Insect Bwchem Molec Bwl Vol 23, No 2, pp 297-302, 1993 0965-1748/93 $6 00 + 0 00 Printed m Great Britain All rights reserved Copyright © 1993 Pergamon Press Ltd

Subcellular Location of the A 12 Desaturase Rules Out Bacteriocyte Contribution to Linoleate Biosynthesis in the House Cricket and the American Cockroach CHARLOTTE E. BORGESON,* GARY J. BLOMQUIST*

Received 13 May 1992; revised and accepted 27 August 1992

Subcellular membranes from cricket whole-body homogenates and cockroach fat body homogenates were separated by differential and isopycnic centrifugation. Marker enzymes, phosphatidylcholine glyceride transferase, Na+/K+-ATPase and H+-ATPase, were assayed to establish the identity of the endoplasmic reticulum, plasma membrane and mitochondria, respectively. Electron microscopy of the separate membrane fractions revealed distinct morphological differences in each fraction. The A 12 desaturase activity was found to comigrate with the lightest fraction, the endoplasmic reticulum, while the endosymbiotic bacteria were recovered in the cell debris and mitochondrial fractions. These results provide definitive evidence that the A 12 desaturase activity in Acheta domesticus and Periplaneta americana is of insect origin and not due to endosymbiotic bacteria. This is also the first reported separation of a microsomal fraction from insects into endoplasmic reticulum and plasma membranes.

Essential fatty acid Llnoleic acid Insect hpld biosynthesis Endosymblotic bacteria

INTRODUCTION

A number of insect species have been shown to biosyn- thesize linoleic acid, 18 : 2(n-6), ( (Z,Z)-9,12-octacosa- dienoic acid) de novo (Cripps et al., 1986), a fatty acid long considered an essential nutrient for animals. Early reports of linoleate biosynthesis in insects (Louloudes et al., 1961; Strong, 1963; Mauldln et al., 1972) were discounted due to criticisms of the chromatographic techniques employed to isolate 18:2(n-6) and the possi- bility that linoleic acid was produced in insects by endosymbiotlc bacteria (Downer, 1978). By the early 1980s, radiochromatographic techniques and mass spectrometry showed that the physiologically important n-6 isomer of 18:2 was being formed in insects (Dwyer and Blomquist, 1981; Blomquist et al., 1982). In ad- dition, experiments in which tissues were incubated with substrate m axenic media lent strong support to the hypothesis that in certain insect species, especially those that do not contain intracellular symbiotic bacteria (e.g. Acheta domesticus), the A 12 desaturase responsible for the synthesis of 18.2 from 18:1 is of insect origin (Borgeson et al., 1990). However, in those insect species that do house symbiotic microorganisms, the posslbtlity

*Department of Blochemtstry, Umverslty of Nevada, Reno, NV 89557-0014, U S A

remained that microorganisms were responsible for the A ~2 desaturase activity.

One way to determine whether an insect relies on endosymbiotic bacteria for the synthesis of 18:2(n-6) would be to render the insect asymbiotic and then assay in vivo for continued linoleic acid biosynthesis. Further corroborating evidence would be obtained if the bacteria could be isolated and assayed for the presence or absence of the A 12 desaturase. Many attempts have been made to obtain endosymbiont-free insects, as well as to culture the bacteria, but none have been demonstrably success- ful (Brooks, 1970). A different strategy to rule out the contribution of microorganisms to the biosynthesis of 18:2(n-6) in insect tissue is to separate subcellular membrane fractions and subsequently determine the locatton of A 12 desaturase activity. If the activity is shown to be limited to the endoplasmic reticulum, then the activity must be of insect origin as bacteria do not have internal subcellular membranes. Furthermore, during this separation of subcellular membranes, the fate of the endosymbionts in the preparation can be deter- mined and assayed with respect to A 12 desaturase ac- tivity. In this paper, we present the results of isopycnic separation of intracellular membranes from the cricket and the cockroach and show that the A 12 desaturase comigrates with the marker enzyme for the endoplasmic reticulum. Furthermore, electron micrographs of the cell fractions indicate that m these preparations the

297

298 CHARLOTTE E BORGESON

symbiotic bacterta are found m the 14,000g pellet, a fractton showmg no desaturase activity.

