Digestion, emphasizing trypsin activity, in adult simuliids (Diptera) fed blood, blood-sucrose mixtures, and sucrose

J. Insect Physiol., 1968, Vol. 14, pp. 205 to 222. Pergamon Press. Printed in Great Britain

DIGESTION, EMPHASIZING TRYPSIN ACTIVITY, IN ADULT SIMULIIDS (DIPTERA) FED BLOOD, BLOOD-SUCROSE MIXTURES, AND SUCROSE Y. J. YANG Department

and D. M. DAVIES

of Biology, McMaster (Received 30January

University, Hamilton, Ontario, Canada 1967; revised 21 August 1967)

Abstract-In vitro trypsin activity was demonstrated from six simuliid species in three genera. Trypsin activity in the black-flies was confined mainly, if not completely, to the midgut. Specifically different levels of the enzyme activity were found in sugar-fed Simulium venustum, Cnephia dacotensis, and Prosimulium decemarticulatum. There was no trypsin activity from the salivary glands. The pH optimum for trypsin activity in both S. venustum and S. rugglesi females were identical at 8.4. The phenomenon of an increased trypsin activity after feeding on whole blood, blood-sucrose mixtures, or erythrocyte-sucrose mixtures was demonstrated. The blood-sucrose mixtures in the crop of female black-flies stimulated steady enzyme activity, as the mixture was dispatched slowly to the midgut for digestion. A low environmental temperature depressed midgut secretion in S. venusturn females. In this species the males had the same trypsin activity as sugar-fed females but failed to increase the enzyme activity after feeding on a blood-sucrose mixture. Presumably in simuliids, as the amount of sucrose added to a blood preparation decreases, the larger is the proportion of the meal going to the midgut but the size of the meal is reduced. No pepsin-like enzyme was detected in either sex of P. &scum.

INTRODUCTION

IN RECENTyears many bloodsucking insects have been studied for their proteolytic enzymes. Most of these investigations have dealt with mosquitoes and fewer with Glossina, Stomoxys, and others. Of the proteolytic enzymes facilitating the breakdown of the complex blood proteins, trypsin is a primary enzyme which attacks the proteins by breaking the centrally located peptide bonds. Reported information indicates that one of the enzymes frequently found in these insects is a proteinase, which is usually active at neutral or alkaline pH, thus resembling vertebrate trypsin more than other known proteinases (WIGGLESWORTH,1965). Bloodsucking Simuliidae, which occur throughout the world, are incriminated as important vectors of disease organisms or are in themselves of considerable economic importance due to the vicious bites which they inflict upon man and other animals, particularly livestock. Reports of research on digestive enzymes in the Simuliidae are few. WANSON(1950) was the first to mention that in Simulium damnosum Theob. blood is digested by proteolytic enzymes. He reported briefly 205

206

Y. J. YANGANDD. M. DAVIES

that S. damnosum had very weak amylase but had strong tryptase and peptidases which were active at an alkaline pH of 8~5,‘and capable of digesting small fragments of coagulated egg yolk in vitro. RUBTZOV (1958) believed that the epitheiial cells of the anterior section of the black-fly midgut secreted digestive enzymes only at the moment the blood entered the midgut. However, this was his impression through observations of a granular secretion in the midgut epithelial cells while dissecting blood-engorged flies. Such a granular secretion in the midgut of a nonblood-fed black-fly, S. jenningsi Mall. (as nigroparvum Twinn) was reported by Cox (1938). BRUES (1946) described in his text that a minute haemorrhagic spot, which was frequently seen after the bite of a black-fly, resulted from the action of a powerful proteolytic enzyme contained in the saliva. However, this is yet to be demonstrated. The present work was initiated to investigate, in some species of Canadian black-flies, the presence and nature of trypsin activity, especially in relation to blood digestion.

