Animal Biology, Vol. 53, No. 1, pp. 1-16 (2003)Ó Koninklijke Brill NV, Leiden, 2003.Also available online - www.brill.nl

Ultrastructure of last larval instar fat body cells ofPachycondyla (= Neoponera) villosa (Formicidae:Ponerinae): cytochemical and chemical analysis

FERNANDO JOSÉ ZARA1;¤, FLÁVIO HENRIQUE CAETANO 2;¤,AIVLÉ CECILIA G. CABRERA 3, KLAUS JAFFÉ 3

1 Faculdade de Ciências Matemáticas, da Natureza e Tecnologia da Informaçao – UNIMEP, CampusTaqvaral – Rod. do Açúcar km 156, 13400-911, Piracicaba, São Paulo, Brazil

2 Departamento de Biologia, Instituto de Biociências de Rio Claro – UNESP, P.O. Box 199,13506-900, Rio Claro (SP), Brazil

3 Laboratorio de Comportamento, Universidad Simón Bolívar (USB), Caracas, Venezuela

Abstract—The ultrastructure of the fat body cells (trophocytes) of the last larval instar of Pachy-condyla (D Neoponera) villosa is presented. The cytoplasm is restricted to the cell periphery and tothe smaller strips among the vacuoles, protein granules, lipid droplets, and around the nucleus. Cyto-chemically, the presence of basic amino acids in the protein granules and in the nuclei was observedby using the ethanolic phosphotungstic acid technique (EPTA). The lipid droplets stained for unsatu-rated lipids. This result was further con� rmed by gas chromatography and mass spectrometry, wherethe unsaturated fatty acids were identi� ed as oleic and linoleic acids together with saturated fattyacids such as palmitic and stearic acid. Carbohydrates (glycogen) were also detected in the fat body.The glycogen is present as ¯ particles distributed among the lipid droplets and sometimes attached tothem.

Keywords: chromatography; cytochemistry; fat body; larva; Pachycondyla villosa; ultrastructure.

INTRODUCTION

The fat body takes on distinct arrangements in different insect orders (Keeley, 1978).In larvae the fat body is disposed in layers, sheets or strands which permit goodcontact between this tissue and the haemolymph (Chapman, 1975; Goiten, 1989).The cells appear as compact masses, or as loose aggregates, freely dispersed in thehaemocoel, covered by a thin basal lamina (Chapman, 1975; Locke, 1984; Dean et

¤Corresponding authors; e-mail: fjzara@rc.unesp.bror fcaetano@rc.unesp.br

2 Fernando José Zara et al.

al., 1985; Goiten, 1989). In the larvae of Pachycondyla (D Neoponera) villosa, thefat body is arranged in a single layer between epidermis and digestive tract, and itscells are grouped in clusters (Caetano et al., 2002).

The fat body is the main site of nutrient storage and the intermediate metabolismcentre of the insects (Wigglesworth, 1972; Locke, 1984; Gullan and Cranston,2000). Its functions are multiple and somewhat similar to that of vertebrates’ liver,since both tissues store excess nutrients, remove toxins from the blood, and serveas a site for biosynthesis of metabolites that are then circulated (Kilby, 1963;Wigglesworth, 1972; Locke, 1984; Padki and Narasubhai, 1993; Gillot, 1995). Themain compounds found in the fat body cells are proteins, lipids and carbohydrates(Gullan and Cranston, 2000). Proteins are stored as granules and are the sourceof amino acids during metamorphosis. Lipids (triacylglycerides) are stored eitheras a source of energy for the metamorphosis or as diacylglycerides, which areliberated in the larval haemolymph for the maintenance of normal metabolism. Thecarbohydrates, which are the principal source of immediate energy, are stored in thefat body as glycogen, which is rapidly used for the energy requirements of otherlarval tissues (Keeley, 1985; John and Muraleedharan, 1993). The ultrastructureof the larval fat body has been described for many insect orders, although inHymenoptera it has been studied only in Melipona quadrifasciata anthidioides(Cruz-Landim, 1983, 1985) and Apis mellifera (Goiten, 1989). In A. melliferalarvae, the fat body cell has an irregular shaped nucleus, with a great quantityof chromatin blocks and nucleolar material. The cytoplasm is restricted to theperiphery of both cell and nucleus, and as islands among the lipid droplets. Thepresence of glycogen and protein granules was not observed in A. mellifera (Goiten,1989). In M. q. anthidioides, however, the cytoplasm does not appear as islands, andprotein granules and stored glycogen can also be seen (Cruz-Landim, 1983).

