Fat body in some genera of leaf-cutting ants (Hymenoptera: Formicidae). Proteins, lipids and polysaccharides detection

Page 1

Fat body in some genera of leaf-cutting ants (Hymenoptera: Formicidae).

Proteins, lipids and polysaccharides detection

Gislaine Cristina Roma a, Maria Izabel Camargo Mathias a,*, Odair Correa Bueno a,b

a Instituto de Biociencias, Departamento de Biologia, UNESP, Universidade Estadual Paulista,

Avenida 24 A no 1515, CEP 13506-900 Rio Claro, SP, Brazilb Instituto de Biociencias, Centro de Estudos de Insetos Sociais, UNESP, Universidade Estadual Paulista,

Avenida 24 A no 1515, CEP 13506-900 Rio Claro, SP, Brazil

Received 13 September 2005; received in revised form 28 October 2005; accepted 28 October 2005

Abstract

The comparative histochemical analysis of the fat body of workers belonging to the basal species Cyphomyrmex rimosus and Mycetarotes

parallelus and to derived species Acromyrmex disciger and Atta laevigata revealed that this tissue is constituted mainly by cells denominated

trophocytes and oenocytes. The trophocytes of all species studied here were characterized mainly by the proteins and lipids synthesis and storage,

being the derived species the ones who have presented higher quantity of lipids in the trophocytes when compared to the trophocytes of basal

species. In workers M. parallelus and A. laevigata, besides proteins and lipids, there has being observed the presence of polysaccharides, however,

in C. rimosus and A. disciger these elements were detected in lower quantities. The histochemical studies of the oenocytes of basal and derived

species revealed significant presence of proteins as well as lipids in these cells. In the oenocytes of derived species A. disciger and A. laevigata a

higher quantity of lipidic inclusions has being observed, when compared to the basal species.

q 2005 Elsevier Ltd. All rights reserved.

Keywords: Ants; Attini tribe; Workers; Fat body; Proteins; Lipids; Polysaccharides

1. Introduction

The leaf-cutting ants are considered one of the mean pests

from Neotropical region, which attack several kind of

vegetation; consequently they are an important object for

research (Holldobler and Wilson, 1990).

The ants belonging to the Attini tribe are known as fungus

growing ants since they lived in symbiosis with fungi that is the

basis of feeding from colony. The fungus growing genera

Cyphomyrmex, Mycetophylax, Mycocepurus, Myrmicocrypta,

Mycetagroicus, Apterostigma, Sericomyrmex, Mycetosoritis,

Mycetarotes and Trachymyrmex are considered basal ants into

the Attini tribe, whiling Atta and Acromyrmex are considered

derived (Holldobler and Wilson, 1990). Pseudoatta is

considered a parasite (Bolton, 2003).

The workers of the monomorphic species are similar in their

morphology; however, in the colony of polymorphic species

0968-4328/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.micron.2005.10.012

* Corresponding author. Tel.: C55 19 35264135; fax: C55 19 35264136.

E-mail address: [email protected] (M.I.C. Mathias).

the workers can have different sizes and body shapes. In these

species, the workers are separate within castes, for instance,

minor and media workers and soldier (Holldobler and Wilson,

1990).

The fat body tissue is regarded as being of great significance

for the insects either in larval or in adult phases (Butterworth

and Bodenstein, 1967; Paes de Oliveira and Cruz-Landim,

2003). The main function of insect fat body is the pilling up of

reserves. Meanwhile, it has several other functions such as:

being the main intermediate metabolism spot; protein, lipids

and carbohydrates synthesis; storing and neutralizing sub-

stances which are not used by the insect; influence on cuticle

changes and in diapause. They also function in the

accumulation of excretion material and in vitellogenic

production, which is an essential protein for the composition

of insect eggs, being found more frequently in females rather

than in males (Kilby, 1963; Chapman, 1998).

The fat body is a filling tissue, which is distributed in the

body of the insect; it usually presents on two forms: the

perivisceral region, located around the digestive tract, might

involving other organs such as ovaries, and the parietal region,

located adjacently to the integument (Chapman, 1998; Gullan

and Cranston, 2000).

Micron 37 (2006) 234–242

www.elsevier.com/locate/micron

Page 2

G.C. Roma et al. / Micron 37 (2006) 234–242 235

The trophocyte is the main cellular type of the fat body of

insects, found during all life stages. The shape, appearance and

volume of the trophocytes varies widely depending on the

developmental stage and nutritional state of the insect (Locke,

1984), however, a marked characteristic of this type of cell is a

cytoplasm filled with several vacuoles. Few inclusions are

present at these regions of the cytoplasm (Cruz-Landim, 1983).