MATERIALS AND METHODS

Chemicals

[1-J4C]Oleic acid (18:1) (52 mCi/mmol) was obtamed from New England Nuclear, Dupont, Boston, Mass. Oleoyl-chlortde was obtained from Nuchek Preps Inc., Elysian, Mmn. [1-14 C]Oleoyl-CoA was synthesized es- sentially as described by Bergstrom and Reitz (1980). CoA, NADPH, NADH, Tris and sucrose were obtained from Sigma. Acetonitrile (HPLC grade) was obtamed from Fisher. All other chemicals were of reagent grade; all solvents except diethyl ether were redistilled before use .

Preparauon of membranes

Crickets were obtained from Fluker's Cricket Farm, Baton Rouge, La. Male penultimate stadium cricket nymphs were crushed wtth a mortar and pestle in 0.25 M sucrose, 0.1 M Tris-HC1 buffer, pH 7.2, and the resul- tant homogenate was filtered through cheesecloth. Cock- roaches, Periplaneta americana, were reared in metal garbage cans and fed Purma dog chow and water ad libttum. Fat bodies were dissected from adult males and homogenized with a Potter glass-Teflon homogenizer m the same buffer. The homogenates were centrifuged for 5 mm at 500g, then for 10 min at 1400g, yielding the low speed pellet. The cricket supernatant was centrifuged for 20 mm at 14,000 g, yielding the mttochondrial pellet. The resultant supernatant was layered onto a linear sucrose gradient (20-42% sucrose [w/v]), and centrifuged for 12 h at 100,000g. The cockroach 1400g supernatant was layered onto a linear sucrose gradient (20-48%) and also centrifuged at 100,000g for 12h Fracttons were collected. Aiiquots were assayed for A ~2 desaturase ac- ttvity immediately. A portion of each sample was frozen in liquid N 2 and kept at - 8 0 ° C for subsequent marker enzyme and protein assays.

Enzyme assays

ATPase acttvities were assayed by momtormg phos- phate release in the presence and absence of vanadate and azide as descrtbed (Bowman and Bowman, 1988) with some modification. The reaction mtxtures con- tained 5mM MgC12, 5mM ATP, 10mM NH4C1, 10mM Tris, 100mM NaC1 and 50mM KCI Other addmons are noted in the figure legends. The reacttons were terminated and free phosphate determined as descrtbed (Bowman and Bowman, 1988). Phosphatidyl- choline glyceride transferase was detected by the incor- poration of [methyl-~4 C]CDP-choline into phosphat~dyl- choline as descrtbed (Lord et al., 1972). The A j-~ desat- urase activtty was determined as described by Cripps et al. (1986) with some modtficatton. The fractions were incubated wtth 14nmol [1-J4C]oleoyl-CoA (400,000 dpm), 2.4 mM NADPH and 0 1 M Tris-HC1 (pH 7.2) m a final volume of 1 ml in a shaking water bath

and G A R Y J BLOMQUIST

at 30°C for 30 min. The reactions were terminated by the addition of 5 ml of chloroform : methanol (2: 1).

Ltptd analysis

Total lipids from the m 12 desaturase reactions were extracted accordmg to Bhgh and Dyer (1959), with polar lipids, diacylglycerols, free fatty acids and trlacylglyc- erols in the lower chloroform phase and the acyl-CoAs in the upper aqueous phase. The aqueous phase was washed three ttmes with chloroform and the chloroform phases were pooled for each sample. The chloroform phase was removed under a stream of nitrogen m a sand bath at 55°C and brought to l ml with chloro- form:ethanol (2:1). An ahquot was assayed for radio- activity m 10 ml dtphenyloxazol (0.4%) m toluene in a Beckman liquid scmttllation counter. Solvent was re- moved under a stream of nttrogen and the remaining sample was separated into liptd classes by thin layer chromatography on slhca gel H plates developed in solvent system I: hexane:diethyl ether:formic acid (80: 20' 2). The polar hpids were hydrolyzed and methyl- ated with BF3-methanol as descrtbed (Crtpps et al., 1986) or with dtazomethane. The fatty acid methyl esters were separated by radto-high-performance hqutd chromatog- raphy (radio-HPLC) on a Supelco LC-8 (5/~) reverse- phase column with acetomtri le:H20 (80:20) as the mobtle phase. Radiolabeled methyl esters were detected by a Radto-matic Instruments Flo-one/Beta flow- through liquid scmttllatton counter with Scmt~ Vers LC as the scmtillation cocktad.