In this work, the ultrastructure of the fat body of the last larval instar of P.villosa was studied. The contents of unsaturated lipids, proteins rich in basic aminoacids and glycogen were analysed using cytochemical techniques, in addition to thechemical analysis (gas chromatography and mass spectrometry) of lipids present inthis tissue.

MATERIAL AND METHODS

Animals

The larvae of Pachycondyla (D Neoponera) villosa ants were collected fromlaboratory colonies maintained at the Department of Biology, UNESP — Campusde Rio Claro, São Paulo, Brazil. The last larval instar of P. villosa was determinedby measurement of the cephalic capsule according to Zara and Caetano (2001).

Larval fat body of Pachycondyla villosa 3

Ultrastructure and cytochemistry

The fat body cells were removed and � xed for 2 h at 4±C in 2.5% glutaraldehydein 0.1 M cacodylate buffer, pH 7.2. Then, the material was separated into differentcategories, one of which was submitted to the routine technique for transmissionelectron microscopy (TEM) and the others were used for cytochemical techniques.

For the detection of unsaturated lipids, the Imidazole-buffered osmium tetroxidetechnique was used, in accordance with Angermüller and Fahimi (1982) and Soares(1998). After � xation, the material was washed for 10 min in 0.1 M imidazole buffer,pH 7.5 and then post-� xed for 30 min in 2% osmium tetroxide in 0.1 M imidazolebuffer at pH 7.5 (in dark room), dehydrated in ethanol, and embedded in Epon.

For detection of proteins rich in basic amino acids, the samples were submittedto the ethanolic phosphotungstic acid (EPTA) technique, according to Benchimol(1998). After having been � xed, the samples were dehydrated in ethanol-series (70-100%), and afterwards transferred to an ethanol solution (100%) containing 2%phosphotungstic acid (PTA) for 2 h, washed in ethanol absolute, and embedded inEpon. The control consisted of incubation of some samples in pyridine for 90 minat 37±C.

For glycogen detection, the Afzelius (1992) technique was used after the routinefor TEM. The ultra-thin sections were incubated in tannic acid solution (1%) andtreated afterwards with uranyl acetate at 4%. Unstained ultra-thin sections of all thematerial submitted to cytochemical techniques were examined and photographed ina TEM Philips CM 100 operating at 80 kV.

Chemical analysis

The fat body of P. villosa last larval instar was dissected directly in hexane and thesamples were enclosed in glasses with Te� on caps. The samples were injected in the5890 series II Hewlett-Packard gas chromatograph with � ame ionisation detectorcontaining a fused silica 5% phenyl-methyl-silicone capillary column (30 m £0.25 mm ID). Helium was used as the carrier gas, and the head pressure was 10psi. The oven was programmed at 50±C for 2 min, then using an 8±C/min rate to280±C, which was maintained for 25 min. Both injector and detector temperaturewere held at 280±C. For GC-MS analysis the Autosystem 2000 Perkin Elmer gaschromatograph was used, coupled to the mass spectrometer Qmass 910 for electronimpact ionisation with 70 eV for 40-500 daltons. Compounds were identi� ed byinterpretation of their mass spectra and also by comparison with the NIST-EPA-NIH library (1994).

RESULTS AND DISCUSSION

Besides trophocytes, the fat body in Pachycondyla (D Neoponera) villosa larvaecontains other types of cells; urate cells and oenocytes (Zara and Caetano, pers.

4 Fernando José Zara et al.

Figure 1. TEM general view of the fat body cells, showing many lipid droplets (Ld), vacuoles (V),protein granules (P), and the peripherical and perinuclear cytoplasm (head arrows). N, nucleus. Scalebar D 5 ¹m.

Figure 2. Detail of fat body cells cytoplasm. Rr, rough endoplasmic reticulum; m, mitochondria; Ld,lipid droplets; P, protein granules; V, vacuoles. Scale bar D 1 ¹m.

Larval fat body of Pachycondyla villosa 5

comm.). In this work, we only discuss the principal and most widespread cell type;the fat body cell or trophocyte. The fat body cells of P. villosa have a tendencyto be slightly round, and contain lipid droplets, vacuoles and protein granules(� g. 1). These cells do not show ultrastructural differences between those near thealimentary canal (perivisceral fat body) and those near the epidermis (parietal orperipheral fat body).

Cytoplasm free of deposits is restricted to the periphery of the cell, a small portionamong the lipid droplets, vacuoles and around the nucleus (� g. 1, read arrows and2), similarly to that observed in M. q. anthidioides (Cruz-Landim, 1983).

