Ultrastructural analysis of the fat body in workers of Attini ants (Hymenoptera: Formicidae)

© Koninklijke Brill NV, Leiden, 2009 DOI 10.1163/157075609X437745

Animal Biology 59 (2009) 241–262 brill.nl/ab

Ultrastructural analysis of the fat body in workers of Attini ants (Hymenoptera: Formicidae)

Gislaine C. Roma 1 , Odair C. Bueno 1,2 , Maria I. Camargo-Mathias 1,*

1 Departamento de Biologia 2 Centro de Estudos de Insetos Sociais

Instituto de Biociências, UNESP, Universidade Estadual Paulista, Avenida 24 A n° 1515, Rio Claro, SP, Brasil, CEP: 13506-900, C. P. 0199

Abstract In the present study a comparative morphological analysis of the fat body cells of ant workers of the basal Attini species Cyphomyrmex rimosus and Mycetarotes parallelus , and the derived species Acromyrmex disciger and Atta laevigata was conducted. Th e results revealed that the fat body is located mainly in the abdomen around organs (perivisceral) and near the integument (parietal). Th e main cells observed are spherical or polygonal trophocytes with a slightly rough surface. Th e oenocytes, another cell type found, are closely associated with trophocytes, and present a spherical or polygonal shape and a smoother surface. Th e mor-phometric analysis showed that the area of trophocytes and oenocytes of C. rimosus and M. parallelus is signifi cantly smaller when compared to those of A. disciger and A. laevigata . In the cytoplasm of parietal and perivisceral trophocytes and oenocytes, electronlucent droplets (probably lipids) and electrondense granules (probably proteins) indicate the participation of these cells in the storage of these elements, while digestive vacuoles, residual bodies, and multivesicular bodies suggest a role in intracellular digestion. In perivisceral trophocytes and oenocytes of C. rimosus , the presence of mitochondria, lamellar rough endoplasmic reticulum, and Golgi complex suggests that these cells synthesize proteins. Based on these data, no signifi cant diff erences were observed between the fat body cells of basal and derived ants, except regarding the larger size of trophocytes and oenocytes of the derived species A. disciger and A. laevigata . © Koninklijke Brill NV, Leiden, 2009

Keywords Trophocyte; oenocyte; morphology; morphometry; ultrastructure

Introduction

Th e Attini tribe consists of approximately 217 ant species that grow symbiotic fungi, the staple food of their colonies (Hölldobler and Wilson, 1990 ). Th ese species are distributed between basal genera, such as Cyphomyrmex, Mycetophylax, Mycocepurus, Myrmicocrypta, Mycetagroicus, Apterostigma, Sericomyrmex, Mycetosoritis, Mycetarotes

*) Corresponding author; e-mail: [email protected]

242 G.C. Roma et al. / Animal Biology 59 (2009) 241–262

and Trachymyrmex , and derived genera, such as Atta and Acromyrmex . Th e latter are commonly named leaf-cutting ants and are serious pests considered dominant herbiv-ores in the neotropics (Hölldobler and Wilson, 1990 ; Bolton, 2003 ). Th e genus Pseudoatta consists of parasite species (Bolton, 2003 ).

Th e fat body is a fi lling tissue that besides fi lls the cavities of the body is the site of intermediary metabolism (group of chemical reactions that occurs in the fat body to provide the necessaries of the own cell or of the organism) of several compounds. It also plays roles in the synthesis and storage of proteins, lipids, and carbohydrates (Palli and Locke, 1988 ), and is characterized by sheets or string of cells, usually one or two cells thick, or even forming small nodes in the hemocele connected to one another or to other tissues by tracheal branches (Keeley, 1985 ; Rosell and Wheeler, 1995 ; Chapman, 1998 ).

Trophocytes are the main cells of the insect fat body. Th ese cells may give rise to other cell types, as a result of a functional specialization, such as urate cells or urocytes, mycetocytes, and chromatocytes (Dean et al., 1985 ; Chapman, 1998 ).

Oenocytes, another cell type that constitutes the fat body, play roles in lipid meta-bolic processes (Keeley, 1985 ; Chapman, 1998 ).

Given the scarce data available on the internal morphology of ants of the basal and derived genera of the Attini tribe, the present study comparatively described the mor-phology, morphometry, and ultrastructure of parietal and perivisceral trophocytes and oenocytes of workers of the basal species Cyphomyrmex rimosus Spinola and Mycetarotes parallelus Emery and media workers of the derived species Acromyrmex disciger Mayr and Atta laevigata Smith, attempting to new information that could contribute to the understanding of their physiology and biology, and on diff erences that could lead to phylogenetic implications of this tribe.

Material and methods

Th e present study used the parietal and perivisceral fat body from workers of Cyphomyrmex rimosus and Mycetarotes parallelus (monomorphic species) and media workers of Acromyrmex disciger and Atta laevigata (polimorphic species), collected out-side the nests in the fi eld and immediately submitted to the techniques. Th e individuals were anesthetized by cooling in a freezer and dissected on Petri dishes with physiologi-cal saline solution for insects (NaCl 7.5 g/L, Na 2 HPO 4 2.38 g/L, KH 2 PO 4 2.72 g/L).

Scanning Electron Microscopy (SEM)

Th e parietal and perivisceral fat body from workers was removed, fi xed in Karnovsky for 24 hours and processed by Critical Point Drying, sputtered with gold and exam-ined by PHILIPS 505 SEM.

Morphometry

Th e morphometric analysis of trophocytes and oenocytes was performed using median histological sections. For the present study were utilized 05 individuals of each species.