When these cells are found with a large quantity of reserve

material in their cytoplasm, the nucleus may take the irregular

shape (Chapman, 1998; Roma et al., 2005).

The cells of the fat body are considered as functional

differentiations of the trophocytes, which means, structural

modifications resulted from an adaptation to a determined

specific activity as the mycetocytes, chromatocytes, urate cells

and hemoglobin cells (Dean et al., 1985).

Other cellular type, the oenocyte was found associated with

the fat body, being spherical or egg-shaped bearing a nucleus

which is centrally located and which is smaller than the one of

the trophocytes. Granulations, vacuoles and inclusions are

structures, which are less observed in the cytoplasm

(Camargo-Mathias and Caetano, 1996).

The present study aimed to realize a comparative

histochemical analysis of the fat body cells to detect proteins,

lipids and polysaccharides in workers of C. rimosus Spinola,

M. parallelus Emery, A. disciger Mayr, and A. laevigata Smith,

species belonging to the Attini tribe, since the information

about the fat body on these insects are scarce or absents and

paying attention to differences to lead philogenetic impli-

cations in this tribe.

2. Material and methods

The present study used the fat body from workers of C.

rimosus and M. parallelus (monomorphic species) and media

workers of A. disciger and A. laevigata (polimorphic species),

collected in the field and immediately used to the histochemical

tests, however, some of them were maintained for approxi-

mately one month in the artificial nests in the Center for the

Study of Social Insects (CEIS) of UNESP, Rio Claro, SP,

Brazil. The individuals were anesthetized by cooling in a

freezer and dissected on a Petri dishes containing physiological

saline solution (NaCl 0,13 M, Na2HPO4 0,017 M, KH2PO4

0,02 M, pH 7.2) for insects.

2.1. Histochemistry

The fat body from workers of C. rimosus, M. parallelus,

A. disciger and A. laevigata was removed and fixed in

paraformaldehyde 4% and 0.9% NaCl in 10% phosphate

buffer (0.1 M—pH 7.5), Bouin’s solution and calcium

formol, depending on the technique employed. The

material was then dehydrated in an ascending series of

ethanol solutions (70, 80, 90 and 95%) for 15 min in each

bath. Following dehydration, the material was transferred

to a resin solution (JB-4 Polaron Instruments/Bio Rad) in

absence of catalyst for 24 h in the refrigerator. The

samples were later placed in plastic moulds previously

filled with resin and catalyst, and sealed with a metal

support. The polymerization was completed at room

temperature in 90 min. The blocks were sliced (4 mm)

using a Sorvall JB-4/Bio Rad microtome, with a dry glass

knife. Sections were collected and transferred to a room

temperature water bath before being placed on cleaned

glass slides. After dried, sections were stained following

standard histochemical procedures and finally placed in an

incubator.

The following histochemical tests were carried out to detect:

2.1.1. Proteins—Bromophenol Blue according to Pearse

(1985)

The material was fixed in paraformaldehyde 4% and 0.9%

NaCl in 10% phosphate buffer (0.1 M—pH 7.5) for 24 h,

sectioned and stained with Bromophenol Blue for 1 h.

Following these procedures, sections were rinsed in acetic

acid 0.5% for 5 min and immersed in tertiary butyl alcohol for

5 min, cleared in xylol and mouted in Canadian Balsam.

2.1.2. Acid and neutral polysaccharides—PAS/Alcian Blue

according to Junqueira and Junqueira (1983)

The material was fixed in Bouin for 6 h and the sections

were stained with 1% Alcian Blue solution pH 2.5 for 30 min,

immersed in 1% periodic acid for 5 min and then they were

submitted to Schiff’s reagent in the dark for 30 min and rinsed

in water during 10 min and contra-stained with Hematoxylin

for 5 min. Sections were then mounted in Canadian Balsam.

2.1.3. Lipids—Sudan Black B according to Pearse (1985)

The material was fixed in 10% formol-calcium for 6 h,

stained with Sudan Black B for 20 min, followed by rinsing in

distilled water and contra-stained with 1% Neutral Red for

2 min. After dried, the material was mounted in glycerin–

gelatin.