The nucleus has an irregular shape, from spherical to elongated, probably due tocompression by products stored in the cytoplasm (� gs 1, 3). The nucleus containsa large amount of chromatin and numerous nucleoli (� gs 1, 3) and is very similarto that observed in A. mellifera (Goiten, 1989) and in M. q. anthidioides (Cruz-Landim, 1983). According to Wigglesworth (1972), Cruz-Landim (1983, 1985),Dean et al. (1985), Goiten (1989) and others, the presence of a large amountof chromatin and nucleolar material can be indicative of polyploidy and highproduction of rRNA, and it can also occur in P. villosa. However, to con� rm thepolyploid condition, cytomorphometric analyses would be necessary, such as thosecarried out on salivary glands of M. q. anthidioides by Silva de Moraes (1972).Moreover, divisions were not observed in the fat body cells of P. villosa, so theconsiderable increase in this tissue during the larval development seems due only tothe increase of the cell size, which grows as a result of endomitosis, as suggestedfor M. q. anthidioides (Cruz-Landim, 1983, 1985). Another point that supports theoccurrence of endomitosis is the fact that, after the material is stored in these cells,their presence would be a mechanical hazard for cellular multiplication. Thus, thepolyploid state reached by endomitosis can also occur in the fat body of P. villosa.

Many mitochondria were found and a well developed rough endoplasmic retic-ulum (Rr) (� g. 2). There were also many Golgi complexes similar to the ones ob-served in M. q. anthidioides (Cruz-Landim, 1983) and in Achaea janata (John andMuraleedharan, 1993). According to those observations, the cytoplasm of the cellsseems to be involved mainly in protein synthesis. According to Dean et al. (1985)and Gullan and Cranston (2000), these proteins are synthesised and liberated to thehaemolymph and later absorbed by other tissues. Secretion vesicles being liberatedfrom the fat body cells were not observed, although some were noticed in the inter-cellular space. However these vesicles can also be membranous debris (� g. 4).

In trophocytes, various large vacuoles which possess a lower electrondensity whencompared to the lipid droplets can be seen (� gs 1-3). These vacuoles are very similarto those observed in the larval fat body of Lepidoptera, as is the case in Calpodes,Manduca, Phormia and Hyalophora (Dean et al., 1985), but are different from theones present in the Coleoptera Leptinotarsa, which are smaller and have a regulardistribution in the cytoplasm (Mcdermid and Locke, 1983). Using biochemicaltechniques, it was demonstrated that in Calpodes and Manduca these vacuolescontain tyrosine and tyrosine glucoside, respectively, and reach their maximum

6 Fernando José Zara et al.

Larval fat body of Pachycondyla villosa 7

accumulation in the last instar, decreasing drastically during metamorphosis. Thedisappearance of tyrosine coincides with a need for phenolic precursor in epidermalcells for cuticular tanning (Mcdermid and Locke, 1983; Chapman, 1998). Therefore,the presence of these vacuoles in the last larval instar of P. villosa can be anultrastructural indication of tyrosine accumulation, or other phenolic precursorsin the fat body for cuticle sclerotisation. However, to solve such questions it isnecessary to carry out further biochemical analyses, similar to those of Mcdermidand Locke (1983).

Many protein granules were found in the fat body cells (� gs 1-4). The presenceof these protein granules in the larval fat body is common in insects, although thesewere not observed in the last larval instar of A. mellifera (Goiten, 1989). The proteingranules are formed from sequestered haemolymph proteins, which are accumulateduntil metamorphosis is accomplished (Locke, 1984; Dean et al., 1985). In P. villosa,the protein granules are composed of multiple regions with variable electrondensitywhich has been shown using TEM (� g. 4). With the EPTA technique we found thatgranules display positive reaction to proteins, meaning that the granules containbasic amino acids such as lysine, arginine and histidine (� gs 5, 6) (see Lehningeret al., 1993). The occurrence of basic amino acids in the granules was not seen inconsulted literature, but its presence in this investigation is also con� rmed by thepositive reaction observed in the nucleus (� g. 5), where the histones are situated(characteristically basic proteins), rich in lysine and arginine (Lehninger et al.,1993), and by the negative reaction observed in the control group (� g. 7). Theseproteins must have been accumulated in the granules to be used in tissue rebuildingduring metamorphosis, as suggested by Price (1973), Locke (1984), Dean et al.(1985) and Gulland and Cranston (2000).