G.C. Roma et al. / Animal Biology 59 (2009) 241–262 243

For each individual, were obtained area measurements (μm 2 ) of 10 parietal trophocytes and 10 perivisceral ones, and 10 parietal oenocytes and 10 perivisceral ones, with a LDMB LEICA microscope with the software Leica Qwin. Th e statistical test applied was the Tukey (p <0,05) (Siegel and Castellan, 1988) performed in order to verify signifi cant diff erences among the parietal and perivisceral trophocytes and among the parietal and perivisceral oenocytes in all species studied here. Besides this, the statistic test evaluated the signifi cant diff erences among the parietal and perivisceral trophocytes among individuals at the same species. Th e same procedure was applied to the oenocytes.

Ultrastructure - Transmission Electron Microscopy (TEM)

Th e fat body was fi xed in 2,5% glutaraldehyde fi xative solution in 0.1M cacodylate buff er (pH 7.2) for 2 hours, at 4°C. Post-fi xation was performed in 1% osmium tetrox-ide in 0.1M cacodylate buff er 0.1M (pH 7.2) for 2 hours, in the darkness. For contrast, the material was immersed in a solution of 2% uranyl acetate in 10% acetone, for 4 hours, in the darkness. Th en, the material was embedded in Epon resin diluted in acetone (1:1) for 12 hours, included in pure Epon resin and incubated at 60°C for 24 hours. After polymerized semithin sections were obtained with ultramicrotome Sorvall – Porter Blum MT2-B and stained with Azur II (1%) and Methylene Blue (1%).

Grids containing ultrathin sections of the material were contrasted with uranyl acetate and lead citrate for 45 minutes and 10 minutes, respectively. Th en, they were analysed and photographed in a PHILIPS CM 100 Transmission Electron Microscopy.

Results

Scanning Electron Microscopy

Th e morphological analysis of the fat body of workers of 1. C. rimosus and M. parallelus and media workers of A. disciger and A. laevigata shows that this tissue is characterized by a loose clump of cells located among organs (periv-isceral) and adjacent to the integument (parietal) ( Figs. 1B ; 2A , C , G ). In all species examined, parietal and perivisceral trophocytes are the main 2. cell types observed ( Figs. 1A – F ; 2B , D - H ). Th ese cells are mostly spherical in shape, but may be polygonal due to the cell arrangement that compresses cells against each other ( Figs. 1B , F ; 2B , E , G ). Th e surface of trophocytes of all worker species examined is slightly rough 3. ( Figs. 1F ; 2D ). In workers of 4. M. parallelus and A. laevigata , some trophocytes are ruptured, releasing large quantities of cytoplasmic granules of varied size and possibly contents ( Figs. 1D ; 2G ). In parietal and perivisceral fat bodies, round or spherical oenocytes are 5. dispersed among and associated with trophocytes. Oenocytes may also be


Figu

re 1

. Sc

anni

ng E

lect

ron

Mic

rosc

opy

(SEM

) of t

he fa

t bod

y ce

lls fr

om w

orke

rs o

f Cyp

hom

yrm

ex ri

mos

us a

nd M

ycet

arot

es pa

ralle

lus .

(A) D

etai

l of t

he C

. rim

osus

pa

rieta

l fat

bod

y ex

hibi

ting

troph

ocyt

e (t)

and

oeno

cyte

(o).

(B) G

ener

al v

iew

and

(C) d

etai

l of t

he C

. rim

osus

per

ivisc

eral

fat b

ody

show

ing

the o

enoc

yte m

embr

anou

s ex

pans

ion

( ➝ ).

(D, E

) Det

ail o

f the

par

ieta

l fat

bod

y fro

m M

. par

allel

us e

xhib

iting

the

gran

ules

in th

e cy

topl

asm

trop

hocy

te (w

hite

arr

ow).

(F) M

. par

allel

us p

eriv

is-ce

ral f

at b

ody

show

ing

troph

ocyt

e m

embr

anou

s exp

ansio

n ( ➝

) and

the

sligh

tly ro

ugh

surfa

ce o

f the

trop

hocy

tes.


polygonal in shape due to the compression of trophocytes ( Figs. 1A - F , 2B , D , E , G ). Th ese are smaller cells with a smoother surface and present in fewer numbers compared to trophocytes ( Figs. 1A – F ; 2B , D , E , G ). Among tissue cells, membranous expansions from trophocytes surround and 6. bind them to one another ( Figs. 1F ; 2F , H ). Similarly, these expansions may also originate in oenocytes extending to trophocytes and binding these two cell types ( Fig. 1C ).

Morphometry

Th e morphometric analysis of the fat body cells of workers reveals that the areas of parietal and perivisceral trophocytes of C. rimosus and M. parallelus are signifi cantly smaller compared to those of A. disciger and A. laevigata . Similarly, oenocytes of media workers of the derived species are signifi cantly larger than those of basal workers. However, no signifi cant diff erences were observed in the areas of parietal and perivis-ceral trophocytes of ants of the same species. Th e same was obtained for oenocytes.

Th e morphometric results of parietal and perivisceral fat body cells of workers exam-ined in this study are summarized in tables 1 and 2 .

Ultrastructure

Parietal and perivisceral trophocytes of all species exhibit individual or fused basal lamina when two or more adjacent trophocytes are observed. Th e same may also occur between trophocyte and oenocyte ( Figs. 3A , B , N ; 4A ; 5C , E , J ; 6F ; 7C , D ). In parietal and perivisceral trophocytes of A. laevigata , the plasma membrane reticular system (PMRS) ( Figs. 6F ; 7C ), result of membrane invaginations, is observed.