MATERIALS

AND METHODS

Collection of Simuliids Most of the adult black-flies used were reared from larvae and pupae collected in Algonquin Park, Ontario, in the early summer of 1966. The larvae were maintained in plastic containers (7.5 x 7.5 in.) in which water was agitated by air bubbles. Small amounts of baker’s yeast were added as food and the containers were kept at 10 or 15°C. Emerged adults were held at 18-20°C in cardboard cylinders (3.5 x 4.5 in.), the top and bottom of which were covered with nylon screen. The bottom of the cylinder rested on water-soaked absorbent cotton in plastic Petri dishes, and sugar cubes or blood-sugar mixtures in absorbent cotton were supplied on the top screen for the adults. Other black-flies were collected, engorged with blood, from different hosts at the Wildlife Research Station in Algonquin Park, Ontario, during the summer. Most of the blood-fed flies were kept in the cardboard cylinders at 1%2O”C, unless otherwise mentioned, and provided with dry sucrose and water until processed for experiments. Preparation of homogenates To prepare various tissues of the fly as an enzyme source, flies were immobilized by chilling in a deep freezer for lo-20 min, and then dissected in cold Tris-HCl buffer at pH 8. The various tissues to be studied were separated and placed in small vials (10 x 3.5 mm), five to ten similar tissues being pooled, and then deep frozen ( - 30°C) until homogenized. Whole flies or carcasses were homogenized in the buffer using a Pyrex glass tissue-grinder, and after centrifuging at 2500-3000 rev/min for 10 min, additional buffer was added to the supernatant to provide a volume of 0.5 ml or 1-O ml, depending on the number of tissues used. On the other hand, the midguts, crops,

DIGESTION, EMPHASIZING TRYPSIN ACTIVITY, IN ADULTSIMULIIDS

207

salivary glands, or Malpighian tubules were macerated with fine needles under the dissecting microscope, and after making up to 0.5 ml or 1.0 ml by adding buffer, the suspension was used as an enzyme source without centrifugation. All of the homogenates and suspensions were immediately stored in the deep freezer until needed. All glassware which had contacted the enzyme source was acidcleaned. TAME

as substrate for determination of trypsin activity

TAME, p-tosyl-L-arginine methyl ester HCl (Nutritional Biochemicals Corp., Cleveland, Ohio), was chosen as a substrate for trypsin analysis. This synthetic substance has been demonstrated as a most specific and suitable substrate for determination of even traces of trypsin by several workers. SCHWERTet aE. (1948) reported that TAME is rapidly hydrolysable by trypsin (900 times faster than benzoyl-L-arginine amide (BAA)), that it does not undergo spontaneous hydrolysis over a wide pH range, nor is it hydrolysed by chymotrypsin. Later SCHWERT and EISENBERG(1949) a1so d emonstrated that the products are not competitively inhibitory. Considering the above-mentioned advantages, HUMMEL(1959), using TAME as substrate, was able to determine traces of trypsin in the presence of a larger amounts of chymotrypsin, using the spectrophotometric method. SIEGELMANet al. (1962) was the first to apply TAME to determine trypsin in normal and diseased human serum, and developed a useful calorimetric method. RAO and FISK (1965) adapted this method to determine trypsin activity in the female Tampa cockroach. Determination of trypsin activity

The quantity of trypsin activity in the black-fly homogenates was assayed, in duplicate, using the calorimetric method developed by SIEGELMANet al. (1962) and adapted by RAO and FISK (1965). A 50 ~1 portion of homogenate was incubated with 0.2 ml of 0.25 M TAME in 0.8 ml boric acid-borax buffer of pH 8.0 at 37°C for 1 hr. After the incubation, further reaction was stopped by adding 10% trichloroacetic acid (TCA). Following centrifugation at 2500 to 3000 rev/min for 5 min, 0.5 ml of the supernatant was treated with potassium permanganate, sodium sulphite, and chromotropic acid. The mixture, after heating for 15 min in a boiling water bath and cooling for 3 min in running water, was read against water in the Spectronic 20 calorimeter at 580 ml”. The control was treated in the same way as the experimental except that the homogenate was added after the reaction was stopped by TCA. Trypsin, twice crystallized and salt free (General Biochemicals, Ohio), was dissolved in 0.05 M calcium chloride buffer, the pH was adjusted to 2.5 with hydrochloric acid, and this was used as a standard for trypsin activity. All trypsin activity values in Results and Discussion are expressed as pg of pure crystalline trypsin,/lO flies (or tissues) per ml, unless otherwise stated.