3. Results

3.1. Bromophenol Blue (proteins detection)

The Bromophenol Blue technique, which evidences the

protein presence, reveals the parietal and perivisceral tropho-

cytes of workers of C. rimosus with positive cytoplasmic

regions. Moreover, some perivisceral trophocytes are more

positive than others at the same region (Fig. 1(A)–(C)). In the

parietal and perivisceral trophocytes of M. parallelus workers,

the reaction is positive only in the cytoplasm among vacuoles,

which appear in high quantities in these cells (Fig. 1(D) and

(E)).

In media workers of A. disciger a high quantity of

cytoplasmic inclusions is observed as proteic granules; the

perivisceral trophocytes present a higher quantity of more

strongly stained granules than the ones from the parietal region

(Fig. 1(F) and (G)). The trophocytes of the parietal and

perivisceral regions of C. rimosus, M. parallelus and A.

disciger present vacuoles negative to the test (Fig. 1(A) and

(C)–G).

Page 3

Fig. 1. Histological sections of the fat body cells from workers of C. rimosus and M. parallelus and media workers of A. disciger and A. laevigata, stained by

Bromophenol Blue. Detail of C. rimosus parietal fat body (A) and perivisceral one (B,C). Parietal fat body (D) and perivisceral one (E) from M. parallelus. Detail of

A. disciger parietal fat body (F) and perivisceral one (G). Parietal fat body (H) and perivisceral one (I) from A. laevigata. c, cuticle; t, trophocyte; n, trophocyte

nucleus; v, vacuole; o, oenocyte; *, oenocyte nucleus; , vacuoles in the oenocyte cytoplasm. BarsZ20 mm.

G.C. Roma et al. / Micron 37 (2006) 234–242236

In media workers of A. laevigata, the reticular cytoplasm of

parietal and perivisceral trophocytes is positive to the test, not

being observed the presence of vacuoles or granules (Fig. 1(H)

and (I)).

The parietal and perivisceral oenocytes of workers studied

here react strongly to the Bromophenol Blue test, indicating

the presence of high quantity of proteins in these cells

(Fig. 1). In workers of M. parallelus a rough and positive

granulation is observed in the oenocytes cytoplasm of both

regions (Fig. 1(D) and (E)). In the oenocytes of the species M.

parallelus, A. disciger and A. laevigata are observed negative

vacuoles to the test (Fig. 1(D)–(F) (H) and (I)).

The parietal and perivisceral trophocytes and oenocytes

nuclei of workers studied here are strongly positive to the

test (Fig. 1).

3.2. PAS/Alcian Blue (acid and neutral polysaccharides

detection)

The parietal and perivisceral trophocytes submitted to the

polysaccharides detection in workers of M. parallelus and

A. laevigata show positivity to neutral polysaccharides, which

is detected as rough granulation distributed in whole cytoplasm

(Fig. 2(C) and (D), (G), (I), (J)).

Page 4

Fig. 2. Histological sections of the fat body cells from workers of C. rimosus and M. parallelus and media workers of A. disciger and A. laevigata, stained by

PAS/Alcian Blue. Detail of C. rimosus parietal fat body (A) and perivisceral one (B). Parietal fat body (C) and perivisceral one (D) from M. parallelus. Detail of A.

disciger parietal fat body (E) and perivisceral one (F). Parietal fat body (G), (H) and perivisceral one (I, J) from A. laevigata. c, cuticle; t, trophocyte; n, trophocyte

nucleus; o, oenocyte; *, oenocyte nucleus; , granules in the oenocyte cytoplasm. Bars A–F; H, JZ20 mm G, IZ50 mm.

G.C. Roma et al. / Micron 37 (2006) 234–242 237

In workers of C. rimosus and A. disciger, the reaction is

weakly positive in the cytoplasm of parietal and perivisceral

trophocytes, however, it is observed the presence of some

granules strongly stained (Figs. 2(A), (B), (E) and (F)).

The oenocytes from the parietal and perivisceral regions of

ant workers studied here are weakly positive to the applied test,

being observed in C. rimosus, A. disciger and A. laevigata

some cytoplasmic granules strongly stained (Fig. 2(A), (B),

(E)–(I)). In A. laevigata these granules are observed in higher

quantity (Fig. 2(H)). In workers of M. parallelus, positive PAS

granules are absent in the oenocytes cytoplasm (Fig. 2(C)

and (D)).

The trophocyte and oenocyte nuclei of C. rimosus,

M. parallelus, A. disciger and A. laevigata do not react to the

acid and neutral polysaccharides test (Fig. 2).

