Download DIGESTIVE PROTEASES OF TWO SPtrCIES OF WASPS OF THE

t r e s s .P r i n t e d i n G r e u t B r i t a i n
I n s e c t B i o c h e m . ,1 9 ' 7 7 ,V o l . 7 , p p . 1 9 1 t o 1 9 6 .P e r g u n t c uP
DIGESTIVE PROTEASESOF TWO SPtrCIES
OF WASPS OF THE GENUS VESPLTLA
DoNl't-o E. GnocnN and JavEs H. HuNr
Louis,St. Louis.
Departmentof Biology.Univcrsityof Missouri-St.
M o . 6 3 1 2 1U. . S . A .
(Rat'eiretl7 Mttrr:h 1977)
wcre examined for digestrveprotease activiAbstract Adults of Vespttlaqermanica ancJ /. nrctr:ulilrons
ties. All castemembers of both speciesthat were analyscd posscssedtrypsitl. chymotrypsin, carboxypeptidase A. and carboxypeptidase B-like activilies. with one cxception: worker V'. nnt'ulilrorr.slacked
carboxypeptidaseB activiil'. Adults of these two spcciescan in all likelihood digest protein. The trypsin
antl chymotrypsin-likc enzymesof the two specieswere also examined with specific inhibitors to determine their similarity to bovine enzymes.The wasp enzymcs posscsstr serine and n histidine in their
active centers;the tiypsin-like enzyrnehas a specificityfor basic amino acids, whereas the chymotrypsinl i k e e n z y m es p e c i f i e sa r o m a t i c a m i t r o a c i r l s .T l t c s e \ \ ' l l s pc n z v m e sl l r c t h c r c f o r es i r n i l a r t o b o v i n c t r y p s i n
and chymotrypsin in these respects.Methodological and interprctative differencesare noted between
the present study an{ an earlier publication on absence of digestive proteases tn Vespa orientttlis-
contain free amino acids ancl the reported absence
of digestive proteases in Vespa orientalis led us to
Ir Has been shown that male Vespula receive part initiate this study on the digestive proteases of adult
of their diet from the saliva of larvae through trophal- Vespula. Freely available amino acids in natural
1963, 1964).The saliva is nutri- liquid foods may have obviated the need for protein
laxis (McrNrAGNER,
tious, containing sugars. proteins. and free amino digestion cnpabilities in wasps in general. If adult
acids(MascHwtrz, 1966fu WtLSoN,l91l). There have Vespu orientalis lack digestive proteases. il similar
been differencesof opinion as to the importance o1' c o n d i t i o n m a y w e l l b e l o u n d i n o t h c r w a s p s p e c i e s .
larval saliva as a food source for adult wasps. MonThe research presented here was pursued as a test
tagner and Maschwitz consider the saliva to be only of this possibility.
INTRODUCTION
a colony food reserve. However, IsHAY and knN
(1968)considerit to be the sole source of amino acid
nutrition for adult Vespaorientalis.karl. e/ a/. (1968)
say that V. orientalis lacks digestive proteases.That
result,if indeed true, indicates that adult tr".orientalis
must receiveall of their amino acid nutrition as free
amino acids in solution. These reports took on new
significancein light of the findings of BarEn and
BaxEn (1973, 1975),who found free amino acids to
be regularly occurring constituents of floral nectar.
Nectars of all flowering plant taxa that have bcen
examined, including morphologically primitive
species,hzrveat least some free amino acids. Barcln
and Barln (1975)correlate amino acid presenceand
abundancepatterns with the primary pollinators (i.c.
bees.hawk moths, birds. etc.) of the plant taxa, and
they suggestpossiblelong-term co-evolutionaryselective pressureson both plant and pollinator. It thus
seemslikely that adult wasps that visit flowers may
have always had free amino acids available as a part
of their diet. The possibility is further enhanced by
noting that all liquid foods of adult Hymenoptera
that have been examined contain free amino acids.
including honeydew from Homoptera (Wav, 1963)
and insect haemolymph (Wvarr, 1961).