208

Y. J. YANG AND D. M. DAVIES RESULTS

Trypsin activity in various tissues of Simuliidae Since preliminary experiments, using homogenates of whole black-fly bodies as an enzyme source, demonstrated a detectable amount of trypsin activity in sugarfed flies, an assay was made to determine whether the trypsin activity was limited to the midgut or was present elsewhere in the fly. Forty sugar-fed females of S. venustum Say, 6 to 7 days after emergence from the pupae, were dissected and various tissues assayed for the enzyme activity (Table 1). Little or no trypsin activity was detected in the salivary glands, TABLE I-TRYPSIN

ACTIVITY(pg/ml) DRY

IN VARIOUSTISSUESOF S. venustum FEMALES*FED SUCROSE

AND

WATER

Tissues analysedt No. test 1 2 3 4 Mean

Midgut 2.9 3.7 3.0 3.2 3.20

1M.t.

s.g.

0 0 0 0

0 0 0

-

-

R.f. 2.1 1.4 1.6 1.5 1.65

Carcass 0.1 0 0,2 0 -

W.f. 4.6 4.5 5.0 4.9 4.75

* Females collected in September. t M.t. : Malpighian tubule; S.g. : salivary gland; R.f. : residual fluid; W.f. : whole fly.

Malpighian tubules, or carcasses. The trypsin activity found in the residual fluid (mainly diluted haemolymph) after dissection was believed to be derived from the midgut contents which may have been squeezed out while separating other tissues in the dissecting fluid. The midgut always showed the highest activity, suggesting that most of the trypsin, if not all, is located in the midgut. When two suspensions of pooled hindguts (15 hindguts/ml for each pool) were tested, one pool showed no activity, while the other pool showed a trace (less than 0.1 pg trypsin/ml), presumably contamination from midgut contents. Fifty females of S. venustum which fed on a mixture of human blood and sucrose solution were dissected at different times after the meal, and the trypsin activity was determined from various tissues (Table 2). The carcasses including the salivary glands and Malpighian tubules, and also the crops which were filled with the blood and sucrose mixture, showed at most only a trace of the activity, again believed to be a contamination from the midgut contents. Trypsin activity in the midgut, however, sharply increased after the meal, compared with the activity in sugar-fed females (Table 1). Noting the trypsin activity in the residual fluid of sugar-fed S. venusturn females in Table 1, more careful dissections were made of thirty females of S. vittatum Zett., a mammalophilic species autogenous

DIGESTION, EMPHASIZING TRYPSIN ACTIVITY, IN ADULT SIMULIIDS TABLE 2-TRYPSIN

209

ACTIVITY &g/ml) IN VARIOUS TISSUES OF S. senustu?n FEMALES FED A BLOOD-SUCROSE MIXTURE (3 : 1 RATIO) Tissues analysed

Time after meal Midgut

(hr) 6 9 12 28 48 70 96 123

for the first gonotropic (Table when

3). fully

Carcass

5.7 8.4 7.7

>O.l >O.l 0

0.5 0 0.2

9.8 8.3 -

0 >O*l -

0 0

cycle, and various

In this species trypsin

compared

with

Crop

with activity

grown

eggs,

TABLE 3-TRYPSIN

10.0 9.0 8.0 10.1 7.4

-

tissues were tested for the enzyme

activity in the residual

in the midgut.

showed

Whole fly

The

activity

fluid was almost negligible,

carcasses,

which

included

ovaries

no activity.

