes show a massive decomposition of their content. This process starts in only slightly modified situation (high proportion of lipid droplets; glycogen and protein vacuoles or biocrystals always scarce or absenting). In some individuals we observed a rapid penetration of lipid droplets into central vacuole (Fig. 13), but lipid droplets may dissolve directly in the cytoplasm (Figs. 14–16), especially in depleted adipocytes. The whole sequence is finished by the stage where almost all space of adipocytes is filled with large but empty central vacuole and the cytoplasm is reduced on thin layer on the cell margin. During this process some mitochondria dramatically increases in size (compare the sizes of mitochondria in Fig. 15), and reach rounded shapes (enlarged mitochondria reach up to 2.5 mm in diameter; see Figs 15 and 16). Consequently heavy electron dense spots start to occur within their matrix (Fig. 15), their cristae disappear and the inner space becomes very dense to form granules (see Fig. 16) enclosed in membrane (these represent protein area in Fig. 21). The granules are subsequently degraded either by formation of myelin figures that appear inside the granule (Fig. 15) or by emergence of lucent gaps inside the granule. Lipid droplets frequently appear inside the granules during both processes (Fig. 15). Further steps represent typical autophagic vacuoles (Fig. 16), i.e. lucent vacuoles with various proportions of heavy dense particles. Differences in fat body structure between

individuals (total numbers of samples studied are four heads, four thoraxes, and six abdomens for males, and four heads, four thoraxes, and five abdomens for females) are much more important than sexual differences. The urocytes reveal decreasing of glycogen amount till its disappearance. Some urocytes may contain only urate granules and lucent cytoplasm and some of them are probably dead (as urocyte at Fig. 16). 3.9. Female neotenic reproductives Adipocytes of neotenic females lack central vacuole and contain lower proportion of lipids, glycogen and proteins (only vacuoles) as well (Fig. 17). Voluminous cytoplasm contains predominantly large proportion of RER and free ribosomes (Figs. 18 and 19). Three basic forms of RER were observed: cisternae of RER organized parallely (Fig. 19) or into whorls (Fig. 18) and parallely oriented tubules of RER (Fig. 18). Other organelles comprise Golgi apparatus filled with dense material and numerous mitochondria. 3.10. Male neotenic reproductives Adipocytes contain large amounts of lipid droplets, glycogen and proteins in vacuoles or biocrystals (Fig. 20). Other aspects of neotenic male fat body correspond to abovementioned general scheme. 3.11. Composition of triacylglycerols Different amounts of TAGs were found in studied castes: nymphs 250 mg/indiv., pseudergates 100 mg/indiv., soldiers

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Figs. 17–20. Adipocyte ultrastructure in neotenic reproductives of P. simplex. (17) Adipocytes in female neotenic reproductive. Scale bar represents 5 mm. (18) Cytoplasm of adipocyte in female neotenic reproductive showing RER organized into whorl and into tubules. Note the region containing large amount of free ribosomes. Scale bar represents 2 mm. (19) Central part of adipocyte in female neotenic reproductive with RER organized into flat cisternae. Note glycogen rosettes surrounding the lipid droplets. Scale bar represents 1 mm. (20) Central part of adipocyte in male neotenic reproductive. Scale bar represents 2 mm. g, glycogen; ld, lipid droplet; m, mitochondria; n, nucleus; r, free ribosomes; rer, rough endoplasmic reticulum; v, electron dense protein vacuole.

30 mg/indiv. TAGs were composed of fatty acids (FAs) with 14–24 carbon atoms (CN) and 0–2 double bonds (DB). Oleic acid was the most abundant FA, forming 50% of all FAs (GC). Stearic, palmitic and linoleic acid formed roughly 10% (each) of all FAs. Other acids were present at concentration of 1.5% or less. Among them those with odd number of carbons (15, 17 and 19), both saturated and unsaturated, were present. Using HPLC/MS more than 40 TAGs with ECN (equivalent carbon number; ECN = CN-2DB) ranging from 42 to 56 and having up to six double bonds were detected. The most abundant TAGs Table 1 The main TAGs from the whole body extract of P. simplex TAG

OOO OPO OLO OOS PLO + OPoO LLO OPS MaOO MoOO SOS

Relative concentration (%) Nymphs

Pseudergates

Soldiers

16.6 11.3 15.0 9.8 7.2 6.7 3.4 2.4 3.1 2.9

13.6 10.5 11.4 9.5 6.3 5.0 3.9 3.0 3.1 3.6

13.2 13.9 8.4 10.6 6.6 2.7 5.4 5.8 4.4 3.8

FA residues in TAGs are abbreviated as follows (in alphabetic order): linoleic (L); margaric (Ma); myristoleic (Mo); oleic (O); palmitic (P); palmitoleic (Po) and stearic (S).