In the trophocytes and oenocytes of all species studied here

are not detect acid polysaccharides in these cells (Fig. 2).

3.3. Sudan Black B (total lipids detection)

The histochemical test for total lipids detection applied in

the trophocytes and oenocytes of workers of C. rimosus,

M. parallelus, A. disciger and A. laevigata evidences

the presence of positive cytoplasmic inclusions (Fig. 3).

Page 5

Fig. 3. Histological sections of the fat body cells from workers of C. rimosus and M. parallelus and media workers of A. disciger and A. laevigata, stained by Sudan

Black B. Detail of C. rimosus parietal fat body (A) and perivisceral one (B). Parietal fat body (C) and perivisceral one (D) from M. parallelus. Detail of A. disciger

parietal fat body (E) and perivisceral one (F). Parietal fat body (G) and perivisceral one (H) from A. laevigata. t, trophocyte; n, trophocyte nucleus; , lipidic

inclusion in the trophocyte cytoplasm; o, oenocyte; *, oenocyte nucleus; , vacuoles in the oenocyte cytoplasm; , lipidic inclusion in the oenocyte

cytoplasm. Bars A–D; G–HZ20 mm E–FZ50 mm.

G.C. Roma et al. / Micron 37 (2006) 234–242238

In A. laevigata large inclusions are found around the nucleus

and the smaller ones appear distributed at the periphery of the

trophocytes (Fig. 3(G) and (H)). In C. rimosus, M. parallelus

and A. disciger the inclusions are distributed uniformly in the

whole cytoplasm (Fig. 3(A)–(F)).

In the derived species A. disciger and A. laevigata it is observed a

higher quantity of cytoplasmic inclusions in the trophocytes if

compared to the ones found in the basal species (Fig. 3).

The parietal and perivisceral oenocytes of media workers of

A. disciger and A. laevigata present lipidic inclusions

distributed in lower quantities when compared to the ones

found in the trophocytes cytoplasm of these species (Fig. 3(E)–

(H)). In workers of C. rimosus and M. parallelus it is observed

some lipidic inclusions in the cytoplasm of oenocytes,

however, in lower quantities when compared to workers of

species A. disciger and A. laevigata (Fig. 3(A)–(D)). In

workers of C. rimosus and A. laevigata, it is observed negative

vacuoles to the test (Fig. 3(A), (G) and (H)).

The trophocyte and oenocyte nuclei of all species studied

here are stained in red due to the contra-stain procedures with

Neutral Red (Fig. 3).

4. Discussion

The fat body of ant workers belonging to the species C.

rimosus and M. parallelus and media workers A. disciger and

Page 6

G.C. Roma et al. / Micron 37 (2006) 234–242 239

A. laevigata is found mainly in the gaster around the organs,

such digestive tract and ovaries, as well as close to the cuticle

(Roma et al., 2005), what matches the data found in the

literature regarding insects in general (Chapman, 1998; Gullan

and Cranston, 2000). The main cells observed in the fat body of

all species studied here were the trophocytes and oenocytes,

which are in close association with each other, corroborating

the results for virgin females of Pachycondyla striata ants

(Thiele and Camargo-Mathias, 2003).

Trophocytes are also called adipocytes, however, this

nomenclature is not appropriated since these cells are not

single lipid deposits, they present also a complex metabolism

and functions including protein and carbohydrate storages

besides its own lipids, previously absorbed from the

haemolymph. We adopted the term trophocyte in the present

work, since it refers to cells with reserves of nutrients, which

matches the histochemical results observed in workers

analyzed here and also in the trophocytes of minor workers

of Camponotus festinatus (Rosell and Wheeler, 1995) and

virgin females of P. striata (Thiele, 2001). This terminology is

still restricted for other functions presented by this cell as

synthesis and secretion of substances besides the detoxification

and excretion functions (Kilby, 1963; Isaac and Bowens,

1982).

For a better visualization, the histochemical tests results of

the fat body cells of workers analyzed here was summarized

comparatively in the Table 1.

The presence of proteins in the trophocytes cytoplasm was

detected in the workers of four species, data also registered in

minor workers of C. festinatus (Rosell and Wheeler, 1995;

Wheeler and Martinez, 1995). In C. rimosus, some perivisceral

trophocytes have presented higher quantity of proteins than

others of the same region, indicating probably that in these

cells the synthesis of proteins does not occur synchronically

and temporally in all cells. However, in all other studied

species, has not being observed any difference in the proteins

synthesis and storage among the trophocytes of the same

region.