In parietal and perivisceral trophocytes, electronlucent droplets of varied shapes and sizes, probably containing lipids, are present ( Figs. 3C - E ; 4B , C ; 5C , F ; 6A , B , D , F , G ). In addition to these, very electrondense granules are observed in parietal tro-phocytes of M. parallelus , and perivisceral trophocytes of A. laevigata ( Figs. 4B ; 6E , H ), probably containing proteins.

Th e ultrastructural analysis of trophocytes of all species examined also shows the presence of digestive vacuoles ( Figs. 3G , H ; 6A , D , G ), characterized by structures delimited by membranes, containing compounds being digested, which may be cell organelles or endocytized molecules.

In perivisceral trophocytes of C. rimosus , mitochondria ( Figs. 3F , N ), lamellar rough endoplasmic reticulum ( Figs. 3H , N ), and Golgi complex ( Fig. 3F ) forming the syn-thesis apparatus of these cells, in addition to free ribosomes ( Fig. 3F ).

Th e nuclei of parietal and perivisceral trophocytes of A. disciger and A. laevigata are very irregular ( Figs. 5A , B , D ; 6A ). In C. rimosus and M. parallelus , nuclei of tro-phocytes are star or oval shaped ( Figs. 3A , C , E ).

Parietal and perivisceral oenocytes of the species examined exhibit individual or fused basal lamina in certain regions of adjacent oenocytes. Th e same is observed between oenocyte and trophocyte ( Figs. 3M , N ; 5J ; 7A , C , D ).

246 G.C. Roma et al. / Animal Biology 59 (2009) 241–262 Ta

ble

1.

Com

paris

on o

f ave

rage

are

a (μ

m 2 )

and

stand

ard

devi

atio

n of

the

troph

ocyt

es o

f the

par

ieta

l and

per

ivisc

eral

fat b

ody

of w

orke

rs o

f C. r

imos

us a

nd M

. par

allel

us a

nd

med

ia w

orke

rs o

f A. d

iscig

er a

nd A

. lae

viga

ta , t

hrou

gh T

ukey

test

(p <

0,05

).

C. r

imos

usM

. par

allel

usA.

disc

iger

A. la

evig

ata

12

12

12

12

Aver

age

area

(μm

2 )1.

774,

1 ±

720,

41.

770,

4 ±

647,

21.

480,

2 ±

346,

51.

647,

6 ±

431,

93.

876,

6 ±

1265

,64.

026,

8 ±

1174

,84.

664,

6 ±

1897

,74.

772,

1 ±

1830

,4

C. r

imos

us1

—ns

nsns

**

**

2ns

—ns

ns*

**

*

M. p

aral

lelus

1ns

ns—

ns*

**

*

2ns

nsns

—*

**

*

A. d

iscig

er1

**

**

—ns

**

2*

**

*ns

—*

*

A. la

evig

ata

1*

**

**

*—

ns

2*

**

**

*ns

—

(1) p

arie

tal t

roph

ocyt

es

(2) p

eriv

iscer

al tr

opho

cyte

s (*

) ave

rage

are

a w

ith si

gnifi

cant

diff

eren

ces a

mon

g th

e gr

oups

(p <

0,05

) (n

s) n

o sig

nifi c

ant d

iff er

ence

s am

ong

the

grou

ps

G.C. Roma et al. / Animal Biology 59 (2009) 241–262 247 Ta

ble

2.

Com

paris

on o

f ave

rage

area

(μm

2 ) an

d sta

ndar

d de

viat

ion

of th

e oen

ocyt

es o

f the

par

ieta

l and

per

ivisc

eral

fat b

ody

of w

orke

rs C

. rim

osus

and

M. p

aral

lelus

and

med

ia

wor

kers

of A

. disc

iger

and

A. l

aevi

gata

, thr

ough

Tuk

ey te

st (p

<0,

05).

C. r

imos

usM

. par

allel

usA.

disc

iger

A. la

evig

ata

12

12

12

12

Aver

age

area

(μm

2 )51

5,2

±15

5,9

596,

1 ±

199,

657

9,9

±15

8,2

601,

8 ±

134,

11.

926,

9 ±

452,

41.

636,

7 ±

484,

11.

622,

2 ±

349,

31.

591,

9 ±

387,

7

C. r

imos

us1

—ns

nsns

**

**

2ns

—ns

ns*

**

*

M. p

aral

lelus

1ns

ns—

ns*

**

*

2ns

nsns

—*

**

*

A. d

iscig

er1

**

**

—ns

**

2*

**

*ns

—*

*

A. la

evig

ata

1*

**

**

*—

ns

2*

**

**

*ns

—

(1) p

arie

tal t

roph

ocyt

es

(2) p

eriv

iscer

al tr

opho

cyte

s (*

) ave

rage

are

a w

ith si

gnifi

cant

diff

eren

ces a

mon

g th

e gr

oups

(p <

0,05

) (n

s) n

o sig

nifi c

ant d

iff er

ence

s am

ong

the

grou

ps


Figu

re 2

. Sc

anni

ng E

lect

ron

Mic

rosc

opy

(SEM

) of t

he fa

t bod

y ce

lls fr

om m

edia

wor

kers

of A

cro m

yrm

ex d

iscig

er an

d At

ta la

evig

ata .

(A) G

ener

al v

iew

of t

he A

. disc

iger

pa

rieta

l fa

t bo

dy

(fb)

and

(B)

deta

il of

th

e tro

phoc

ytes

(t)

an

d oe

nocy

tes

(o).

