LYY (IYYL) ESTERASE IS XZYMES IN TRIBOLIUM ...
LYY (IYYL)
ESTERASE IS XZYMES IN TRIBOLIUM CASTANEUM
LEOPTERA, TENEBI IONXDAE) : P0TENTIA.L USES IN ETHOLO’GICAL
AND ECOLOGICAI STUDIES OF STORED-PRODUCT
INSECTS
Eric HAUBRUGE (t), Bernard WATHELET (2), Jean-Louis H:EMPTINXE (r), Dogo SECK (r),
Jean-Cl .ude GILSON (1) & Charles GASPAR (1)
( 1 Unit of generai and applied Zoology,
(3) Unit of bit ogical Chemistrv, Faculty of Agricultural Sciences,
2 Passa e des Déportés, B-5030 Gembloux (Belgium)
SIJMMARY. - A n estera : polymorphism in Triboliwn castanerrm has been detected
and studied during ontogen sis. The presence of a blochemical marker throughout
development offers many po! ;ibilities to follow the deveiopment of pest infestations in
storageU,systems and to analyz flow of genes conferring resistance to insecticides into this
closed ecosystem.
*..
RÉSUMÉ. - Poi.vmorph me des esrérases chez Tribolium castaneum (Coleoptera,
Tenebrionidae) : possibilité d’ô ‘ilisarion en éthologie et en tkologie des insectes des denrées
entreposées. - Un polymorp isme au niveau des estérases chez Tribolium castanewn a
été mis en évidence et a. été ~livi au cours de l’ontogénèse. La présence d’un marqueur
biochimique à tous les stades I e développement de TT: casraneum offre de nombreuses pos-
sibilités pour comprendre la m ;ration et le mouvement d’insectes dans la filière de stockage
ainsi que 1’ impact de ces phét amènes sur l’évolution de la résistance aux insecticides chez
les ravageurs des denrées entre osées.
INTRODUCTIOI’
stored. Its region of origin is unknown as are its
natural habitats. In addition to its economic
Resistance to the stored-proc tct protectants
importance, this insect is largely studied because
is a worldwide phenomenon.. Mala lion resistance
it is easy and. cheap to rear in the lahoratoty.
in the flour beetle (Tribaliurn
:asranerrm) is
The developmental time is fairly short (25 days
virrually universai and has been ecorded in at
at 32” C’and 70% RH) and reproduction takes
least 70 count,ries (CHAMP & DYTI 1976). CHAMP
place the whole year round without diapause.
$2 CAMPBELL-BROW;\\I
(1970) founc that resistance
As a result of the development of many bio-
to malarhion in T. casranewn c1
;
govemed by
chemical techniques, some biochemical markers
a single autosomal semidominant gene.
cari now be utilized for studying the biology and
7: cûstanetlm is a cosmopolit m pest of cereal
ethology of insects. In resolving problems as-
mills and generally of any place
where flour is
sociated with insect pests in agriculture, bio-
2 %
I LGInS JOURNAL OF BOTANY 125
chemical characters have prov
t o b e veq
minutes for polymerization and then the concentration
successfull to reveal and to moni 7 resistance
to
gel (5% acrylarnide) wâs prepared with the following
insecticides in Myzru persicrre (WC loptera, Aphi-
ingredients : 0.8 ml of acrylamide stock solution, 3.6 ml
didae) (DEVONSHIRE 1977) and L!e.~ pipiens
of distilled water, OS mi of Tris-HC1 bufFer (1.25 M,
(Diptera, Culicidae) (P
pH 6.8), 7 pl (of TEMED and 15 pl
ASTEUR et
1981) as well
of ammonium
as to unravel the reproductive be .viour of Dia- : persulphate.
brotica virgifera
PAGE EL:crrophoresis. Vertical electrophoresis
(Coleoptera,
qtsomelidae)
was’ performed at 4’ C under 20 mA and 120 V for
(RWD er al. 1988). Detection o
xerase polir-
approximatively 2 hours until the tracking dye (bro-
morphism in rl: castanewn offers
lany possibil-
mophenol blue) bas mi_orated to the end of the gel.
ities to understand the develo
nent of pest
Esleruse sroinin,a. After completion of electro-
infestations
in storage svstems ai
to study the
phoresis. the g-1 was cquilibrated for 10 minutes in
malathion resistant gene flow i
3 t h i s closed
the dark, in a Tris-I-iCl buffer solution (0.1M. pH 7)
ecos-tem.
containing 10 mM EDTA. Then the buffer was replaced
by a solution clantaining 200 mg of l-naphryl acetate.