ACTIVITY (pg/ml)

IN VARIOUS TISSUES OF S. oittattlm FEMALES* FED

DRY SUCROSE AND

WATER

Tissues analysed t No. test

Midgut

S.g.

R.f.

Carcass

W.f.

1 2 3 Mean

1.2 2.1 1.9 1.7

0 0 -

0.5 0 0.8 0.4

0 0 0 -

2.2 2.0 1.4 1.9

from

the midgut

* Females collected in September. t For details, see Table 1. When

the trypsin

were totalled was

good

which trypsin

agreement

fed on blood activity

The activity

activities

and compared

foregoing was

in the overall and sucrose

between

in the

interfered

little with

remaining

experiments,

performed

with homogenates

14

trypsin

mixture

the midgut

experiments

located

and other tissues tested separately

with the ones from whole

analysing

(Table

indicated and

trypsin

that that

flies.

in S.

1 and 3), there

wenustum

females in

fly.

most use

activity

for trypsin

of whole

flies (Tables Also

2), there was little difference

and the whole

midgut

determining

activity.

of

of

the

detectable

homogenates

in the midguts.

activity

of the black-fly

trypsin

of whole Therefore, midgut,

fly the were

210

Y. J. YANG AND D. M. DAVIES

The eflect of incubation time and enzyme concentration

To determine the relation between incubation time and enzyme activity, homogenates of blood-fed females of two different ornithophilic species, Prosimulium decemarticulatum (Twinn) and Simulium rugglesi Nicholson and Mickel, were used (Fig. 1). With a homogenate of P. decemarticulatum females (about 4 days after

Incubation

time (min.)

FIG. 1. Relationship between trypsin activity of female black-fly homogenates and incubation time at 37°C. A-A, P. decemarticulatum, 5 flies/ml; @-a, P. decemarticulatum, O-5 fly/ml; O-O, S. rugglesi, 1 fly/ml.

blood-feeding on a bantam chicken), the hydrolysis of the substrate increased rapidly for the first hour of incubation and then remained more or less constant. This indicated that there was insufficient substrate left after 60 min of incubation to be hydrolysed by the highly active homogenate. When this homogenate was further diluted, however, the enzyme activity increased linearly for at least the first 2 hr of incubation. A homogenate of 5’. rugglesi females (6-18 hr after bloodfeeding on a Pekin duck) showed a linearity during the 2 hr incubation period. A tenfold increase in the concentration of the homogenate of P. decemarticulatum females resulted in a similar increase in trypsin activity (e.g. Fig. 1 at 30 min), suggesting a linear relation over this range of concentration.

DIGESTION,

EMPHASIZING

TRYPSIN

ACTIVITY,

IN

c 5

6

7

B

9

ADULT

5

lo

211

SIMULIIDS

I

1

1

6

7

8

’ 9

’

10

PH

PH

2. The effect of pH upon the trypsin activity of S. venusturn and S. rugglesi homogenates. Buffers and symbols: e-0, citric acid/HPO,; O-O, Tris aminomethane/HCl; A-A, boric acid/borax.

FIG.

I

Substrate

/ Subslrote FIG.

3.

2

3

cwcentrotion,

2

concenfrotion,

4

mole x IO?

4

3

mole

5

reaction

6

tube

5

x10-?reaction

6

tube

The effect of substrate concentration upon the trypsin activity S. venusturn and S. rugglesi homogenates at pH 8.0 and 37°C.