(C)

Gen

eral

vi

ew

of

the

A.

disci

ger

periv

iscer

al

fat

body

an

d (D

) det

ail,

exhi

bitin

g th

e inn

er as

soci

atio

n be

twee

n tro

phoc

ytes

(t) a

nd o

enoc

ytes

(o) a

nd th

e slig

htly

roug

h su

rface

of t

he tr

opho

cyte

s. (E

, F) D

etai

l of t

he A

. lae

viga

ta

parie

tal f

at b

ody,

show

ing

the t

roph

ocyt

e mem

bran

ous e

xpan

sion

( ➝ ).

(G) G

ener

al v

iew

and

(H) d

etai

l of t

he A

. lae

viga

ta p

eriv

iscer

al fa

t bod

y ex

hibi

ting

the g

ranu

les

in th

e tro

phoc

yte

cyto

plas

m (w

hite

arr

ow) a

nd th

e tro

phoc

yte

mem

bran

ous e

xpan

sion

( ➝ ).


Figu

re 3

. El

ectro

n m

icro

grap

h of

the f

at b

ody

cells

from

wor

kers

of t

he b

asal

spec

ies o

f Cyp

hom

yrm

ex ri

mos

us. (

A-D

) Det

ail o

f the

C. r

imos

us p

arie

tal t

roph

ocyt

es (t

) ex

hibi

ting

the

fusio

n of

bas

al la

min

a (b

l) fro

m a

djac

ent t

roph

ocyt

es. O

bser

ve th

e in

ner a

ssoc

iatio

n be

twee

n tro

phoc

ytes

and

cut

icle

(c),

star o

r ova

l sha

ped

nucl

eus

(n) a

nd th

e el

ectro

nluc

ent d

ropl

ets (

d). (

E-H

) C. r

imos

us p

eriv

iscer

al tr

opho

cyte

s sho

win

g th

e ov

al sh

aped

nuc

leus

(n),

Gol

gi c

ompl

ex (G

c) a

nd d

iges

tive

vacu

oles

(d

v). (

I-L)

Par

ieta

l oen

ocyt

es (o

) exh

ibiti

ng th

e rou

nd sh

aped

nuc

leus

(*) a

nd g

lyco

gen

depo

sits (

gl).

(M-P

) Per

ivisc

eral

oen

ocyt

es fr

om C

. rim

osus

. Obs

erve

the f

usio

n of

bas

al la

min

a (b

l) fro

m a

djac

ent o

enoc

ytes

(o) a

nd/o

r am

ong

oeno

cyte

s and

trop

hocy

tes (

t). ➝

= tr

opho

cyte

nuc

leol

us, m

i = m

itoch

ondr

ia, r

= fr

ee ri

boss

omes

, lre

r = la

mel

lar r

ough

end

opla

smic

retic

ulum

, rb

= re

sidua

l bod

ies.


Figu

re 4

. El

ectro

n m

icro

grap

h of

the

fat b

ody

cells

from

wor

kers

of t

he b

asal

spec

ies o

f Myc

etar

otes

para

llelu

s . ( A

, B ) D

etai

l of t

he M

. par

allel

us p

arie

tal t

roph

ocyt

es

exhi

bitin

g tw

o in

divi

dual

bas

al la

min

a (b

l) of

adj

acen

t tro

phoc

ytes

(t).

( C

, D )

M. p

aral

lelus

per

ivisc

eral

tro

phoc

ytes

. ( E-

H )

M. p

aral

lelus

par

ieta

l oen

ocyt

es (

o).

Obs

erve

the f

usio

n of

bas

al la

min

a (bl

) of t

wo

adja

cent

oen

ocyt

es w

ithou

t cyt

opla

sm fu

sion

and

the t

rach

eole

s (tr

) pre

senc

e. ( I

, J ) M

. par

allel

us p

eriv

iscer

al o

enoc

ytes

. m

i = m

itoch

ondr

ia, r

b =

resid

ual b

odie

s, gr

= e

lect

rond

ense

gra

nule

s , d

= e

lect

ronl

ucen

t dro

plet

s, dv

= d

iges

tive

vacu

oles

, * =

oen

ocyt

e nu

cleu

s.


Figu

re 5

. El

ectro

n m

icro

grap

h of

the f

at b

ody

cells

from

wor

kers

of t

he d

eriv

ed sp

ecie

s of A

crom

yrm

ex d

iscig

er . (

A-C

) Par

ieta

l tro

phoc

ytes

(t) e

xhib

iting

the i

rreg

ular

nu

cleu

s (n)

and

the t

wo

indi

vidu

al b

asal

lam

ina (

bl) f

rom

adja

cent

trop

hocy

tes.

(D-F

) Det

ail o

f the

A. d

iscig

er p

eriv

iscer

al tr

opho

cyte

s. O

bser

ve th

e nuc

leus

, the

bas

al

lam

ina a

nd el

ectro

nluc

ent d

ropl

ets (

d). (

G-I

) Par

ieta

l oen

ocyt

es (o

) fro

m A

. disc

iger

. Obs

erve

the r

ound

shap

ed n

ucle

us (*

), th

e mul

tives

icul

ar b

odie

s (m

b), o

enoc

ytes

nu

cleo

lus

( ➝ )

and

dige

stive

vac

uole

s (d

v).

(J,

K)

A. d

iscig

er p

eriv

iscer

al o

enoc

ytes

sho

win

g th

e tw

o in

divi

dual

bas

al l

amin

a of

the

adj

acen

t tro

phoc

ytes

and

oe

nocy

tes.