MATERIAL AND ME-I ODS
200 mg of 2-naphryl acetate. 20 ml of acetone and
80 ml of Tris-HC1 buffer (O.IM. pH 7). This mixture
was agitated for 5 minutes in the dark. Then 100 mg
Insrcts. Seven strains of the flou1
eetle were used
of Fast Carnet! RR sait was added and agitation was
in this srudy. Two strains specific
y resistant to
continued for 30 ninures. Finslly, the ccl is transierred
maiathion were obtained from the ?
:u:al Resource
to 7Cc acetic acid for 24 hours as a tixation step.
Insritute i n Chatham. England. The,
I~:X- bern col-
lected in storage systems in The
nilippines and
Botswana and kept in the labaratoq
f this Institute
f o r several generations. One malatt ri non-specific
RESULTS
resisrant strain (CTCl2) was obtnined
,rn the cultures
o f rhe Departmenc o f Zoology, ( 7rge S . \\VI~E
Adults or six out of the seven populatiorPs
Faculty of Life Sciences. Tel Aviv Uni
rsity. Tel Aviv.
possess two distinct isoqme groups labelled 1
Israel. Finally four populations suscepc
le tc) malathion
and 6 on fïgcre 1 : insects of Belgium (lane I),
bave been collected in storage place!
)y the authors
Israël (lane 2). Botswana (lane 3), The Philippines
a! Jamagne (Belgium). Bordeaux (F
nce). Abidjan
(lane 4), Senegal (lane 5) and Ivory Coast (lane
(Ivory Coax) and Nioro (Senegol). e insecrs were
6). However the two strains specificallu resistant
reared wirh whole wheat flour enric
j with brewer
to malathion that are located at the third and
yeast nnd kept in the darkness at 30 C and 60% of
fourth fanes. displau a weaker activity for the
relative humidity.
Erqw7e preparntion.
esterases of the group I than the samples of lanes
100 adult insects of each
strain were respectively ground in a lortar containing
1, 2. 5 and 6. (figure 1).
I ml of phosphate buffer (0.05 M, pH 7.2) with 1OmM
The French population (lane 7) which is
EDTA. The mixture was filtered through glass w001 :
susceptible to malathion, hns a different esterase
the tiltrate was stored in Eppendorf tl bes at do C. and
isozyme pattern. .L\\ highly stained group of bands
centrifuged nr 20.000 g. 4O C for 15 minutes. Finally
a’ppears at,the position 4 (figure 1).
a sample of 20 FI was poured in a gel weli.
The activitv of the esterase isozymes has also
Gel eiecrrophorcsis preparorion. Non denaturing
been ahalyzed throughout rhe developmental
discontinuaus
polyacrylamide gel electrophoresis
stages of 7: costunellnz of the Philippines and rhe
(PAGE) was used to examine various esteraxe isozymes.
French breeds (figure 2). For insects from The
The gels have a thickness of 0.75 mrr. The separation
Philippines, it appears that the activity of the
gel (7.51~ acrylamide) wxs firstly prepared Ivith 3.8 ml
esterases 1, 3 and 4 is ver? low during the egg
of acrylamide stock solution (containing 29.lCc acry-
instar but increases during the larval stage. In
lamide and 0.9% N,.~‘-rnerhrlene-bis-~~crylamids).
8 ml
of disrillrd water. 3 ml of Tris-HC1 buffer (1.975 $1.
pupae the esterases are again weakly stained while
pH 8.8). 10 b1l of N.N,N’.X’-retrameth~leth~lenediamine
they reappear strongly in adults. The French
(TEMED). 50 ~1 of IOC, ammonium pcrsulphate and
insecrs have a different pattern : esTerases 1 and
74 111 of 0.5yc Triton X-100. This tïr’r gel was left 60
3 are hardly visible during all stages of devel-
2%
ELGIAN JOURNAL OF BOTANY 12.5
* i’ ‘cg
.
opment but esterase 4 is very ac s’e from egg to
millions tons of stored grain (S~LOT 1990).