The function of the present proteins in the trophocytes of the

workers analyzed here remains still uncertain. However, it can

be suggested that part of this proteic material is the rest of

insect immature phases (Dutkowski, 1974), since it is known

that during the larval development of such insects, certain

proteins synthesized by the trophocytes are released in the

haemolymph and when the insects go into pupate these proteins

can be reabsorbed by the same cells to be used as amino acid

source during the formation of new tissues in the adult (Dean et

al., 1985; Keeley, 1985; Gullan and Cranston, 2000).

The oenocytes of the ant species of this study also present

proteins in their cytoplasm, which matches data found for

oenocytes of bee workers (Cruz-Landim, 1985) and virgin

females of P. striata (Thiele, 2001). However, the presence of

protein in the oenocytes is not frequently registered in the

literature, therefore the participation of these elements in these

cells have not being definitively established, since the functions

performed by the oenocytes still generate discussions among

the researchers. For the species analyzed here as well as for the

other insects, it can be suggested that the oenocytes could be

involved in the synthesis of lipoproteic material, which will be

placed in the internal epicuticle of these insects for cuticle

maintenance process. Besides that, it can works like a source of

material for repairs in eventually damaged cuticles (Dielph,

1975; Hepburn, 1985).

The histochemical tests for polysaccharides detection

applied in the fat body cells of workers studied here have

revealed the presence of high quantities of neutral poly-

saccharides in the trophocytes of A. laevigata workers. This

fact indicates the possible production of these elements by

these cells or their capture of haemolymph in high rates. These

data would be expected due to the behaviour presented by these

workers as well as for all remaining species of leaf-cutting ants.

These insects cover long distances during the foraging,

building trails with hundreds or thousands of workers, specially

the genus Atta, going until the vegetal that will be inspected

and cut (Fowler, 1978). In Atta genus the foraging distances

varying from 21 m in A. capiguara (Fowler et al., 1986) to

235 m in A. cephalotes (Lewis et al., 1974).

These data suggest that the polysaccharides present in the

trophocytes of A. laevigata, as well as in leaf-cutting ants in

general, could be utilized as energy source, especially for

muscles of the legs during the food search for the colony.

Concerning M. parallelus, there was also found a high

quantity of polysaccharides in the trophocytes, indicating that

these elements would be also used as energy source for

foraging, however, for this species as well as for the other

basal ones, the covered distances in the search of resources

are lower than the distances achieved by the derived species,

with distances varying from 5 cm to 7 m around the nest

(Weber, 1972). Therefore, it is also suggested that the

trophocytes of M. parallelus workers could be involved in

the synthesis of lipids from non-lipidic precursors, as the

polysaccharides present in these cells. These data was also

reported for other insects such as Hyalophora cecropia

(Lepidoptera: Saturniidae) and Locusta migratoria (Orthop-

tera: Acrididae) (Candy and Kilby, 1975; Keeley, 1985)

A low quantity of polysaccharides has being observed in the

C. rimosus trophocytes. In this genus, the foraging area

normally does not go through from 20 to 40 cm around the nest

(Weber, 1972), which suggest that for these insects the needs of

energy for foraging are lower than in those species that cover

longer distances. Regarding the A. disciger workers, as

observed in A. laevigata, it would be expected to find high

quantity of polysaccharides in the trophocytes, since the ants of

this genus also build long trails with distances varying from

1.5 m for A. balzani to 110 m for A. crassispinus (Fowler et al.,

1986). However, only a fine cytoplasmic granulation has being

observed suggesting the low production of this element, or that

part of this material stored in the trophocytes is remains of the

youth phases of these insects that was not totally used during

the metamorphosis (Keeley, 1985). It is also suggested that in

the trophocytes of this species there is no polysaccharide

storage, therefore, these elements would be used immediately

by the insect, for instance, the trehalose, which is produced by

the trophocytes and instantaneously released in the

Page 7

Tab

le1

His

toch

emic

alre

sult

so

fth

efa

tb

od

yfr

om

the

wo

rker

so

fC

.ri

mosu

s,M

.p

ara

llel

us,

A.

dis

cig

eran

dA

.la

evig

ata

Cel

l

stru

cture

Pro

tein

sP

oly

sacc

har

ides

Lip

ids

C.

rim

osu

sM

.para

llel

us

A.

dis

ciger

A.

laev

igata

C.

rim

osu

sM

.para

llel

us

A.

dis

ciger

A.

laev

igata

C.