Figu

re 6

. El

ectro

n m

icro

grap

h of

the f

at b

ody

cells

from

wor

kers

of t

he d

eriv

ed sp

ecie

s of A

tta la

evig

ata .

(A-D

) A. l

aevi

gata

par

ieta

l tro

phoc

ytes

exhi

bitin

g th

e irr

egu-

lar n

ucle

us (n

) and

elec

tronl

ucen

t dro

plet

s (d)

. (E-

H) P

eriv

iscer

al tr

opho

cyte

s (t).

Obs

erve

the e

lect

rond

ense

gra

nule

s (gr

) pre

senc

e and

the f

usio

n of

the b

asal

lam

ina

(bl)

of tw

o tro

phoc

ytes

. dv

= di

gesti

ve v

acuo

les,

m =

mem

bran

ous r

emna

nts,

mi =

mito

chon

dria

, whi

te a

rrow

= p

lasm

a m

embr

ane

retic

ular

syste

m (P

MR

S).


Figu

re 7

. El

ectro

n m

icro

grap

h of

the

fat b

ody

cells

from

wor

kers

of t

he d

eriv

ed sp

ecie

s of A

tta la

evig

ata .

(A-C

) Par

ieta

l oen

ocyt

es (o

) fro

m A

. lae

viga

ta sh

owin

g th

e fu

sion

of th

e ba

sal l

amin

a (b

l) am

ong

oeno

cyte

s an

d tro

phoc

ytes

(t),

end

ocyt

ic v

esic

les (

ev)

and

plas

ma

mem

bran

e re

ticul

ar s

yste

m (

PMR

S) (

whi

te a

rrow

). (D

-G)

Periv

iscer

al oe

nocy

tes s

how

ing

the o

val s

hape

d nu

cleus

with

irre

gular

edge

s (*)

, the

fusio

n am

ong

basa

l lam

ina

of o

enoc

yte a

nd tr

opho

cyte

s and

dig

estiv

e vac

uoles

(dv)

.


In M. parallelus , parietal oenocytes show basal lamina with long prolongations with cytoplasm in their interior. Th ese structures, termed here membranous expansions, connect oenocytes to oenocytes, and oenocytes to trophocytes, without fusion of the cytoplasm of the involved cells ( Fig. 4E ).

In parietal oenocytes of A. laevigata the plasma membrane reticular system (PMRS) is also present ( Fig. 7B ).

In general, the cytoplasm of parietal and perivisceral oenocytes contains electronlu-cent droplets ( Figs. 3I , M , O ; 4G , J ; 5I , J ; 7A ), digestive vacuoles ( Figs. 3P ; 4H , I ; 5H ; 7F , G ), residual bodies ( Figs. 3K ; 4G ) and multivesicular bodies ( Figs. 5I , K ). In parietal oenocytes of A. laevigata , endocytic vesicles are also observed ( Fig. 7B ). In C. rimosus , these cells present lamellar rough endoplasmic reticulum ( Fig. 3P ), mito-chondria ( Figs. 3K , N ), and free ribosomes ( Fig. 3J ), in addition to abundant glycogen deposits ( Fig. 3L ). Th e nuclei of these cells are round or oval shaped ( Figs. 3I ; 4F ; 5G , H ; 7D ), and in some of them, the edges are irregular ( Figs. 3I ; 5G ; 7E ).

In the parietal fat body of M. parallelus , several tracheoles are also present ( Fig. 4F ). Comparative analyses of the ultrastructural features of parietal and perivisceral oen-

ocytes of workers of C. rimosus and M. parallelus and media workers of A. disciger and A. laevigata are summarized in tables 3 and 4 .

Discussion

Th e morphological, morphometric, and ultrastructural study on the fat body of work-ers of C. rimosus and M. parallelus (basal species) and media workers of A. disciger and A. laevigata (derived species) revealed that this tissue is located mainly in the abdomen around organs (perivisceral) and right underneath the integument (parietal), as described for gynes and queens of these same species (Roma et al., 2006b ) and insects in general (Chapman, 1998 ; Gullan and Cranston, 2000 ).

Th e morphometric analysis of the fat body cells of the examined species showed that the areas of parietal and perivisceral trophocytes are signifi cantly larger in workers of derived species compared to those of basal ones, confi rming the obtained by Roma et al. ( 2006b ) for gynes and queens of these same species. Th is suggests that during the evolution of these insects, there was a gradual increase in the volume of trophocytes of A. disciger and A. laevigata , probably due to greater presence of substances, such as proteins and lipids, and consequently an increase in their body size.

Regarding parietal and perivisceral oenocytes of workers, the morphometric results confi rm those above described for trophocytes, as oenocytes of basal species are signifi -cantly smaller than those of derived ones, also leading to an increase in the volume of cells of Attini ants during evolution.

No signifi cant morphometric diff erences were found between the areas of parietal and perivisceral trophocytes inside each species. Th e same was obtained for oenocytes. Th is suggests that in basal species as well as derived ones, there is not a functional com-partmentalization between trophocytes and oenocytes, parietal as well as perivisceral,


confi rming the reported by Ignatti ( 2001 ) and Paes de Oliveira and Cruz-Landim ( 2003 ) for other insect species.