adult. As a consequence,
the la :r esterase is a
h’loreover intensive control of theses pests wi:h
specific biochemical mark foi the population
pesticides such as phosphin and malathiol t;g-
fro m Bordeaux.
gered the deve!opment of resistant strains. As
trade of cereals in the world implies circulation
DISCUSSICN
of freight from one stornge place to another it
allows dispersion of pest from dinerent geogaphic
PAGE electrophoresis enab s rapid resolu-
areas and results sometirne in the introduction
tion of the different groups of i xymcs as well
of susceptible strain in si- 2s already contaminated
as direct detection of esterase p .ymorphtim o f
with insects with single locus mechanism resist-
7: caxaneum.
ance to pesticides. Theorctically such introduction
T%e seven populations of 7: xsloneL<m that
wilI increases the frequcilcy of sensible alleles in
ha!,e been examined in this stud are sorted out
the population. However heterozygotes for the
in two groups in respect of thek e: :rase isozymes.
resistance
characrer Will aiso appear tha,t growth
On the one hand there are two gr ups of isozyme
faster than resistant homozygotes (GEORGHIOU &
in the insects from Belgium, 1s tël, Botswana,
TAYLOR 1977, COMINS 1977). Beyond these
The Philippines, Senegal and Iv ry Coast . On
generai statements,
dispersion of pest bas attracted
the orher hand the French breed ; c!xracterized
very little attention and it is therefore difficult to
by an other pattern of bands’(fi
.!re 1). C~~IEL~
predict what could’ be the influence of such
er ul. (1977) and S VERDLOV el
1. (1976) have
transfers on the developnient of resistancr ir; pests
also observed two dLfferen: estera
: pattcms with
of stored prcducts. The Frexh breîd chat is
four groups of isozymes. More
cently COHEN
biochemicaly marked by the Ps;erase isozyme 4
(1952) has made an electrophor
:ic analysis o f
throughout ail the Iife stages wiil be a valuable
19 strains of 7: casruneum and h did not detect
matez-ial to improve our knowledge of population
any orher esterase polymorphism n this species.
ecology of th- pests living in storcd product?,
It is aiso obvious that the ïrst group o f
Experîmental designs combining the French strain
six populations is not homoger O:IS regarding
with a second breed biochemically different from
tkie activity of the isozyme gro p n u m b e r 1 :
the fkst Will proba.bly dic.c:ipate some clouds over
thc esterase of the two populati
ns specifïcally
the population ecology of X cnsranerlm in silos
resistant to mnlathion are less s ined than the
of cereals.
correcnonding enzyme in the flc r bectle from
BeigiLlm. I s r a ë l , Seneeai
2nd
Ivory
Coast.
BEE.LiAN & SCHMIDT (1982) ha%
observed the
We thanks Dr P. Golob from the NRI in Chathsm
same resuhs for malathion specifïc
:sistant strains
(En$md) and Professor D. Wool from the University
of Plcxiia interpunctefl~ (Lepidopl -7. Pyralidae).
ol Tel ,\\viv (I:;ra?i) for sending us a strain of 7:
‘The- attributed this phenomenvr :o a mutation
The srudies on insecticide wistance was
at an esterase gene. The mutai:t
xerase has an
supported by the ,orant 1.525I.91F of the National
aitered substrate specificity ir.:lL ing increased
Lund for Scienrlc Research (5elgi~m).
xtil:iry toward malathion.
Studies of biochemical mut nts Lhat have
REFERI’XCES
hcen performed on 1 cast~new* a very limited :
BCNA‘I R.?V. SC SClt~flliT 3.X.. 1982. - Biochcmical
YEH cP: SCHEINBERG (1972:) ix\\
observed an
and genetic aspects of maiathion-specifïc res?-
kohol dehydrogenase polymor lisrn ; BREM-
ance i n the Indian meal m o t h (Lepidop:
51 ER E! ni. (1980) have repofl:ed OI xmylase poly-
Pyralidae). J. Eccn. En[. 75 : 945-949.
morphism.
BKESIVER T.A.. ~IXRSON
M.D., POPE G . J .
Insecrs and mites are resp)r ible for adul-
ANDEKSOS R.M.. 1983. - Genetic polumor-
terarion o f alimentary prodlxts : :d they cause
phism of arnylase in three species of T%oiiw~.
:;mri;; gosses estimnted at nbcrut
30% o f 1soo
Com,D. Biochm. ard PhJsioi. 11B : 755-758.