rim

osu

sM

.para

llel

us

A.

dis

ciger

A.

laev

igata

Tro

pho

cyte

12

12

12

12

12

12

12

12

12

12

12

12

Cyto

pla

smC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CK

KK

KK

KK

K

Gra

nu

les

or

incl

usi

ons

Xx

xx

CC

CC

CC

xx

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

Vac

uole

sK

KK

KK

Kx

xx

xx

xK

KK

KK

KK

KK

KK

K

Nu

cleu

sC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CK

KK

KK

KK

KK

KK

KK

KK

K

Oen

ocy

te1

21

21

21

21

21

21

21

21

21

21

21

2

Cyto

pla

smC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CK

KK

KK

KK

K

Gra

nu

les

or

incl

usi

ons

Xx

CC

CC

CC

xx

xx

CC

CC

CC

xx

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

Vac

uole

sX

xK

KK

KK

Kx

xx

xx

xx

xx

xx

xx

xK

K

Nu

cleu

sC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CK

KK

KK

KK

KK

KK

KK

KK

K

1,

par

ieta

lfa

tbody;

2,

per

ivis

cera

lfa

tbody;

CC

C,

stro

ngly

posi

tive;

CC

,in

term

edia

teposi

tive;

C,

wea

kly

posi

tive;

K,

neg

ativ

e;x

,n

ot

ob

serv

ed.

G.C. Roma et al. / Micron 37 (2006) 234–242240

haemolymph to be used for other tissues (Candy and Kilby,

1975; Keeley, 1985).

The low quantity of polysaccharides found in the

trophocytes of C. rimosus and A. disciger could still indicate

that other elements, besides these, would be used as energy

source necessary to the accomplishment of tasks performed by

these insects, as for instance, molecules of proteic and lipidic

nature (Keeley, 1985).

The oenocytes analysis of ants workers studied here has

showed these cells were weakly positive for polysaccharides,

excepting few granules strongly stained in C. rimosus,

A. disciger and A. laevigata. These data have suggested that

these elements have being produced and used by the own cell

for chemical reactions as catabolism or biosynthesis of several

cell molecules as reported for other insects (Candy and Kilby,

1975).

The histochemical test for lipids detection has revealed the

presence of these elements as cytoplasmic inclusions in the

trophocytes of workers of C. rimosus, M. parallelus,

A. disciger and A. laevigata. This fact matches the data

found for virgin females of P. striata ants (Thiele, 2001). On

the other hand, in queens of Apis mellifera during the transition

the newly emerged to oviposition phase, there was not

observed any lipidic deposit in the trophocytes (Cruz-Landim,

1985).

The presence of lipids in the fat body trophocytes confirms

one of the primordial functions of this tissue in insects, which is

the material storage, mainly of lipidic nature (Cruz-Landim,

1983). The lipidic material would be used by the workers

studied here as energy source by the trophocytes during the

foraging.

Further these functions, it is suggested that the lipids present

in the trophocytes of C. rimosus, M. parallelus, A. disciger and

A. laevigata could be also forming complexes with proteins,

since these elements were also observed in these cells. The

presence of lipoproteic elements in the trophocytes was

reported in larvae of Diatraea grandiosella (Lepidoptera:

Pyralidae) (Shelby and Chipendale, 1996), specifically the

lipophorin, a lipoprotein synthesized by the trophocytes that

participates in the cuticular hydrocarbon transport in the

haemolymph from its synthesis place, probably in the

oenocytes, until the epidermal cells, from where these elements

are secreted and transported through porous and wax channels

to the surface of epicuticle of the insect (Blomquist and

Dillwith, 1985). These processes could be occurring in the

species of ants studied here, since in these individuals, as well

as in insects in general, there is the cuticle impermeabilization

(Keeley, 1985).

The oenocytes of the workers studied here also present

lipidic inclusions, however, they are smaller than the ones

found in the trophocytes of these same species, which

matches the data obtained for the oenocytes of Gryllus

bimaculatus crickets (Romer, 1974) and virgin females P.

striata ants (Thiele, 2001). It is suggested that the presence

of lipids in the oenocytes of the species studied here, as well

as in the insects in general, is correlated to the synthesis of

waxes and hydrocarbons, deposited on the epicuticle for

Page 8

G.C. Roma et al. / Micron 37 (2006) 234–242 241

surface impermeabilization, thus avoiding the loss of water

and ions (Wigglesworth, 1970; Blomquist and Dillwith,

1985). Furthermore, the cuticular hydrocarbons acts in the

recognition among castes and species, since the species of

ants studied here, as well as other social insects, depend on

the sensorial olfactory or tactile perception obtained through

chemical compounds with a high tenor information and low

volatility as the cuticular hydrocarbons, which occurs on the

surface of the insect tegument (Blomquist and Dillwith,

1985).