Electron mtcroscopy

Cell debrts and mttochondrial fracttons obtained by differential centrlfugatton described above and endo- plasmlc reticulum and plasma membranes obtained from sucrose gradients above were washed with cacodylate buffer, than fixed for 2h m 3% glutaraldehyde/

0 75

0 50

[ ~ Mlto ATPose

PM AT'Pose

PCGTase

>~ 0 25

L 0 00 1K xg 12K xg 40K xg lOOK xg

Pellets recovered from differential centnfu(Jotion

F I G U R E 1 Distribution of marker enzymes following differential centnfugatlon of cricket homogenate Achv]ty of mltochondnal H +- ATPase was measured as the difference between reactions in the

absence and presence of 5 mM azlde, with 10 pM vanadate m both, at pH 8 3 Peak mltochondnal ATPase activity was 0 025 pmol P, mm -) mg i Actw]ty of plasma membrane Na+/K+-ATPase was measured as the difference between reactions m the absence and presence of 10pM vanadate, with 5 mM azlde m both. at pH 7 0 Peak plasma membrane ATPase was 0065/1mol P, mm i mg ] PCGTase

measured as described m the Materials and Methods section had a maximal activity of 5 60 pmol mm- L mg-

A t2 DESATURASE ACTIVITY 299

cacodylate buffer. The fractions were washed again with cacodylate buffer, then post-fixed for 1 h in 2% osmium tetroxide/cacodylate buffer. The fractions were washed again with cacodylate buffer, then suspended in low temperature agar, 2% aq. The fractions were dehydrated in acetone:propylene oxide series, then infiltrated with resin. After polymerization, the ultrathin sections were cut w~th a Sorval Porter-Blum ultramicrotome, stained with 8% uranyl acetate and 2% lead citrate and exam- ined at 80 kV.

RESULTS

Differential centrifugation separation of membranes

Initially, whole penultimate mstar male crickets were homogenized in 0.1 M Tris buffer and the homogenate was subjected to differential centrifugation. Pellets re- sulting from these centrifugations were assayed for activities of various enzymes found to be specific for mitochondria, plasma membranes and endoplasmic reticulum in other systems. The results of assays on a typical preparation indicate that azide-sensitive H ÷- ATPase activity, found predominantly in the 12,000g pellet and vanadate-sensitive Na+/K+-ATPase activity, found in the higher speed pellets, were good candidates for marker enzymes for mitochondrial and plasma mem- brane fractions, respectively (Fig. 1). Cyanide-insensitive N A D P H :cytochrome c reductase, an enzyme specific to the ER in plants (Lennarz, 1970), rat liver (Beaufay et al., 1974), fungi (Borgeson and Bowman, 1983) and the tobacco hornworm (Weirich and Adams, 1984), proved to be an unreliable marker in fractions from the cricket. Phosphatidylcholine glyceride transferase (PCGT), the last enzyme in one pathway of phospha-

1.6t

E,.L[ ~ m ~ o . 8 ~

i~o.4 0.0 ¢

~ 4

g ~ r, 2

I <1

/ O, ooo.o/ O.oo/ I I I I I I I I

A , 2 o _ t . . . .

&xA

-'---'- ~ ,

2 4 6 8 10 12 14 16 18 20 Froction Number

0.2

~7 ;? 0.1c~ ' ~'~-

?i 0

0.0

FIGURE 3 ATPase, PCGTase and A ~2 desaturase actwmes m frac- tions after sucrose density fractmnatlon of cockroach fat body hom- ogenate After isopycmc centnfugatmn of the 1400g supernatant on a sucrose gra&ent, PCGTase actwlty (A) was separated from Na+/K+-ATPase actwlty (O) A lz desaturase actwlty (A) comlgrated

with the PCGTase

tidylchohne biosynthesis, is specifically found on the ER in many organisms (Borgeson and Bowman, 1983; Lord et al., 1973; Van Golde et al., 1971). In the cricket subcellular fractions, PCGT activity was found in the high speed fractions, suggesting that it would be useful as a marker for ER in the cricket as well. The data in Fig. 1 show that the 12,000 g pellet was enriched for mitochondria; however, separation of ER and PM was not accomplished as indicated by approximately equal amounts of both Na+/K+-ATPase and PCGT in each of the two higher speed pellets.