ESTERASE IS XZYMES IN TRIBOLIUM CASTANEUM
LEOPTERA, TENEBI IONXDAE) : P0TENTIA.L USES IN ETHOLO’GICAL
AND ECOLOGICAI STUDIES OF STORED-PRODUCT
INSECTS
Eric HAUBRUGE (t), Bernard WATHELET (2), Jean-Louis H:EMPTINXE (r), Dogo SECK (r),
Jean-Cl .ude GILSON (1) & Charles GASPAR (1)
( 1 Unit of generai and applied Zoology,
(3) Unit of bit ogical Chemistrv, Faculty of Agricultural Sciences,
2 Passa e des Déportés, B-5030 Gembloux (Belgium)
SIJMMARY. - A n estera : polymorphism in Triboliwn castanerrm has been detected
and studied during ontogen sis. The presence of a blochemical marker throughout
development offers many po! ;ibilities to follow the deveiopment of pest infestations in
storageU,systems and to analyz flow of genes conferring resistance to insecticides into this
closed ecosystem.
*..
RÉSUMÉ. - Poi.vmorph me des esrérases chez Tribolium castaneum (Coleoptera,
Tenebrionidae) : possibilité d’ô ‘ilisarion en éthologie et en tkologie des insectes des denrées
entreposées. - Un polymorp isme au niveau des estérases chez Tribolium castanewn a
été mis en évidence et a. été ~livi au cours de l’ontogénèse. La présence d’un marqueur
biochimique à tous les stades I e développement de TT: casraneum offre de nombreuses pos-
sibilités pour comprendre la m ;ration et le mouvement d’insectes dans la filière de stockage
ainsi que 1’ impact de ces phét amènes sur l’évolution de la résistance aux insecticides chez
les ravageurs des denrées entre osées.
INTRODUCTIOI’
stored. Its region of origin is unknown as are its
natural habitats. In addition to its economic
Resistance to the stored-proc tct protectants
importance, this insect is largely studied because
is a worldwide phenomenon.. Mala lion resistance
it is easy and. cheap to rear in the lahoratoty.
in the flour beetle (Tribaliurn
:asranerrm) is
The developmental time is fairly short (25 days
virrually universai and has been ecorded in at
at 32” C’and 70% RH) and reproduction takes
least 70 count,ries (CHAMP & DYTI 1976). CHAMP
place the whole year round without diapause.
$2 CAMPBELL-BROW;\\I
(1970) founc that resistance
As a result of the development of many bio-
to malarhion in T. casranewn c1
;
govemed by
chemical techniques, some biochemical markers
a single autosomal semidominant gene.
cari now be utilized for studying the biology and
7: cûstanetlm is a cosmopolit m pest of cereal
ethology of insects. In resolving problems as-
mills and generally of any place
where flour is
sociated with insect pests in agriculture, bio-
2 %
I LGInS JOURNAL OF BOTANY 125
chemical characters have prov
t o b e veq
minutes for polymerization and then the concentration
successfull to reveal and to moni 7 resistance
to
gel (5% acrylarnide) wâs prepared with the following
insecticides in Myzru persicrre (WC loptera, Aphi-
ingredients : 0.8 ml of acrylamide stock solution, 3.6 ml
didae) (DEVONSHIRE 1977) and L!e.~ pipiens
of distilled water, OS mi of Tris-HC1 bufFer (1.25 M,
(Diptera, Culicidae) (P
pH 6.8), 7 pl (of TEMED and 15 pl
ASTEUR et
1981) as well
of ammonium
as to unravel the reproductive be .viour of Dia- : persulphate.
brotica virgifera
PAGE EL:crrophoresis. Vertical electrophoresis
(Coleoptera,
qtsomelidae)
was’ performed at 4’ C under 20 mA and 120 V for
(RWD er al. 1988). Detection o
xerase polir-
approximatively 2 hours until the tracking dye (bro-
morphism in rl: castanewn offers
lany possibil-
mophenol blue) bas mi_orated to the end of the gel.
ities to understand the develo
nent of pest
Esleruse sroinin,a. After completion of electro-
infestations
in storage svstems ai
to study the
phoresis. the g-1 was cquilibrated for 10 minutes in
malathion resistant gene flow i
3 t h i s closed
the dark, in a Tris-I-iCl buffer solution (0.1M. pH 7)
ecos-tem.
containing 10 mM EDTA. Then the buffer was replaced
by a solution clantaining 200 mg of l-naphryl acetate.