Thc consistencywith which liquid foods of wasps
r.n7
. i3 A
l9l
MATERIALS
AND
METHODS
R e m g r , uo
l f'ntidOuts
Wasps were rcmoved under carbon dioxide anesthesia
from the vacuum cleaner in which they were collected and
transferred to several plastic containers. The live wasps
were removed individually, sexed, and the midguts were
removed. While holding a wasp with one pair of forceps,
the apex of the abdomen was grasped firmly wlth another
pair and the latter gently pulled. This movement usually
removed the entire digestive tract from the abdomen. The
midgut was severeclfrom the remainder of the digestive
tract and transferred to a test tube suspended in an acetone dry ice mixture. This resulted in freezing of the midgut within 15 sec after removal from the living wasp. The
test tube containing the midguts was stored at * 60"C until
use.
Midguts wcre collected from lJu workers and 58 males
ol V. clarmanicaand from 365 workers, 26 males, and 28
rons nest conqueens of V. mucrlilrnns. Also, the Iz.n'tucul|f
taincd capped queen cclls from which 78 more queens
eclosed. The midguts from these queens were also collectcd.
Elzl:nte d.s)^dJrs
T r y p s i u .F o r a n a s s a y2 t o 4 V . q e r n r u n i c am i d g u t s w c r e
p l a c e d i n 1 m l o f 0 . 0 0 1N H C I a n d l c a c h e d i o r 1 5 m i n a t
DoxaLo E. GnocaN lNIl Jalrns H. HuNr
Table 1. Midgut trypsin activities in adult castes of two Vespulaspecies.Midgut weights given are the averagesover
all assays,enzyme activity values are averagesof 2 6 replicates
(me)
Trypsin
specificactivity
(units/mgprotein)
Trypsin
(units/midgut)
Trypsin
(units/mgmidgut)
6.6
4.1
0.87
0.36
0.46
0.17
0.070
0.041
0.14
0.087
0.16
0.055
0.017
0.094
0.021
0.010
0.010
Weight of
midgut
Species
caste
V. qernunica
Worker
Malc
V. nruculifrons
Worker
Male
Queen
2.1
1.8
l 0 .t
37"C. For the smaller V. rnctc'ulifi'on.s
worker, 10 to 20 midguts were used.The extract was then centrifuged at 2500g
l o r l 0 m i n a t r o o m t c m p e r a t u r e ,a n d t h e s u p e r n a t e w a s
used as the enzyme source. The reaction mix had a final
volume ol 3.0 ml and contained 40 mM Tris HCI buffer
( p H 8 . 1 ) ,l 0 m M C a C l 2 ,a n d 0 . l m l o f m i d g u t c x t r a c t .T h e
reaction was initiated by the addition of p-toluer-resulfonylt--arginine methyl ester (TAME) to a final concentration
of I mM. The changc of absorbance at 247 nm was
measured as an index ol enzymc activity (Hulrlrtr-, 1959).
Chvntcttrvpsiu.Midguts were extracted as described
above. and enzymc activity was determincd in a 3.0 ml
reaction mix containing 0.1 ml of midgut extract, 40 mM
Tris HCI buffer (pH 7.8). and 5 mM CaCl.. The reaction
was initiated by the addition ol benzoyl-l-tyrosine ethyl
ester (BTEE) in 50?; methanol (WiW) to a final concent r a t i o n o l 0 . 5 m M . T h e c h a n g e i n a b s o r b a n c ca t 2 5 6n m
was measured (Hutuur.l. 1959).
CarhoxvpeptidaseA. Midguts wcrc cxtractcd in l0%
LiCl under thc conditions described for trypsin above. A
rcaction mix was formed by adding 2.9 ml ol freshly prepared I mM hippuryl-r--phenylalanine(HPA) dissolved in
25 mM Tris HCI buffer (pH 7.5) and containing 500mM
NaCl to 0.1 ml ol enzyme extract. The change in absorbance at 254nm was used as an index of enzyme activity
(For-n and Scsrnvr,rr. 1963).