were triolein (OOO), dioleoyl-linoleoyl-glycerol (OLO), dioleoyl-palmitoyl-glycerol (OPO), and dioleoyl-stearoyl-glycerol (OOS). Only minor differences in TAG composition existed among castes (Table 1). 4. Discussion The general development of the fat body well corresponds with the previous observations. Basic features of adipocytes and urocytes are similar to other termite and cockroach species (Gharagozlou, 1965; Han and Bordereau, 1982a,b; Grasse´, 1982; Hyatt and Marshall, 1985; Polver et al., 1986). Oenocytes and mycetocytes are lacking in the fat body of P. simplex in contrary to some termite species (Grasse´, 1982; Sacchi et al., 2000). The results on ultrastructure of so-called ‘‘endolophocytes’’ presented by Gharagozlou (1965) are not satisfactorily comprehensive, e.g. we miss information of function, nature of secretion produced or evacuation of secretory products from the cells (to the interior of tracheae?). ‘‘Endolophocytes’’ may even represent cells of other organ. The columnar ‘‘royal fat body’’ adipocytes (as described by Gharagozlou, 1965) are not in principle different from other adipocytes. The structure of the fat body in P. simplex was compared among the head, thorax and abdomen (both peripheral and perivisceral) and no regional differences were found in contrary to several species, in which regional differentiation of the fat

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body occurs (reviewed in Dean et al., 1985; Haunerland and Shirk, 1995 or e.g. Grasse´, 1982; Wang and Haunerland, 1992; Maas et al., 2001); up to 11 different cell types belonging to the fat body was recognized (Jensen and Børgesen, 2000). Even in egg-laying (neotenic) females are all adipocytes equivalent in contrary to other termite species (Gharagozlou, 1965; Han and Bordereau, 1982a,b). Ontogenetical changes are manifested only in adipocytes, urocytes do not reveal changes during ontogeny. The only exception is urocytes of alate imagoes whose glycogen reserves are utilized. Urocytes of P. simplex are of very similar structure in comparison to other termite (Gharagozlou, 1965; Grasse´, 1982; Han and Bordereau, 1982b) and cockroach (Polver et al., 1986) species. Urocytes always contain single type of granules in contrary to the situation in cockroaches, in which up to four different types of granules were found; the urate granules of P. simplex correspond to the so-called urate spherule (Hyatt and Marshall, 1985). In comparison to drastical changes occurring during ontogeny of certain insect (for a review see Haunerland and Shirk, 1995; Locke, 1998; or e.g. Larsen, 1976; de Priester and van der Molen, 1979; Willott et al., 1988), observed changes in P. simplex are rather mild and consist in gradual changes in particular inclusions content; no autophagic activity was observed except for the imagoes. The changes in the structure of adipocytes during ontogeny lie predominantly in varying proportion of storage inclusions (summarized in Fig. 21). Proportion of glycogen is high in the larvae, moderate in pseudergates, soldiers, presoldiers, and neotenics and rare or absenting in nymphs and imagoes. Lipid reserves show opposite trend; lower amount is present in larvae, moderate amount in pseudergates, soldiers, presoldiers, and neotenics and high amount in nymphs. This seems to be a more general trend; decrease of amount of lipid reserves causes increase in glycogen content and vice versa (Oguri and Steele, 2003; Lorenz, 2003). All these results strongly differ from those published by Gharagozlou (1965) in Kalotermes flavicollis, in

Fig. 21. Proportion of fat, glycogen rosettes and proteins in the extranuclear cytoplasm of adipocytes in all studied castes and developmental stages.