10

E 6

8 ~ 4 O- _

~ 2 ,4

o

8

~ 4

0.3

ER 0.2 o

:- 0.0

A ~ &12 De$oturmme

A -z~

\ ,a,da\ a-"-A. A A A A - \ /

2 4 6 8 10 12 14 1'6 18 - 20 Fraction Number

FIGURE 2 ATPase, PCGTase and A t2 desaturase actwmes m frac- tions after sucrose density frachonatlon of whole cncket body hom- ogenate. After ~sopycmc centnfugatlon of the 14,000g supernatant on a sucrose gradient, PCGTase actlvlty (&) was separated from Na+/K+-ATPase actwlty (O) A ~2 desaturase actwlty (A) comlgrated

with the PCGTase

lsopycnic separation of subcellular membranes

In cricket preparations, the plasma membrane was successfully separated from endoplasmic reticulum and mitochondria when supernatant from the 14,000g cen- trifugation was applied to a linear sucrose gradient (20-42%) and centrifuged at 100,000 g for 12 h (Fig. 2). In addition to the marker enzymes, Na+/K+-ATPase and PCGT, A '2 desaturase activity was assayed in frac- tions from this gradient. Fractions with the highest A 12 desaturase activity corresponded to those with highest PCGT activity.

The protocol outlined above for cricket preparations did not successfully separate the membrane fractions from cockroach fat body preparations. After the 14,000g centrifugation, more than 50% of the PCGT activity was recovered in the 14,000 g pellet, which also contained most of the mitochondria, while nearly all of the plasma membrane ATPase was recovered m the pellet at the bot tom of the sucrose gradient. However, if the 14,000 g centrifugation was eliminated and the super- natant from the low speed centrifugation (1400g) was applied to a 20-48% gradient and centrifuged at

300 CHARLOTTE E. BORGESON and GARY J. BLOMQUIST

100,000g for 12 h, good separation of the ER and the PM was achieved (Fig. 3). Again, A 12 desaturase activity comigrated with PCGT activity.

pellet [Fig. 5(B)] and the mitochondrial pellet (Fig. 5(C)], but not in the plasma membrane or endoplasmic ret~cu- lum pellets [Fig. 5(D) and 5(E)].

Electron microscopy of subcellular membranes

Fat body cells and subcellular membrane fractions from both the cricket and the cockroach were examined by electron microscopy. Fat body cells from the cricket showed no evidence of the dark electron-dense bac- terolds [Fig. 4(A)], whereas fat body cells from the cockroach clearly housed many of the endosymblotic bacteroids (Fig. 5(A)]. Membrane fractions from the whole bodies of cricket [mitochondria in Fig. 4(B), plasma membranes in Fig. 4(C) and endoplasmic reticu- lure in Fig. 4(D)] show discrete morphological differ- ences and no sign of any bacterolds. In contrast, m membrane fractions from the cockroach fat body, the electron-dense bacteria are found in both the cell debris

. . . . ~ i =:~= ~7,~ ~

DISCUSSION

The contribution of endosymbiotic bacteria to the metabolism of the host insect remains largely unknown To determine whether the endosymb~onts are revolved m a particular biochemical pathway, many early studies attempted to nd the insect of the bacteria and, simul- taneously, to culture these bacterm. Activities of the enzymes in question could then be assayed in the two separate milieus to determine whether the bacteria were involved. In this way malic dehydrogenase (Tarver and Pierre, 1967), lsocitrate dehydrogenase (Dubowsky and Pierre, 1967), guanine aminohydrolase (Pierre, 1965) and urate oxldase (Pierre, 1964) were presumed to be