Curhoxl'peptitluseB. Midguts were prepared as describcd
lor carboxypeptidaseA. A reaction mix was prepared by
adding 2.9 ml of freshly prepared I mM hippuryl-t-arginine (HA) in 25 mM Tris HCI buffer (pH 7.65) and
containing 100mM NaCll to 0.1 ml of enzyme extract. The
change of absorbance at 254 nm was measured (For-r ct
n/.. 1960).
I nhibition experiments
Inhibitors of trypsin and chymotrypsin, N-a-p-tosyl-llysine chloromethyl ketone (TLCK) (ScHocr-r-naNand
Snew, 1963), phenyl-methyl-sulfonylfluoride (PMSF)
(Goro, 1965), and l-1-tosylamine-2-phenyl-ethylchloromethyl ketone (TPCK) (Sirlw et ul., 1965)were added to
the reactions. The final concentrations of the inhibitors
w e r e : T L C K , 0 . 2 0 . 4 m M ; P M S F , 0 . 9 5 -1 . 9 m M ; T P C K ,
0.2 mM. The sequenceof addition of the components was:
enzyme source to the bull'er followed by the inhibitor. The
enzyme was pre-incubated for 20 min with PMSF or for
onc hr with TLCK or TPCK beforc addition of the substrate. When inhibitors were added. the volumc of Tris
buffer was decreasedby an equivalent amount in order
to maintain a constant volume.
Protein anall,sis
Protein was determincd as described by Lownv cr a/.
( 1 9 5 1 )u s i n g c r y s t a l l i n eb o v i n e a l b u m i n a s a s t a n d a r d .
RESULTS
Diplestiueproteaseactiuity o'f the V. germanica nitlplut
Both castesexamined of V. qerntanic:apossessedthe
digestive proteases trypsin, chymotrypsin, carboxypeptidaseA, and carboxypeptidaseB. (We did not
conclusivelyshow that any of the enzymeswe studied
were identical to mammalian proteaseenzymes.They
rurc.however. directlv comparable. and for the sake
of simplicity in presentingthis rcport they are inferred
to be the same.) Trypsin was the predominant
Table 2. Midgut chymotrypsin activities in trdult castesof two Vespuluspecies.Enzyme activity values
are avcragesof 2 6 replicates
Species
CASTC
V. germunicct
Worker
Male
V. muculilrons
Worker
Male
Queen
Chymotrypsin
specificactivity
(units/mgprotein)
Chymotrypsin
(units/midgut)
0 . 13 0
0.072
0.086
0.026
0.013
0.0064
0.076
0.081
0.126
0.037
0.016
0.079
0.014
0.0085
0.0079
Chymotrypsin
(units/mg midgut)
Digestive proteasesof two specicsof wasps
o.t4
o.t2
o.ro
o.o8
o.06
o04
(D
O'
c
o
-o
o.o2
o
o
!
7
Minutes
Fig. 1. The trypsin-like miclgut activities of male (a) ancl worker (b) and the chymotrypsin-like midgut
activities of male (c) and worker (d) Vespula gerntanit:u.The response is shown of these enzymes
and TPCK (n--x).
(o-o)
ro pMSF (o
o), TLCK (A-A),
enzyme,with specificactivity 6.1 and 5.0 times greater
than the next most abundant enzyme, chymotrypsin,
in workers and males respectively(Tables l, 2). The
reaction rates for trypsin r.verelinear over a 7 min
time period (Figs. l(a), 1(b)).
Chymotrypsin was present in significant amounts
in both worker and male V. germanir:a(Table 2). The
specificactivity of chymotrypsin was 0.130 and 0.072
for workers and males respectively.The reaction rates
for this enzyme were not linear; the rate of the first
2 min exceededthe rate of the last 5 min (Figs' 1(c),
1(d)).
Botlr worker and male /. gernmnicapossessedmidgut carboxypeptidaseA activity, but the amount of
activity was less than either trypsin or chymotrypsin
(Tables l, 2, and 3). The specific activity of this
enzyme was 7.3 times less than trypsin activity and
1.5 times less than the chymotrypsin specificactivity
in males and 15.0 and 2.2 times less than those
enzymesin workers. The specificactivity of carboxypeptidase A in workers exceededthat of males by
a factor of 1.2 (Table 3).