of

212

Y. J. YANG ANDD. M.

DAVIES

The efiects of pH and substrate concentration

The pH optima for trypsin activity in females of S. venustum fed a bloodsucrose mixture and of S. rugglesi blood-fed on a duck were determined by preparing homogenates of whole flies, ground up in cold distilled water which had been neutralized with diluted NaOH and/or HCI. The homogenate was then adjusted with buffer solutions to produce a concentration of about four females/ml for S. venustum or of about 5 females/ml for S. rugglesi. In three buffers used, the trypsin in the two species showed similar activity, being active between pH 8 and 8.5 with a maximum activity at pH 8.4 (Fig. 2). The effect of substrate concentrations are shown in Fig. 3. From the data obtained from substrate concentrations below the point at which the enzyme is saturated, the values of the Michaelis-Menton constant (K,) were calculated, applying the Lineweaver-Burk double reciprocal plot of the activity and substrate concentration. The K, for the S. venusturn homogenates was 2.4 x 10e3 M and for the S. rugglesi homogenates 3.1 x 1O-3 M. The trypsin activity in sucrose-fed Simuliidae after emergence

Adults of S. venusturn, Cnephia dacotensis (Dyar and Shannon) and P. decemarticulatum, being supplied only with dry sucrose and water, were tested for trypsin activity at intervals after the flies had emerged from pupae. These species, belonging to three simuliid genera, have different feeding behaviours. S. venusturn, an anautogenous, mainly mammalophilic species, is known as the most serious simuliid pest of humans in Ontario. C. dacotensis, a non-blood feeder with eggs mature in newly emerged females, is highly parasitized by mermithid nematodes (DAVIESet al., 1962). P. decemarticulatum is anautogenous and known specifically as a bird feeder (BENNETT, 1960). Each of the three species tested showed a more or less constant level of trypsin activity during 1 to 24 hr after emergence from the pupae (Fig. 4). It was obvious that sucrose stimulated no trypsin activity since these flies began feeding on sugar within a few hours after emergence.

Ti \ 5.0 z

4.0-

Q. ‘\

; .fl .-z

3.0-

5

2.0.

c-

’

00’

l.O-

A---_____

--_(y

_-

-

c

‘2

_

__A-__

e

_

l -_

----0’

.

___-A._._

-

”

---_

___------__a

>

c 0

4

8

12

-0

16

20

-

-1

24

Age in hours FIG. 4. Trypsin activity in females of S. venusturn, C. dacotensis,P. decemarticulatum at different intervals after emergence from the pupae. Flies were provided with dry sugar and water during the experimental period. O-O, S. venusturn; @-a, C. dacotensis; A-A, P. decemarticulutum.

213

DIGESTION,EMPHASIZINGTRYPSIN ACTIVITY, IN ADULTSIMULIIDS

Trypsin activity in male and female Simuliidae Since it is only the female black-fly

which is capable of piercing

and sucking

blood

from vertebrates and which must digest this food to supply nutrients for oiigenesis, it was of interest to compare the trypsin activity in the two sexes. Before the females took blood, their enzyme activity was practically that of the males in the three species tested (Table females of C. dacotensis, a non-blood TABLE 4-TRYPSIN

identical with

4), and that of males and of

feeder, were also similar.

ACTIVITY IN MALESAND FEMALESOF SIMULIIDSPECIES Trypsin activity &g/ml) Species

s. vt?nustum S. P. P. C.

vittatum decemarticulatum fuscum dacotensis

Male

Female

3.3 (5)* -

343 (5) 1.9 (3) 1-o (5) 0.9 (5) 2.1 (5)

l-2 (3) 0.5 (5) 1.8 (3)

* Average from number of tests in parentheses.

Trypsin activity after blood feeding The subsequent experiments

were undertaken in order to determine how much

the trypsin activity increases in black-flies which blood-fed

a chicken were analysed at different increasing

after a blood meal.

S. rugglesi females

on a duck and P. decemarticulatum females which blood-fed times after the blood meal.

The

trypsin activity in the two species after a blood meal differed

In S. rugglesi there was a gradual increase up to 18 hr followed

on

pattern of (Fig.

by levelling

5). off,

whereas in P. decemarticulatum there was a sharp increase before 5 hr and then the activity remained more or less constant for at least 24 hr.