Among parietal fat body cells of M. parallelus , the presence of several tracheoles may facilitate respiratory and oxidative processes occurring in the fat body (Chapman, 1998 ). Th is has also been reported for gynes and queens of Pachycondyla striata (Th iele and Camargo-Mathias, 2003 ) and queens of Atta sexdens (Ignatti, 2001 ). In addition, the ramifi cations of tracheoles in between fat body cells may help support this tissue, as it is extremely slack. Th e membranous expansions of trophocytes and oenocytes of the species examined in this study could also be playing this role, as well as binding fat body cells, as observed by Cruz-Landim ( 1976 ) in Apis mellifera bees. As a result of this arrangement, basal lamina may fuse, although the plasma membrane and the cyto-plasm of adjacent cells remain separated.

Roma et al. ( 2006b ) reported that these membranous expansions consisted of only membranous extensions. In the present study, however, cytoplasm was present in these expansions, indicating that they are extensions of the cell itself.

Th e ultrastructural analysis of the fat body cells showed fused basal lamina in certain regions or individual basal lamina between two trophocytes, between oenocytes and trophocytes, or between two oenocytes. In both cases, they may act as a selective per-meable barrier, controlling the transport of material in both directions (intra and extra-cellular) in oenocytes and especially in trophocytes, which are specialized in the synthesis and export of specifi c proteins that frequently cross the basal lamina. On the other hand, fused basal lamina may play a role in maintaining cells together, and con-sequently providing more cohesion to the tissue.

As already reported in the literature for other insects, the plasma membrane reticular system (PMRS) was present in parietal and perivisceral trophocytes of A. laevigata . Th is structure is a result of membrane invaginations forming channels that penetrate the cytoplasm, and consequently increase the surface area for absorbing compounds, mainly proteins, from the hemolymph. Th ese data confi rm those obtained by Ignatti ( 2001 ) for trophocytes of A. sexdens .

Th e present study supports the hypothesis that perivisceral trophocytes of workers of C. rimosus are responsible for an intense protein synthesis, since they have the entire apparatus to perform this activity. Th is is contrary to the previously proposed by Roma et al. ( 2006a ) that proteins observed in trophocytes of workers of these species are a leavings not used during the metamorphosis.

Th e presence of electrondense granules, possibly containing proteins, in trophocytes of other species analyzed here also suggests that these cells could be a site of protein synthesis, or that proteins are being absorbed from the hemolymph.

In addition, the ultrastructural analysis of parietal and perivisceral trophocytes revealed the presence of electronlucent droplets in the cytoplasm, probably lipid source. Th is has been reported for trophocytes of other insects by Kilby ( 1963 ), which sug-gested, similarly to Roma et al. ( 2006a ), that these structures could provide the energy needed during foraging activities of workers. Based on the literature (Zara et al., 2003 ) fat body cells, especially trophocytes, are thought to be the center of the intermediary


ble

3.

Ultr

astr

uctu

re fe

atur

es o

f the

par

ieta

l and

per

ivisc

eral

fat b

ody

cells

from

wor

kers

of C

ypho

myr

mex

rim

osus

and

Myc

etar

otes

para

llelu

s

Spec

ies

Stru

ctur

es

Trop

hocy

tes

Oen

ocyt

es

Pari

etal

Peri

visc

eral

Pari

etal

Peri

visc

eral

Bas

al L

amin

a- i

ndiv

idua

l and

/or f

used

to th

e ot

her t

roph

ocyt

es

an

d/or

oen

ocyt

es

(F

igs.

3A, B

)

- ind

ivid

ual a

nd/o

r fus

ed

to

the

othe

r tro

phoc

ytes

and/

or o

enoc

ytes

- ind

ivid

ual a

nd/o

r fus

ed to

the

othe

r tro

phoc

ytes

and/

or o

enoc

ytes

- ind

ivid

ual a

nd/o

r fus

ed

to

the

othe

r tro

phoc

ytes

and/

or o

enoc

ytes

(Fig

s. 3M

, N)

C.

rim

osu

sC

ytop

lasm

- ele

ctro

nluc

ent d

ropl

ets

fro

m d

iff er

ent s

hape

s

and

sizes

, som

e of

them

app

aren

tly

un

derg

oing

fusio

n

(Fig

s. 3C

, D)

- ele

ctro

nluc

ent d

ropl

ets

(F

ig. 3

E)- d

iges

tive

vacu

oles

(Fig

s. 3G

, H)

- mito

chon

dria

(Fig

. 3F)

- Gol

gi c

ompl

ex (F

ig. 3

F)- f

ree

ribos

omes

(Fig

. 3F)

- lam

ella

r rou

gh e

ndop

lasm

ic

re

ticul

um (F

igs.

3F, H

)

- ele

ctro

nluc

ent d

ropl

ets

(F

ig. 3

I)- r

esid

ual b

odie

s (Fi

g. 3

K)

- mito

chon

dria

(Fig

. 3K

)- f

ree

ribos

omes

(Fig

. 3J)

- gly

coge

n de

posit

s

(Fig

. 3L)

- ele

ctro

nluc

ent d

ropl

ets

(F

igs.

3M, O

)- m

itoch

ondr

ia (F

ig. 3

N)

- lam

ella

r rou

gh en

dopl

asm

ic

re

ticul

um (F

ig. 3

P)- d

iges

tive

vacu

oles

(Fig

. 3P)

Nuc

leus

- sta

r or o

val s

hape

d

(Fig

s. 3A

, C)

- sta

r or o

val s

hape

d w

ith

ev

iden

t nuc

leol

us

(F

ig. 3

E)

- ova

l sha

ped

with

irre

gula

r

edge

s (Fi

g. 3

I)- r

ound

or o

val s

hape

d

(Con

tinue

d )


ble

3. (C

ont.)