CarboxypeptidaseB activity was also found in the
midguts of worker and male V. germanica,but its concentration was very low (Table 4). The specificactivity
in workers was approximately equal to that in males,
but the workers contained approximately twice the
amount of total enzyme (Table 4). In males, trypsin
specificactivity was l8 times as great and chymotrypsin activity was 3.6 times its great as carboxypeptidase
Table 3. Midgut carboxypeptidaseA activities in adult castesof two Vespuluspecies.Enzyme activity values are averages
of 2-6 reolicates
Species
caste
Carboxypeptidase A
specific activity
(units/mg protein)
CarboxypeptidaseA
(units/midgut)
V. gerntartica
Worker
Male
V. muculilrorts
Workt:r
Male
Queen
Carboxypeptidase A
(units/mg midgut)
0.058
0.049
0.030
0.014
0.0038
0.0028
0.044
0.044
0 . 18 6
0.0088
0.011
0.097
0.0036
0.0062
0.0091
DoN.q,Lu E. GnoclN
t94
aNo JalrES H. HuNr
Table 4. Midgut carboxypeptidaseB activities in adult castesof two Vespulaspecies.Enzyme activity values are averages
of 2 6 replicates
Species
caste
V. gerrnutica
Worker
Male
V. mactilifrons
Worker
Male
Quecn
Carboxypeptidase B
specific activity
(units/mg protein)
CarboxypeptidaseB
(units/midgut)
CarboxypeptidaseB
(units/mg midgut)
0.02r
0.020
0.011
0.0056
0.0013
0.0012
0.000
0.0046
0.082
0.000
0.00r2
0.043
0.000
0.0007
0.0043
B; carboxypeptidaseA specificactivity exceededcarboxypeptidaseB by a factor of 2.5. In workers the
values were 41, 6.2, and 2.8 respectively.
obviously differs in some respects from bovine chymotrypsin, however, as the latter is inhibited by
PMSF from time zero.
TLCK did not significantly inhibit the male or
Effectsof inhibitorson V. germanica,trypsin and chy- worker V.germanicachymotrypsin (Figs. 1(c),1(d)).In
motrypsin
order for TLCK to have an inhibitory effect an
Severalinhibitors were used in order to compare enzyme must possessa substrate specificity for basic
the V. gennanica trypsin enzyme to bovine trypsin. amino acids. Like bovine chymotrypsin, the wasp
PMSF was used to test the enzyme for the presence chymotrypsin does not.
TPCK does inhibit male and worker V. qermanicu
of a serinegroup in its active center. The trypsin in
both worker and male V. germanicctwas inhibited by
chymotrypsin (Figs. 1(c),1(d)).Therefore,this chymo1.9mM PMSF (Figs. 1(a),1(b))and resemblesbovine trypsin and the bovine enzyme are similar in that
trypsin in this respect.The range of inhibition varied both have a histidine group in their active center and
from 63L to 86L in extractsof both male and worker both have specificities for aromatic amino acids.
However. as was noted for PMSF. TPCK did not
midguts.
TLCK will inhibit an enzyme that has a histidine immediatelycauseinhibition. Ten minute incubations
were usually required before inhibition could be
in its active center and a specificity for basic amino
acids.The trypsin in the worker and male wasps was demonstrated.
inhibited by 0.4 mM TLCK (Figs. 1(a),1(d)),indicating similarity between the V germanicatrypsin and Mitlptut proteaseuctiuity oJ'Y. maculifrons
The trypsin-like activities of V. maculifrons were
bovine trypsin. The range of inhibition observedwas
similar to but less than those of V. qermanica Qable
43/, to 6l/".
The effects of TPCK on the V. germanica trypsin
1).The reaction rates were linear over time (Figs. 2(a),
are shown in Figs. 1(a)and 1(b).As can be seen,TPCK
2(b),2(c)),and the specificactivity of the worker trypsin
did not inhibit this enzyme. In order for TPCK to was approximately 1.6 times greater than that of
inhibit an enzyme it must possessa histidine in its males.