Additionally, the lipids synthesized by the oenocytes could

act as precursors of pheromones (Blomquist and Dillwith,

1985) used in the trail demarcation by the workers of

A. disciger and A. laevigata.

Finally, based on the comparative analysis of the

histochemical tests obtained for the trophocytes and oenocytes

of the basal species of C. rimosus and M. parallelus, and

derived species of A. disciger and A. laevigata, it can be

inferred that no difference in the lipids and proteins synthesis

and storage has been detected among the trophocytes and

oenocytes of the parietal and perivisceral regions. However,

some researchers have described that the parietal region would

be mainly involved with the lipids synthesis and storage, while

the perivisceral region would be specialized in the proteins

synthesis and storage (Dean et al., 1985). However, it has being

observed differences related to the polysaccharides synthesis

and storage among the species studied here, since M. parallelus

and A. laevigata have presented more significant quantities of

polysaccharides in the trophocytes what has not being observed

in the other two analyzed species. Furthermore, it has being

observed more quantities of lipids in the trophocytes and

oenocytes of derived species when compared to the quantities

of lipids of the basal ones.

Thus through the histochemical tests applied in the fat body

cells of all species studied here, it was not observed significant

differences to lead philogenetic implications between basal and

derived species.

Acknowledgements

FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao

Paulo) Grant no: 04/01768-0 for the financial support. Eduardo

A. Diniz, Lucimeire de Souza Ramos and Itamar Cristina Reis

for the insects suplly. Cristiane Marcia Mileo, Gerson Mello

Souza, Rogilene Aparecida Prado and Rogerio Sueshiro Hatore

for the technical support.

References

Blomquist, G.J., Dillwith, J.W., 1985. In: Kerkut, G.A, Gilbert, L.I. (Eds.),

Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol. 3.

Pergamon Press, Oxford, pp. 117–154.

Bolton, B., 2003. Synopsis and Classification of Formicidae. Memoirs of the

American Entomological Institute, Gainesville.

Butterworth, F.M., Bodenstein, D., 1967. Adipose tissue of Drosophila

melanogaster. The effect of adult internal environment of growth, protein

deposition and histolysis in the larval fat body. The Journal of Experimental

Zoology 164, 251–266.

Camargo-Mathias, M.I., Caetano, F.H., 1996. Ultrastructural cytochemistry of

oenocytes of Pachycondyla villosa ants (Hymenoptera: Ponerinae). Acta

Microscopica 5 A, 6000–6003.

Candy, D.J., Kilby, B.A., 1975. Insect Biochemistry and Function. Chapman &

Hall, London.

Chapman, R.F., 1998. The Insects Structure and Function, fourth ed. Cambridge

University Press. Hodder and Stoughton Educational, New York.

Cruz-Landim, C., 1983. O corpo gorduroso da larva de Melipona

quadrifasciata anthidioides Lep. (Apidae: Meliponidae). Naturalia 8, 7–23.

Cruz-Landim, C., 1985. Modificacoes das celulas do corpo gorduroso de

rainhas de Apis mellifera L. (Hymenoptera: Apinae). Ciencia e Cultura 37,

471–475.

Dean, R.L., Locke, M., Collins, J.V., 1985. Structure of at body. In:

Kerkut, G.A, Gilbert, L.I. (Eds.), Comprehensive Insect Physiology,

Biochemistry and Pharmacology, vol. 3. Pergamon Press, Oxford,

pp. 155–210.

Dielph, P.A., 1975. Synthesis and released of hydrocarbons by the oenocytes of

the desert locust Shistocerca gregaria. Journal of Insect Physiology 21,

1237–1246.

Dutkwoski, A.B., 1974. Fat body of Galleria mellonella during metamorphosis.

Chemical and ultrastructural studies. Folia Histochemica et Cytochemica

12, 269–280.

Fowler, H.G., 1978. Foraging trails of leaf-cutting ants. Journal of the New

York Entomological Society 86, 132–136.