MATERIAL AND ME-I ODS
200 mg of 2-naphryl acetate. 20 ml of acetone and
80 ml of Tris-HC1 buffer (O.IM. pH 7). This mixture
was agitated for 5 minutes in the dark. Then 100 mg
Insrcts. Seven strains of the flou1
eetle were used
of Fast Carnet! RR sait was added and agitation was
in this srudy. Two strains specific
y resistant to
continued for 30 ninures. Finslly, the ccl is transierred
maiathion were obtained from the ?
:u:al Resource
to 7Cc acetic acid for 24 hours as a tixation step.
Insritute i n Chatham. England. The,
I~:X- bern col-
lected in storage systems in The
nilippines and
Botswana and kept in the labaratoq
f this Institute
f o r several generations. One malatt ri non-specific
RESULTS
resisrant strain (CTCl2) was obtnined
,rn the cultures
o f rhe Departmenc o f Zoology, ( 7rge S . \\VI~E
Adults or six out of the seven populatiorPs
Faculty of Life Sciences. Tel Aviv Uni
rsity. Tel Aviv.
possess two distinct isoqme groups labelled 1
Israel. Finally four populations suscepc
le tc) malathion
and 6 on fïgcre 1 : insects of Belgium (lane I),
bave been collected in storage place!
)y the authors
Israël (lane 2). Botswana (lane 3), The Philippines
a! Jamagne (Belgium). Bordeaux (F
nce). Abidjan
(lane 4), Senegal (lane 5) and Ivory Coast (lane
(Ivory Coax) and Nioro (Senegol). e insecrs were
6). However the two strains specificallu resistant
reared wirh whole wheat flour enric
j with brewer
to malathion that are located at the third and
yeast nnd kept in the darkness at 30 C and 60% of
fourth fanes. displau a weaker activity for the
relative humidity.
Erqw7e preparntion.
esterases of the group I than the samples of lanes
100 adult insects of each
strain were respectively ground in a lortar containing
1, 2. 5 and 6. (figure 1).
I ml of phosphate buffer (0.05 M, pH 7.2) with 1OmM
The French population (lane 7) which is
EDTA. The mixture was filtered through glass w001 :
susceptible to malathion, hns a different esterase
the tiltrate was stored in Eppendorf tl bes at do C. and
isozyme pattern. .L\\ highly stained group of bands
centrifuged nr 20.000 g. 4O C for 15 minutes. Finally
a’ppears at,the position 4 (figure 1).
a sample of 20 FI was poured in a gel weli.
The activitv of the esterase isozymes has also
Gel eiecrrophorcsis preparorion. Non denaturing
been ahalyzed throughout rhe developmental
discontinuaus
polyacrylamide gel electrophoresis
stages of 7: costunellnz of the Philippines and rhe
(PAGE) was used to examine various esteraxe isozymes.
French breeds (figure 2). For insects from The
The gels have a thickness of 0.75 mrr. The separation
Philippines, it appears that the activity of the
gel (7.51~ acrylamide) wxs firstly prepared Ivith 3.8 ml
esterases 1, 3 and 4 is ver? low during the egg
of acrylamide stock solution (containing 29.lCc acry-
instar but increases during the larval stage. In
lamide and 0.9% N,.~‘-rnerhrlene-bis-~~crylamids).
8 ml
of disrillrd water. 3 ml of Tris-HC1 buffer (1.975 $1.
pupae the esterases are again weakly stained while
pH 8.8). 10 b1l of N.N,N’.X’-retrameth~leth~lenediamine
they reappear strongly in adults. The French
(TEMED). 50 ~1 of IOC, ammonium pcrsulphate and
insecrs have a different pattern : esTerases 1 and
74 111 of 0.5yc Triton X-100. This tïr’r gel was left 60
3 are hardly visible during all stages of devel-
2%
ELGIAN JOURNAL OF BOTANY 12.5
* i’ ‘cg
.
opment but esterase 4 is very ac s’e from egg to
millions tons of stored grain (S~LOT 1990).
adult. As a consequence,
the la :r esterase is a
h’loreover intensive control of theses pests wi:h
specific biochemical mark foi the population
pesticides such as phosphin and malathiol t;g-
fro m Bordeaux.