Induced feeding of S. venustum females with blood-sucrose mixtures Laboratory generally

in captivity, captivity

breeding

of successive

been unsuccessful.

generations

of any simuliid

species

has

This is partly because these species feed reluctantly

although a few Ontario simuliids can be induced to feed on hosts in

(DAVIES and PETERSON, 1956; WOOD and DAVIES, 1966).

On the other

hand, WENK (1965) was able to induce many females of Boophthora erythrocephala (De Geer), a mammalophilic simuliid, to feed on humans, rabbits, or ‘naked blood in the laboratory.

However,

to induce captive S. venusturn females to feed

on hosts in large numbers has been unsuccessful.

Realizing

that female simuliids

feed on nectar and water in addition to blood in nature, an attempt was made to feed them on blood-sucrose mixtures. S. venustum females were netted as they flew around the authors in Algonquin kept in the laboratory

until used.

The

Park, Ontario,

in early sI_m-Imer and were

contents of a few crops were tested with

Y. J. YANG ANDD. M. DAVIES

214

I3 -

12.

II IO-

Time

after

meal

(hr.1

FIG. 5. Differences in amount of trypsin activity in females of two simuliid species at various intervals after blood feeding on birds. O-O, S. rugglesi fed on duck; l-@, P. decemarticulatum fed on chicken.

100

X Humm

blood

% Chicken blood

80

% Duck

50 blood

FIG. 6. The percentage of unfed, partially fed, and fully fed S. oenustum females in relation to the proportion of blood and saturated sucrose solution in various mixtures presented as food. Confidence limits of the percentages taken at 0.05 x 2 level of significance. -, Unfed; - - - -, partially fed; ................... fully fed.

DIGESTION, EMPHASIZING

215

TRYPSIN ACTIVITY, IN ADULT SIMULIIDS

Benedict’s solution and shown to contain sugar. Whole blood or mixtures of saturated sucrose solution and titrated human, chicken, or duck blood were prewarmed to 40°C and provided for 3 hr to a group of female flies which had been given only water for the previous 24 hr. Of the 639 females used, 326 females were fed on human blood, 148 on chicken blood, and 165 on duck blood. The trends of feeding and lack of feeding are represented in Fig. 6. Evidently engorgement increases as the proportion of sucrose in the blood-sucrose mixture increased. Some flies fed partially on whole human blood but took a full meal only when the sucrose comprised 30 per cent of the blood-sucrose mixture. With duck blood feeding commenced only when some sucrose was added to the blood, and with chicken blood more than 20 per cent sucrose was needed in the mixture to stimulate any feeding. The number of flies fully engorged on the 50 : 50 mixture of either avian blood was much less than with the human blood-sucrose mixture. This is consistent with observations in nature, which indicate that this mammalophilic species seldom feeds on ducks (DAVIES and PETERSON, 1956) and rarely can be induced to feed on chickens, even when held near the skin in screened tubes. When five females, partly or fully fed on each human blood-sucrose mixture, were dissected 5 hr later to check whether the meal was in the crop or in the midgut, most of the meals were found in the crops, except for flies fed on whole human blood in which the meal was found only in the midguts. Distribution of blood-sucrose mixtures ingested by S. venustum females

Although most blood-sucrose mixtures were found in the crops in the above experiment, this provided an insufficient trend of distribution of the mixture in the female alimentary tract, presumably because saturated sucrose solutions were used. Therefore, the following tests were performed to determine specifically what concentration of sucrose in blood was responsible for sending the mixture to the crop or to the midgut in the females. S. venusturn females, 3 days after emergence from pupae in the laboratory, were grouped in the cardboard cylinders. Citrated human blood was serially diluted with 1 M sucrose solution and the TABLE

~-DISTRIBUTION

OF DIFFERENT BLOOD-SUCROSE MIXTURES s. 7%97’2UStUm FEMALES

IN MIDGUT

AND

CROP OF

No. of females having the mixture in Sucrose (moles) in blood 0.5 0.4 0.3 0.2 0.1 0.05 0*02-0~01