The trypsin activities for V. maculiJi'onsqueens are
active center and possessa specificity for aromatic
amino acids. That TPCK did not inhibit the wasp also shown in Table 1. Although the queenscollected
trypsin further suggestsa similarity tretweenthe tryp- in the field and those that eclosed in the laboratory
were analyzed separately, there were no significant
sin of V. plermanicaand bovine trypsin.
Bovine chymotrypsin contains a serinein its active differencesin their activities, and therefore all replicenter and is inhibited by PMSF. As can be seen cates of both groups were averaged. The specific acin Figs. 1(c) and 1(d) the chymotrypsin of both male tivity of queen trypsin exceededthe worker trypsin
and worker V. germanica was inhibited by PMSF, by a factor of 1.1 and was 3.7 times greater than
but the degree of inhibition was neither immediate in males.
Significant amounts of chymotrypsin were also
nor very obvious rn a 7 min experiment.In fact, during the first 3 min of the experiment,the rate of the found in the midguts of male, worker, and queen Z.
inhibited reaction equalledor in many casesexceeded maculifrons(Table 2). As in V. germanica the reaction
the rate of the uninhibited reactiorr(e.-e.Fig. l(d)). In rates were not linear with time, with the first 2 to
most casesthe assayhad to exceed7 min before inhi- 3 min being greater than the last 4 to 5 min (Figs
The chymotrypsin activity was lessthan
bition could be demonstrated.With an extendedincu- 2(d),2(e),2(f)).
bation time, however,PMSF always inhibited V. ger- the trypsin-like activity in all castes.Trypsin specific
manicachymotrypsin. Therefore, V. germcmicachymo- activity exceededthat of chymotrypsin by a factor
trypsin is similar to bovine chymotrypsin in that both of 1.8 in workers, 1.1 in males, and 1.3 in queens.
enzymesare inhibited by PMSF. The wasp enzyme The specific activity of chymotrypsin in queens was
l9-5
Digestive proteasesof two speciesof wasps
o.04
o.03
o.o2
o.ol
o.04
n n a
o.o2
o .o l
o
o
o
c
o
-o
o
o
-o
n n ?
o.02
ool
o
(e)
U.UO
( f )
o
o .0 4
o.o4
n n ?
o.02
o.ol
v.va
0.ol
Minutes
Fig. 2. The trypsin-like midgut activities of male (a), worker (b), and queen (c) and the chymotrypsinof mile (d), worker (e), and queen (f) vespula mctculifions. The response is
lik"e midgut i"iiuiti..
and TPCK (!--tr)'
TLCK (A-A),
shown oithese enzymes (O- f) to PMSF (O-O),
1.6 times greater than in males and l'7 times greater
than in workers.
CarboxypeptidaseA activity was found in the midguts of all castesof V. maculiJions.In queenscarboxypeptidaseA specific activity was greater than chymotrypsin; it was in fact about equal to that of trypsin.
This result is the same when activity is expressedas
units per midgut (Tables 1,2, and 3). In castecomparisonsof carboxypeptidaseA specificactivities,the
qu.en enzyme had the highest activity, followed
equally by workers and males (Table 3).
Carboxypeptidase B activity was found in queen
and male V. nuculiJronsmidguts but not in midguts
of workers. Although males and queens possessed
measurable amounts of carboxypeptidase B, the
valueswere low (Table 4). Of all enzymesexamined,
B occurred in the lowest concencarboxypeptida-se
carboxypeptidase
tration. Of the 6astesthat possessed
B, the queens possessedmore, with the specific activity exceedingthat of males by a factor of 1.7.
Effectsof inhibitorson V. maculifrons trypsittand chynntrypsin
Vespulamaculilronstrypsin-like enzyme of workers,
males and queenswas inhibited by PMSF (Figs. 2(a),
2(b), 2(c)).The results were similar to those obtained
for trypsin in V. germnnica.The range of inhibition
obtained with 1.9mM PMSF in all castesof tr2.muculifi'ons was from 43'A to 100%. The trypsin of this
specieswas also inhibited by TLCK (Figs. 2(a),2(b),
2(c)).The range of inhibition observed in all castes
was 6Ch to 100|i,.TPCK did not inhibit the trypsin
Bovine trypenzymeof this species(Figs.2(a),2(b),2(c)).
sin and the trypsin enzyme of V. mautldrons therefore
are similar in their responseto inhibitors.