Ultr

astr

uctu

re fe

atur

es o

f the

par

ieta

l and

per

ivisc

eral

fat b

ody

cells

from

wor

kers

of C

ypho

myr

mex

rim

osus

and

Myc

etar

otes

para

llelu

s

Spec

ies

Stru

ctur

es

Trop

hocy

tes

Oen

ocyt

es

Pari

etal

Peri

visc

eral

Pari

etal

Peri

visc

eral

Bas

al L

amin

a- i

ndiv

idua

l and

/or f

used

to th

e ot

her t

roph

ocyt

es

an

d/or

oen

ocyt

es

(Fig

. 4A)

- ind

ivid

ual a

nd/o

r fus

ed

to

the

othe

r tro

phoc

ytes

and/

or o

enoc

ytes

- with

mem

bran

ous

ex

pans

ion

whi

ch c

onne

ct

to th

e ot

her o

enoc

ytes

or

tro

phoc

ytes

, how

ever

with

out c

ytop

lasm

ic

fusio

n (F

ig. 4

E)

- ind

ivid

ual a

nd/o

r fus

ed

to

the

othe

r tro

phoc

ytes

and/

or o

enoc

ytes

M.

pa

rall

elu

s

Cyt

opla

sm- e

lect

ronl

ucen

t dop

lets

fro

m d

iff er

ent s

hape

s

and

sizes

, som

e of

them

appa

rent

ly

un

derg

oing

fusio

n

(Fig

. 4B)

- ele

ctro

nden

se g

ranu

les

(

Fig.

4B)

- mito

chon

dria

(Fig

. 4A)

- ele

ctro

nluc

ent d

ropl

ets

fro

m d

iff er

ent s

hape

s

and

sizes

, som

e of

them

app

aren

tly

un

derg

oing

fusio

n

(Fig

. 4C

)- m

itoch

ondr

ia (F

ig. 4

D)

- ele

ctro

nluc

ent d

ropl

ets

(F

ig. 4

G)

- dig

estiv

e va

cuol

es (F

ig. 4

H)

- res

idua

l bod

ies (

Fig.

4G

)

- ele

ctro

nluc

ent d

ropl

ets

(F

ig. 4

J)- d

iges

tive

vacu

oles

(Fig

. 4I)

Nuc

leus

- sta

r sha

ped

- sta

r sha

ped

- rou

nd o

r ova

l sha

ped

(F

ig. 4

F)- r

ound

or o

val s

hape

d


ble

4.

Ultr

astr

uctu

re fe

atur

es o

f the

par

ieta

l and

per

ivisc

eral

fat b

ody

cells

from

med

ia w

orke

rs o

f Acr

omyr

mex

disc

iger

and

Atta

laev

igat

a

Trop

hocy

tes

Oen

ocyt

es

Spec

ies

Stru

ctur

esPa

riet

alPe

rivi

scer

alPa

riet

alPe

rivi

scer

al

Bas

al L

amin

a- i

ndiv

idua

l and

/or f

used

to th

e ot

her t

roph

ocyt

es

an

d/or

oen

ocyt

es

(F

ig. 5

C)

- ind

ivid

ual a

nd/o

r fus

ed

to

the

othe

r tro

phoc

ytes

and/

or o

enoc

ytes

(Fig

. 5E)

- ind

ivid

ual a

nd/o

r fus

ed

to

the

othe

r tro

phoc

ytes

and/

or o

enoc

ytes

(Fig

. 5G

)

- ind

ivid

ual a

nd/o

r fus

ed

to

the

othe

r tro

phoc

ytes

and/

or o

enoc

ytes

,

thin

ner t

he tr

opho

cyte

s

(Fig

. 5J)

A.

dis

cige

rC

ytop

lasm

- ele

ctro

nluc

ent d

ropl

ets

fro

m d

iff er

ent s

hape

s

and

sizes

(Fig

. 5C

)

- ele

ctro

nluc

ent d

ropl

ets

fro

m d

iff er

ent s

hape

s

and

sizes

(Fig

. 5F)

- ele

ctro

nluc

ent d

ropl

ets

(F

ig. 5

I)- d

iges

tive

vacu

oles

(Fi

g. 5

H)

- mul

tives

icul

ar b

odie

s

(F

ig. 5

I)

- ele

ctro

nluc

ent d

ropl

ets

(F

ig. 5

J)- m

ultiv

esic

ular

bod

ies

(

Fig.

5K

)

Nuc

leus

- ext

rem

ely

ram

ifi ed

, with

fi ne

and

long

prol

onga

tions

(Fig

s. 5A

, B)

- ext

rem

ely

ram

ifi ed

, with

fi ne

and

long

prol

onga

tions

(Fig

. 5D

)

- rou

nd o

r ova

l sha

ped,

som

e w

ith ir

regu

lar l

imit

(F

igs.

5G, H

)- e

vide

nt n

ucle

olus

(Fi

g. 5

G)

- rou

nd o

r ova

l sha

ped

(Con

tinue

d )


ble

4. (C

ont.)