gered the deve!opment of resistant strains. As
trade of cereals in the world implies circulation
DISCUSSICN
of freight from one stornge place to another it
allows dispersion of pest from dinerent geogaphic
PAGE electrophoresis enab s rapid resolu-
areas and results sometirne in the introduction
tion of the different groups of i xymcs as well
of susceptible strain in si- 2s already contaminated
as direct detection of esterase p .ymorphtim o f
with insects with single locus mechanism resist-
7: caxaneum.
ance to pesticides. Theorctically such introduction
T%e seven populations of 7: xsloneL<m that
wilI increases the frequcilcy of sensible alleles in
ha!,e been examined in this stud are sorted out
the population. However heterozygotes for the
in two groups in respect of thek e: :rase isozymes.
resistance
characrer Will aiso appear tha,t growth
On the one hand there are two gr ups of isozyme
faster than resistant homozygotes (GEORGHIOU &
in the insects from Belgium, 1s tël, Botswana,
TAYLOR 1977, COMINS 1977). Beyond these
The Philippines, Senegal and Iv ry Coast . On
generai statements,
dispersion of pest bas attracted
the orher hand the French breed ; c!xracterized
very little attention and it is therefore difficult to
by an other pattern of bands’(fi
.!re 1). C~~IEL~
predict what could’ be the influence of such
er ul. (1977) and S VERDLOV el
1. (1976) have
transfers on the developnient of resistancr ir; pests
also observed two dLfferen: estera
: pattcms with
of stored prcducts. The Frexh breîd chat is
four groups of isozymes. More
cently COHEN
biochemicaly marked by the Ps;erase isozyme 4
(1952) has made an electrophor
:ic analysis o f
throughout ail the Iife stages wiil be a valuable
19 strains of 7: casruneum and h did not detect
matez-ial to improve our knowledge of population
any orher esterase polymorphism n this species.
ecology of th- pests living in storcd product?,
It is aiso obvious that the ïrst group o f
Experîmental designs combining the French strain
six populations is not homoger O:IS regarding
with a second breed biochemically different from
tkie activity of the isozyme gro p n u m b e r 1 :
the fkst Will proba.bly dic.c:ipate some clouds over
thc esterase of the two populati
ns specifïcally
the population ecology of X cnsranerlm in silos
resistant to mnlathion are less s ined than the
of cereals.
correcnonding enzyme in the flc r bectle from
BeigiLlm. I s r a ë l , Seneeai
2nd
Ivory
Coast.
BEE.LiAN & SCHMIDT (1982) ha%
observed the
We thanks Dr P. Golob from the NRI in Chathsm
same resuhs for malathion specifïc
:sistant strains
(En$md) and Professor D. Wool from the University
of Plcxiia interpunctefl~ (Lepidopl -7. Pyralidae).
ol Tel ,\\viv (I:;ra?i) for sending us a strain of 7:
‘The- attributed this phenomenvr :o a mutation
The srudies on insecticide wistance was
at an esterase gene. The mutai:t
xerase has an
supported by the ,orant 1.525I.91F of the National
aitered substrate specificity ir.:lL ing increased
Lund for Scienrlc Research (5elgi~m).
xtil:iry toward malathion.
Studies of biochemical mut nts Lhat have
REFERI’XCES
hcen performed on 1 cast~new* a very limited :
BCNA‘I R.?V. SC SClt~flliT 3.X.. 1982. - Biochcmical
YEH cP: SCHEINBERG (1972:) ix\\
observed an
and genetic aspects of maiathion-specifïc res?-
kohol dehydrogenase polymor lisrn ; BREM-
ance i n the Indian meal m o t h (Lepidop:
51 ER E! ni. (1980) have repofl:ed OI xmylase poly-
Pyralidae). J. Eccn. En[. 75 : 945-949.
morphism.
BKESIVER T.A.. ~IXRSON
M.D., POPE G . J .
Insecrs and mites are resp)r ible for adul-
ANDEKSOS R.M.. 1983. - Genetic polumor-
terarion o f alimentary prodlxts : :d they cause
phism of arnylase in three species of T%oiiw~.
:;mri;; gosses estimnted at nbcrut
30% o f 1soo
Com,D. Biochm. ard PhJsioi. 11B : 755-758.