V. ntaculi'fionschymotrypsin was also inhibited by
PMSF (Figs.2(d),2(e),2(f)),but as in V. germanicaIhe
degree of inhibition was negligible in the first few
minutes of the assay. However, inhibition occurred
if the enzymereaction was continued. TLCK did not
chymotrypsin (Figs. 2(d), 2(e),
inhibit V. mucultJi"ons
2(f)). TPCK did inhibit the chymotrypsin of male,
worker, and queen V. maculifrorzs(Figs. 2(d), 2(e),2(f)).
However, the degree of inhibition was low in the initial minutes of the assay but increased with time.
Ten minute incubations were sufficient to demonstrate inhibition. These results were similar to those
noted in V. germanica.
DISCUSSION
Our results demonstrate that adult Vespula of the
two speciesexamined contain considerable digestive
proteise activity. We tested for the presence of four
enzymes(trypsin, chymotrypsin, carboxypeptidaseA,
and carboxypeptidaseB) that are normally associated
with protein digestion in higher animals zrnd found
activilies in Vespulamidguts similar to the activities
of thesefour mammalian enzymes.All castemembers
possessedall four activities, with one exception: Z.
maculifronsworkers lacked carboxypeptidaseB activity. Average total protease units per midgut can
196
DoNero E. GnocaN aNo JanausH. HuNr
be calculated, and although our survey is narrowly
limited, it seemslikely that intraspecific caste rankings are queen
obtained for averagetotal proteaseunits per midgut
in V. ntamlifrons are queen : 0.31, worker : 0.10,
male : 0.05, and in V. lermanica, worker : 0.59,
male : 0.22. Ecological or evolutionary correlatesof
thesecaste differencesare not now apparent.
We assumedthat proteasesextracted from the tr/espula midguts during the leaching process were from
the midgut cavity. There is a possibility,however,that
some of the enzyme activity was intracellular in origin, but we did not examine the midguts for the presence of lysozomes.The inhibition characteristicsof
the chymotrypsin-like enzyme that indicated differencesfrom bovine chymotrypsin are, therefore, suspect in this regard.We further assumed,however,that
regardless of the origin, the proteases that were
presentwere capable of protein digestion.
Our results do not agree with results reported by
IraN er al. (1969)on Vespaorientalis.These workers
concluded that adult V. orientalis do not possess
digestivc proteases.The discrepancy between their
results and ours might be attributed to two factors
other than the speciesinvolved: methodology and interpretation. All of our studies were conducted on
isolated Vespulamidguts. kaN er a/. obtained digestive juices by squeezingthe abdomensof Vespaorientulis and aspirating the vomitus with a pipette. Due
to the proventricular anatomy of wasps the vomitus
that thev exiimined was almost certainly of crop origin, not midgut. Also, kaN et ul. (1969) examined
total body proteaseactivity rather than midgut proteaseactivity. The protease specific activity of total
body protein would be lower than that of midgut
protein. Interpretative differencesmay be noted by
examining the tabular data of kaN et al. (1969).
These data are presented in an irregular manner;
specific activity values for all enzymes in all castes
are not given. Of the values presented,however, we
feel that most do, in fact, indicate positive enzymic
activity. The primary discrepancybetweenour results
from Vespuluand the reported results from Vespa
orientalis may be largely one of data interpretation.
In our assayson Vespulawe found no instance in
which any adult caste member was totally lacking
in midgut proteaseactivity.
ln summary, we have documentcd the presenceof
digcstiveproteasein midguts of all castesof the two
Vespulaspeciesthat were available for study. These
insectsare in all likelihood fully capable of protein
dieestion.
Acknowletlgtements----We are
grateful
to
Davro
O'BnyaNr for his excellenttechnical assistanceand to Roy
R. SNerlrNc for identification of the wasp taxa. For review
of the manuscript we thank Roennr Bor-u.
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