U

ltras

truc

ture

feat

ures

of t

he p

arie

tal a

nd p

eriv

iscer

al fa

t bod

y ce

lls fr

om m

edia

wor

kers

of A

crom

yrm

ex d

iscig

er a

nd A

tta la

evig

ata

Trop

hocy

tes

Oen

ocyt

es

Spec

ies

Stru

ctur

esPa

riet

alPe

rivi

scer

alPa

riet

alPe

rivi

scer

al

Bas

al L

amin

a- i

ndiv

idua

l and

/or f

used

to th

e ot

her t

roph

ocyt

es

an

d/or

oen

ocyt

es

- ind

ivid

ual a

nd/o

r fus

ed

to

the

othe

r tro

phoc

ytes

and/

or o

enoc

ytes

(Fig

. 6F)

- ind

ivid

ual a

nd/o

r fus

ed

to

the

othe

r tro

phoc

ytes

and/

or o

enoc

ytes

(Fig

s. 7A

, C)

- ind

ivid

ual a

nd/o

r fus

ed

to

the

othe

r tro

phoc

ytes

and/

or o

enoc

ytes

(Fig

. 7D

)

A.

laev

iga

taC

ytop

lasm

- ele

ctro

nluc

ent d

ropl

ets

fro

m d

iff er

ent s

hape

s

and

sizes

, som

e of

them

appa

r ent

ly u

nder

goin

g

fusio

n (F

igs.

6A, B

, D)

- ele

ctro

nluc

ent d

ropl

ets

(F

igs.

6F, G

)- e

lect

rond

ense

gra

nule

s

Figs

. 6E,

H)

- ele

ctro

nluc

ent d

ropl

ets

(F

ig. 7

A)- e

ndoc

ytic

ves

icle

s

(Fig

. 7B)

- dig

estiv

e va

cuol

es

(F

igs.

7F, G

)

- dig

estiv

e va

cuol

es

(F

igs.

6A, D

)- m

embr

anou

s rem

nant

s

(Fig

. 6C

)

- dig

estiv

e va

cuol

es

(F

ig. 6

G)

- mito

chon

dria

(Fig

. 6H

)

Nuc

leus

- irr

egul

ar, e

xtre

mel

y

ram

ifi ed

, with

fi ne

and

long

pro

long

atio

ns

(F

ig. 6

A)

- irr

egul

ar, e

xtre

mel

y

ram

ifi ed

, with

fi ne

and

long

pro

long

atio

ns

- rou

nd o

r ova

l sha

ped

- rou

nd o

r ova

l sha

ped,

som

e w

ith ir

regu

lar

ed

ges (

Fig.

7E)


metabolism of insects, providing lipids and proteins for other tissues. Th us, in the species examined in this study, lipids may be mobilized from trophocytes to the hemo-lymph for the maintenance of their metabolism.

Regarding the ultrastructure of oenocytes, a well-developed lamellar rough endo-plasmic reticulum was observed in C. rimosus . Th is suggests that these cells may syn-thesize proteins, supporting cytochemical and ultrastructural studies reporting the presence of protein granules in their cytoplasm (Roma et al., 2008 ). On the other hand, our fi ndings contradict studies reporting that oenocytes are characterized mainly by extensive areas of smooth endoplasmic reticulum, due to the synthesis of ecdyster-oids (Dean et al., 1985 ), as this organelle was not observed in the present study. It is also possible that oenocytes absorb proteins from the hemolymph, since plasma mem-brane invaginations (PMRS) were observed, in addition to endocytic vesicles in the parietal oenocytes of A. laevigata .

Th e cytoplasm of parietal and perivisceral oenocytes of all species examined contained electronlucent droplets that, according to Blomquist and Dillwith ( 1985 ), consist of lipids and may contain hydrocarbons. Th ese compounds are eventually deposited in the insect cuticle for impermeability, used in the maintenance and recognition of castes, in addition to serving as precursors of pheromones used in scent trails, often long, especially in derived species (Blomquist and Dillwith, 1985 ).

In the parietal oenocytes of C. rimosus , abundant glycogen deposits were found, sug-gesting the participation of this compound as a source of energy for several processes, such as the biosynthesis or catabolism of several cell molecules (Candy and Kilby, 1975 ). In workers of C. rimosus examined in this study, glycogen may be providing energy mainly for muscles during foraging activities.

In trophocytes of C. rimosus and A. laevigata and in oenocytes of C. rimosus , M. parallelus , A. disciger , and A. laevigata , many digestive vacuoles of varied shapes and sizes were observed. Th ey might be associated with protein digestion, since part of these elements may have an exogenous origin, from the hemolymph, as mentioned previously. Similarly, digestive vacuoles may also be autophagic, responsible for autodi-gestion, a process that allows fat body cells to maintain well-functioning components (Dean et al., 1985 ).

Th e residual bodies found in parietal oenocytes of C. rimosus and M. parallelus could also play a role in digestive processes of compounds from the hemolymph or cell organelles (Dean et al., 1985 ).

Th e presence of multivesicular bodies in the parietal oenocytes of A. disciger might be involved mainly in the recycling of membranes and digestion of extracellular pro-teins, which are used in several metabolic processes, as reported for other insects (Locke, 1984 ; Dean et al., 1985 ).

Based on the presented data, no signifi cant diff erences were observed between the fat body cells of basal and derived ants, except regarding the larger size of trophocytes and oenocytes of the derived species A. disciger and A. laevigata . Th is suggests that the structural and functional features of this tissue remained unaltered during the evolu-tion of this group of insects.


Acknowledgments

Th is work was fi nanced by the FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo), Grant n° 04/01768-0. We are grateful to CEIS (Centro de Estudos de Insetos Sociais) and Eduardo Arrivabene Diniz for the insects supply and to Antonio Teruyoshi Yabuki, Mônica Iamonte and Rogerio Sueshiro Hatore for the technical support.

References

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Ultrastructural analysis of the fat body in workers of Attini ants (Hymenoptera: Formicidae)

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