Fowler, H.G., Pereira-da-Silva, V., Forti, L.C., Saes, N.B., 1986. Population

dynamics of leaf-cutting ants: a brief review. In: Lofgren, C.S., Vander

Meer, R.K. (Eds.), Fire Ants and Leaf-Cutting Ants—Biology and

Management. Westview Press, Boulder, pp. 123–145.

Gullan, P.J., Cranston, P.S., 2000. The Insects: An Outline of Entomology.

Chapman & Hall, London.

Hepburn, H.R., 1985. Structure of the integument. In: Kerkut, G.A, Gilbert, L.I.

(Eds.), Comprehensive Insect Physiology, Biochemistry and Pharma-

cology, vol. 3. Pergamon Press, Oxford, pp. 1–58.

Holldobler, B., Wilson, E.O., 1990. The Ants. Harvard University Press,

Cambridge.

Isaac, P.G., Bownes, M., 1982. Ovarian and fat-body vitellogenin synthesis in

Drosophila melanogaster. European Journal of Biochemistry 123, 527–534.

Junqueira, L.C.U., Junqueira, L.M.M.S., 1983. Tecnicas basicas de citologia e

histologia. Santos, Sao Paulo.

Keeley, L.L., 1985. Physiology and biochemistry of the fat body. In:

Kerkut, G.A, Gilbert, L.I. (Eds.), Comprehensive Insect Physiology,

Biochemistry and Pharmacology, vol. 3. Pergamon Press, Oxford,

pp. 211–248.

Kilby, B.A., 1963. The biochemistry of the insect fat body. In:

Beament, J.W.L., Treherne, J.E., Wigglesworth, V.B. (Eds.), Advances in

Insect Physiology. Academic Press, New York, pp. 111–174.

Lewis, T., Pollard, G.V., Dibley, G.C., 1974. Rhythmic foraging in the leaf-

cutting ant Atta cephalotes (L.) (Formicidae: Attini). Journal of Animal

Ecology 43, 129–141.

Locke, M., 1984. The structure and development of the vacuolar system in the

fat body of insects. In: King, R.C., Akai, H. (Eds.), Insect Ultrastructure.

Plenum Press, New York, pp. 151–197.

Paes de Oliveira, V.T., Cruz-Landim, C., 2003. Size of fat body trophocytes and

the ovarian development in workers and queens of Melipona quadrifasciata

anthidioides. Sociobiology 41, 701–709.

Pearse, A.G.E., 1985. Histochemistry: Theoretical and Applied. Churchill,

London.

Roma, G.C., Correa-Bueno, O., Camargo-Mathias, M.I., 2005. Comparative

study of the fat body in some genera of the Attini tribe (Hymenoptera:

Formicidae). Sociobiology 45, 449–462.

Romer, T., 1974. Ultrastructural changes of the oenocytes of Gryllus

bimaculatus deg. (Salthatoria, Insecta) during the moulting cycle. Cell

and Tissue Research 151, 27–46.

Rosell, R.C., Wheeler, D.E., 1995. Storage function and ultrastructure of the

adult fat body in workers of the ant Camponotus festinatus (Buckley)

Page 9

G.C. Roma et al. / Micron 37 (2006) 234–242242

(Hymenoptera). International Journal of Insect Morphology and Embryol-

ogy 24, 413–426.

Shelby, K.S., Chippendale, G.M., 1996. Factors regulating lipophorin synthesis

by the larval fat body of the Southwestern corn borer, Diatraea

grandiosella. Journal of Insect Physiology 42, 643–648.

Thiele, E., 2001. Estudo morfo-histologico, morfometrico, histoquımico e

ultra-estrutural do corpo gorduroso de femeas virgens e rainhas de formigas

Pachycondyla striata (Hymenoptera: Ponerinae). Dissertacao (Mestrado

em Biologia Celular e Molecular). Instituto de Biociencias, Universidade

Estadual Paulista, Rio Claro, 104 pp.

Thiele, E., Camargo-Mathias, M.I., 2003. Morphology, ultramorphology and

morphometry of the fat body of virgin females and queens of the ants

Pachycondyla striata (Hymenoptera: Formicidae). Sociobiology 42,

243–254.

Weber, N.A., 1972. The gardening ants. The Attines. American Philosophical

Society, Philadelphia.

Wheeler, D.E., Martinez, T., 1995. Storage proteins in ants (Hymenoptera:

Formicidae). Comparative Biochemistry and Physiology 112, 15–19.

Wigglesworth, V.B., 1970. Structural lipids in the insect cuticle and the

function of the oenocytes. Tissue and Cell 2, 155–179.