On the other hand, the presence of basic proteins stored in granules of P. villosafat body cells can be related to the adult cuticle sclerotisation. In Manduca, thetyrosine is rapidly converted into catecholamine (N -¯-alanyldopamine) at the timeof the moult. After ecdysis, these compounds are oxidised to quinones, which arehighly reactive molecules immediately linked to proteins. Most of the amino groupsof these proteins are involved in peptide linkages to form the protein chain. Thus, theamino group available for linking to quinones is that associated with dibasic acids,usually lysine (Chapman, 1998). Likewise, the basic proteins stored in granules infat body cells of P. villosa during the last instar can be mobilised for linkage toquinones during the moult.

Figure 3. Nucleus (N) showing its irregular shape and large nucleolar mass (Nu). See two largevacuoles (V) beside the nucleus. Arrow, vacuole membrane. Scale bar D 2 ¹m.

Figure 4. Detail of the peripheral and perinuclear cytoplasm with a protein granule (P) presentingdifferent electrondensities (head arrows). A vesicle (S) can be seen in the intercellular space (Es). Rr,rough endoplasmic reticulum; m, mitochondria; N, nucleus; Nu, nucleolus; Ld, lipid droplet. Scalebar D 1 ¹m.

8 Fernando José Zara et al.

Larval fat body of Pachycondyla villosa 9

In the P. villosa fat body cells many lipid droplets are observed and, as aconsequence, this fat body is the main storage tissue, in agreement with theobservations of Kilby (1963). The use of the imidazole-buffered osmium tetroxidetechnique in P. villosa shows that there is a positive reaction for unsaturatedlipids in the lipid droplets (� gs 8, 9), which must have being mobilised for thehaemolymph as diacyglycerol from triacyglycerol, for metabolism maintenanceand/or accumulated for pupation, as suggested by Keeley (1985). Another aspectof this technique is that the mitochondria and cytomembranes become evident (� g.9) according to Angermüller and Fahimi (1982) and Soares (1998).

Chromatography and mass spectrometry techniques were performed to identifythe chemical composition of unsaturated lipids observed in the P. villosa fat bodyby means of imidazole-buffered osmium tetroxide technique. Unsaturated lipidswere found to be present, such as fatty acids oleic acid (C18:1) and linoleic acid(C18:2) and, in addition, saturated ones such as palmitic acid (C16:0) and stearicacid (C18:0) were identi� ed (� gs 10-13, respectively). Hence the results of thecytochemical test for unsaturated lipids combined with chromatography of thefat body cells of P. villosa indicate that the imidazole-buffered osmium tetroxidetechnique has a strong af� nity for unsaturated fatty acids, mainly linoleic and oleicacids, as suggested by Angermüller and Fahimi (1982).

These fatty acids are among the most commonly observed of the products storedin fat body cells of Lepidoptera larvae, as seen in Hyalophora cecropia (Stephenand Gilbert, 1969). Stearic acid (C18:0), however, seems to be the initial product offatty acid synthesis in Hymenoptera parasitoid (Chapman, 1998). These saturatedand unsaturated fatty acids in� uence the growth of the majority of insects. The effectof their de� ciency has been demonstrated in various species and the main symptomsare: impairment of larval growth and development which can, in extreme cases,inhibit adult emergence, wing expansion or even wing malformation, and abnormalgrowth of some body parts. Furthermore, the oleic and linoleic fatty acids connectto the membrane phosphoglycerides, and the absence of these compounds results inthe impairment of membrane function (Downer, 1978).

The carbohydrates are, in the same way as lipids, important metabolites synthe-sised and liberated from the fat body to be utilised as energy resources for adjacenttissues (Keeley, 1985). The carbohydrate glycogen is present in P. villosa fat body;this was detected using the Afzelius technique (� gs 14, 15).

Figure 5. General feature of a fat body cell submitted to the ethanolic phosphotungstic acid (EPTA)technique showing positive reaction inside the protein granules (P) and in the nucleus (head arrows).Scale bar D 1 ¹m.

Figure 6. Detail of a protein granule (P) with positive reaction for basic proteins (head arrows). Scalebar D 1 ¹m.

Figure 7. Fat body cell control for EPTA technique.Ld, lipid droplets; V, vacuole; P, protein granules.Scale bar D 2 ¹m.

10 Fernando José Zara et al.

Figure 8. Partial view of a fat body cell exposed to the imidazole-bufferedosmium tetroxide techniquewith a positive reaction in the lipid droplets (Ld), indicating the presence of unsaturated lipids. P,protein granule; N, nucleus; Nu, nucleolus. Scale bar D 4 ¹m.