which similar trends for lipids, glycogen and proteins were found in all sterile castes: the lowest relative proportion in the first instar larvae and its gradual increasing during larval stages, the highest proportion in nymphs and the lowest in soldiers; in sexuals, low proportion of lipids and glycogen and high of proteins was found. These ontogenetical changes observed in P. simplex are in agreement with the supposed storage function of adipocytes. In larvae, the energy is cumulated in the form of glycogen that is consequently converted into lipids. The ratio between glycogen and lipids remains stable in pseudergates, presoldiers, soldiers and neotenics, and these inclusions provide necessary reserve of energy. The absence of glycogen and high content of lipids in nymphs is probably due to necessity of storing large amounts of energy for metamorphosis and imaginal life till new colony foundation (see below). Hence the energy is stored in the form of lipids, which retain more than twice as much energy per weight unit in comparison to carbohydrate glycogen (Garrett and Grisham, 1999). Situation in sexuals differs from all other castes. Transparent changes occur in imagoes, in which the reserves are going to be spent: glycogen is usually lacking at all, lipids are decomposing in the central vacuole or directly in cytoplasm and even mitochondria are changing into granules and are destroyed by autophagy. Similar transformation and elimination of mitochondria were found in other organs of the same species, e.g. in the labial glands (Sˇobotnı´k and Weyda, 2003), in the epidermal glands in neotenics (Sˇobotnı´k et al., 2003), in the frontal gland (Sˇobotnı´k et al., 2004), and in the midgut (unpubl.). In our opinion, the transformation of mitochondria into dense granules and their subsequent elimination belongs to the equipment of the genus Prorhinotermes and represents unique feature of this genus. Imagoes are not fed before swarming (for a review see Nutting, 1969 or Grasse´, 1984) what fits with our observation: the abdomens of swarming imagoes of P. simplex are very flat what contrasts with the inflated abdomens of dealates in incipient colonies. The imagoes for this study were extracted from the colony shortly before swarming what explains reserve depletion. The statistical differences between imago males and females are probably just a coincidence; these differences are nearly negligible at the entirety of the micrographs. Adipocytes of neotenic males contain very high amounts of all reserves, lipids, glycogen and proteins (biocrystals and protein vacuoles) but functional consequences of such a high amount of the reserves are not clear. Amount of reserves is relatively low in neotenic females as the adipocytes contain very large amounts of RER. Surprisingly, amount of proteins (in vacuoles only) is only moderate therefore we suppose that the proteins continually evacuate from the adipocytes. Structure of adipocytes strongly differs in mature neotenic females of P. simplex and in queens of Termitidae (Grasse´, 1982; Han and Bordereau, 1982a). Adipocytes of P. simplex queens contain lower proportion of protein vacuoles and Golgi apparatuses while amount of lipid droplets and glycogen particles is much higher. On the other hand situation in kings is similar. Differences between queens probably originated in fact that the fat body of queens of Termitidae is secondarily adapted to yolk production only while the fat body of P. simplex queens perform

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concurrently two functions: storage function and yolk protein synthesis. Main part of disintegration of storage products probably runs in the central vacuole. Lipid droplets are commonly observed to enter the central vacuole and disappear there. Central vacuole sometimes contains granular material representing subunits of glycogen rosettes (see Fig. 10). We suppose that rather formation than decomposition of glycogen rosettes takes place here as the entering of glycogen into the central vacuole was never observed (while entering of lipid droplets is a common process) but complete filling of the central vacuole by glycogen rosettes was observed in presoldiers and soldiers and spatially delimited regions of adipocytes containing only glycogen were observed in adipocytes of other castes as well (see Fig. 9). Unfortunately, the direct disappearance of central vacuole by fulfilling with glycogen was never observed. Amounts of TAGs detected in the studied castes corresponds well with the observed structure and the performed picture analysis. The fat body in nymphs is very voluminous and contains as much as 71% of lipids; overall amount of TAGs is 250 mg per indiv. what represents about 5% of fresh body weight (5.3 mg on average; n = 10). The fat body is much smaller in pseudergates, and especially in soldiers. In pseudergates, it contains 52% of lipids, i.e. 100 mg of TAGs per indiv. representing about 2% of fresh body weight (4.9 mg on average; n = 10). In soldiers, the fat body includes 42% of lipids; overall amount of TAGs is only 30 mg per indiv. representing about 0.8% of fresh body weight (3.6 mg on average; n = 10). The most abundant FAs found in P. simplex TAGs contain 18 and 16 carbons. Such FAs predominate in fat bodies of insect from many orders (McGuire and Gussin, 1967; Thompson et al., 1973; Dillwith et al., 1993; Sayah et al., 1997; Canavoso et al., 1998; Kosˇta´l and Sˇimek, 1998; Hoback et al., 1999). Composition of the most abundant FAs in P. simplex is similar to FAs found in the fat body of other termite species, e.g. Macrotermes natalensis and M. goliath (Cmelik, 1969a,b, 1971), or Reticulitermes flavipes (Carter et al., 1972). FAs of P. simplex fat body contain also small amounts of odd-chain FAs. Such FAs are rare, however, they have been reported for several insect species (Howard and Stanley-Samuelson, 1990; StanleySamuelson et al., 1990) including termites (Cmelik, 1971). In this work, TAGs were characterized as well. To the best of our knowledge, this study is the first report on TAGs composition in fat body of termites. Acknowledgement The authors wish to thank to the staff of Laboratory of Electron Microscopy (Institute of Parasitology, Czech Academy of Sciences), to Jitka Pflegerova´ (Laboratory of Digital Imaging in Entomology, Institute of Entomology, Czech Academy of Sciences) for their valuable help during sample preparation, and to Karel Stra´nsky´ (Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences) for GC analysis. Jan Sˇobotnı´k, Robert Hanus and Josef Cvacˇka thank to Z4 055 0506 project realized in IOCB, Prague.

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