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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. REFERENCES B a x r n H . G . a n d B a r e n L ( 1 9 7 3 )A m i n o a c i d s i n n e c t a r and thcir evolutionary significance.Iluture, Loncl. 341. 543 545. Barsn H. G. and Baren f. (1975)Studies ol nectar-constitution and pollinator-plant coevolution. In Coeuolution ol Animals and Plunts (Ed. by Grt-senr L. E. and RavsN P. H.)., pp. 100 140. Univ. of Texas Press,Austin. Folr J. E,., Ptp.z D. A.. Cnnnoll W. R.. and GlaoNr;n J. A. 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