Figure 9. Detail of the cytoplasm showing the Rr and the mitochondrial membrane, conspicuouswhen using imidazole-bufferedosmium tetroxide technique. Scale bar D 0.5 ¹m.

Larval fat body of Pachycondyla villosa 11

Figures 10 and 11. Mass spectra and chromatograms of fat body cells of last larval instar. The � gureshows the oleic and linoleic mass espectra and its chromatographic peaks (arrows).

12 Fernando José Zara et al.

Figures 12 and 13. Chromatographicpeaks and mass spectra of palmitic and estearic acids (arrows),found in the fat body of last larval instar.

Larval fat body of Pachycondyla villosa 13

Figure 14. Fat body cells exposed to the Afzelius technique for glycogen. This picture shows theglycogen (arrow) attached to the lipid droplets (Ld) and among protein granules (P). Scale bar D1 ¹m.

Figure 15. View of the glycogen stored as ¯ particles widespread in the cytoplasm around proteingranules. Scale bar D 1 ¹m.

14 Fernando José Zara et al.

Glycogen is the main carbohydrate stored in insect fat bodies and may constitutefrom 10-25% of this tissue dry weight (Keeley, 1985): in mature honey bee larva,they make up about 33% of dry weight (Gillot, 1995). The presence of glycogenin the fat body is fully reported in the literature (Kilby, 1963; Wyatt, 1967; Wu,1983; Cruz-Landim, 1983, 1985; John and Muraleedharan, 1993, and others). Theglycogen is distributed among lipid droplets and sometimes some particles can beseen attached to the droplets. Such association seems to be normal and this wasalso observed in other larvae such as M. q. anthidioides (Cruz-Landim, 1983),A. janata (John and Muraleedharan, 1993) and A. mellifera adult queens (Cruz-Landim, 1985). The ultrastructural signi� cance of glycogen particles attached tolipid droplets could be related to the glyoxylate cycle. In this cycle, the cleavage ofisocitrate is catalysed by the enzyme isocitrate lyase, to form glyoxylate which,in a reaction catalysed by malate synthase, condenses with acetil-CoA to yieldmalate. The malate serves as a source of oxaloacetate for the gluconeogenesispathway. Gluconeogenesis usually occurs in lipid-rich seed during germination,before developing plants produce glucose by photosynthesis, in membrane-boundorganelles called glyoxysomes (for review see Lehninger et al., 1993). In P. villosa,membrane-bounded organelles were not observed in fat body cells. However,enzymes of the glyoxylate pathway occur in the cytosol of some bacteria, includingEscherichia coli (Lehninger et al., 1993). Hence in P. villosa such enzymes canoccur in the cytosol, involved in glycogen synthesis. The presence of glyoxylatepathway enzymes, mainly isocitrate lyase, has been detected in some invertebratessuch as Ancylostoma ceylanicum, Nippostrongylus brasiliensis (Singh et al., 1992)and Caenorhabditis elegans (Shahid and Mcfadden, 1985; O’Riordan and Burnell,1990) larvae. In insects, the presence of isocitrate lyase activity has been observedin prepupae and pupae of the southern armyworm Prodenia eridania fat body cells(Carpenter and Jaworski, 1962). Thus, these enzymes could occur in P. villosalarvae, but to solve this question an accurate biochemical analysis is needed to� nd out if these larvae use the glyoxylate cycle to produce glycogen. In P. villosa,glycogen (� g. 15) is stored as ¯ particles. The formation of ® particles (rosettes)has not been observed. Distribution in ¯ particles was also observed in M. q.anthidioides, A. janata, and A. mellifera queens. The glycogen present in P. villosafat body can be considered as an important reserve associated with the lipids,and must be the principal immediate energetic metabolite liberated to be used inother tissues. However, glycogen could be an important carbohydrate reserve in thelast larval instar to provide glucose units for chitin synthesis during pupation, asproposed by Gillot (1995) for the developing embryo.

In conclusion, the fat body cells from last larval instar of P. villosa show manycytoplasmic structures related to substance accumulation (tyrosine vacuoles, basicproteins in granules, lipid droplets with saturated and unsaturated fatty acids) tocomplete the post-embryonic growth of this insect.

Larval fat body of Pachycondyla villosa 15

Acknowledgements

This study was supported by FAPESP (Fundação de Amparo à Pesquisa do Estadode São Paulo — Brasil) Proc. 98/00576-8 and Animal Behavior Laboratory of theSimon Bolivar University — Venezuela.

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