FIGURE 4 Transmlsslonelectron mlcrographsof cricket fractions. (A) In a typical unbroken fat body cell, mltochondnaare clearly visible (B) mltochondna; (C) plasma membranes, and (D) endoplasmlc retlculum were obtained from lsopycnlc

eentnfugatlon of whole cricket body homogenate, as shown m Fig 2 Bar scale m all mlcrographs is 1 0/~m

A L2 DESATURASE ACTIVITY 301

FIGURE 5. Transmission electron mlcrographs of cockroach fractions (A) In this unbroken fat body cell, both mltochondna (m) and bacterolds (b) are clearly visible (B) Cell debris, (C) mltochondna, (D) plasma membranes, and (E) endoplasmlc retlculum were obtained from lsopycmc centnfugatlon of cockroach fat body homogenate, as shown m Fig 3 Bar scale m

all mlcrographs is 1 0#m

primarily of bacterial origin. However, these results have been disputed, with claims that the bacteria cultured were actually contaminants and the insects were not truly rendered asymblotic (Brooks, 1970; Brooks and Richards, 1966). Faced with these contradictory reports, the difficulty of unambiguously demonstrating whether the A 12 desaturase activity found in certain insects is due to endosymbionts or insect tissue is apparent. We have therefore endeavoured by other means to show that A IE desaturase activity is due solely to insect tissue.

In the case of the cricket, this is more straightforward as microscopic studies show that they do not contain intracellular bacteriocytes (Ulrich et al., 1981). The micrographs presented in this paper of intact fat body cells from the cricket also show no evidence of intracellu- lar bacteriocytes. This absence is also apparent in the micrographs of the cricket subcellular fractions. On the other hand, in the micrographs of the cockroach fat body intact tissue, bacteriocytes are clearly visible In

earlier studies, these bacteriocytes were released from the fat body by gentle homogenization with a Potter glass-Teflon homogenizer, while the resident bacteria were not disrupted but were precipitated with the mito- chondria during a differential centrifugation (Laudani et al., 1974). Similarly in our preparations, the dark bodies identified as endosymblotic bacteria are recovered in the low speed pellet containing cell debris and nuclell as well as in the pellet from the sucrose gradient where the mitochondria are recovered. Thus, we obtained a preparation of endoplasmic reticulum free of endosym- biotic bacteria.

The enzyme markers we have employed for insect subcellular membranes indicate a distinct separation of endoplasmlc reticulum from plasma membranes and mitochondria in both cricket and cockroach prep- arations. The separation of microsomes from other cellular organelles has been well documented in the literature. However, the data presented here represent

302 CHARLOTTE E BORGESON and GARY J. BLOMQUIST

the first t ime the microsomes have been fur ther separated

into their c o m p o n e n t membranes , that is, the endoplas-

mic ret iculum and the p lasma membrane . Because the

A ~2 desaturase activity in both the cricket and the

cockroach systems comigrates with the endoplasmic

ret iculum marker , this activity is most likely found in the

endoplasmic ret iculum and is thus o f insect origin. The

one possibility that would preclude this assumpt ion is i f

the bacteria were broken dur ing the homogen iza t ion and

the resultant bacterial cell membranes were o f the same

buoyant density as the insect ER. In this case, an enzyme

marker for the bacterial cell membrane would be desir-

able. However , a l though the presence o f dehydrogen-

ases, cy tochrome c oxidase, alanine and g lu tamate

t ransaminases and var ious amino acids synthesizing

enzymes have been detected in intact endosymbio t ic

bacteria (Brooks, 1970), none o f these enzymes have

been specifically ascribed to the cell membrane . It is,

however, very unlikely that many of the bacteria are

disrupted as they are generally considered to be G r a m

positive, and thus possess a cell wall as well as the cell

membrane. Because very few of the mi tochondr ia are

broken in our preparat ions , i.e. found as submi tochon-

drial particles in the gradient, it is likely that most o f the

bacterm remain unbroken as well.

The data presented in this paper const i tute s t rong

evidence that the A t2 desaturase activity in the Amer ican

cockroach as well as in the house cricket is solely

of msect origin, and not due to any con tamina t ing

microorganisms.

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Acknowledgements--This work was supported by the National Science Foundation under grant DCB-8914417 and grant MCB-9206584 We would hke to thank Tom Saylor for his excellent techmcal assistance and Karen McCoy for the electron microscopy