No. females exposed

Crop

25 25 25 25 25 25 50

5 1 1 0 0 0 0

Crop-midgut 10 18 11 2 4 2 0

Midgut 0 1 2 1 5 3 4

216

Y. J. YANG AND D. M. DAVIES

prewarmed blood-sucrose mixtures, soaked in cotton, were placed on the top screen of the cylinders for 5 hr. The flies were then dissected and the distribution of the mixture was examined (Table 5). Although the blood-sucrose mixtures were found in both crop and midgut in six of the seven mixtures tested, the amount of mixture in the crop or midgut showed dissimilarity. With solutions of 0.5 M sucrose in blood, all of the ten females had most of the mixture in the crop, with only traces in the midgut. With 0.4 M sucrose, fifteen of eighteen females had most of the mixture in the crops and three had much of the mixture in the midgut with a little in the crop. With 0.3 M sucrose seven of eleven females had more of the mixture in the midgut and four had more in the crop. In 0.2, 0.1, and 0.05 M sucrose only a small amount of the mixture was ingested by the females and the mixtures were equally distributed in both crop and midgut and no females had the mixture in the crop alone. Only four of fifty females offered 0.01 to 0.02 M sucrose in blood took traces of the mixture and these small meals were found only in the midgut. Trypsin activity in S. venustum females fed a blood-sucrose mixture

After 1 : 1 mixture of human blood and 1 M sucrose solution was given to about l-week-old males and females, previously starved for 24 hr, the partially and fully engorged females were kept at two temperatures : one group at 30 f 4”C, the other at 15 + 1°C. The males, which also readily fed on the mixture (although the amount ingested was much smaller than for females), were kept at 15 + 1°C. Most of this mixture was initially dispatched to the crop, but as this mixture must eventually pass into the midgut for digestion, determinations of trypsin activity in females were made at different times after they fed on the mixture (Fig. 7). The

Time

after

meal

(hr.)

FIG. 7. Trypsin activity in S. venustum adults after feeding a mixture of human blood and sucrose solution.

DIGESTION, EMPHASIZING

TRYPSIN ACTIVITY,

IN ADULT SIMULIIDS

217

trypsin activity in females kept at both temperatures was analysed for seconddegree polynomial curve fitting. Theoretical values of Y were plotted against corresponding X values. At 15°C secretion of trypsin in the females was apparently less active than in the females at 30°C. However, they showed similar patterns of enzyme secretion activity during the test period. It was notable that the trypsin secretion was being activated more than 8 days after the females fed on a bloodsucrose mixture. Although the action of trypsin in the females of 15°C was less than at 3O”C, it was still higher than the activity in sugar-fed females, suggesting that enzyme secretion was stimulated by the blood-sucrose mixture even at the low temperature. The trypsin in males at 15°C was not increased by the bloodsucrose mixture. The trypsin activity was rather decreased when compared with the one in unfed males (see Table 4). Trypsin activity in S. venustum females fed erythrocytes or whole blood

Since the blood-sucrose mixture stimulated increased trypsin activity in S. venusturn females, further experiments were conducted to determine what fraction of the blood was essential for the stimulation of enzyme production by the midgut. One batch of flies was fed on duck erythrocytes which were thrice washed in saline and then suspended in sucrose solution, while another batch was fed on duck whole blood diluted in sucrose solution. (Also a plasma-sucrose mixture (4 : 1) was tried without success.) Trypsin activity was determined in females at different times after they had partially or fully fed on the mixtures (Table 6). Similar experiments were conducted with oxalated cow blood and the enzyme activity was determined (Table 7). Trypsin activity in females fed on duck erythrocytes or cow erythrocytes was only slightly less than that in females fed whole blood. The erythrocytes apparently stimulated the enzyme production almost as much as whole blood. TABLE

6-TRYPSIN

ACTIVITY IN S.

venustum

FEMALES AFTER FEEDING ON DUCK WHOLE BLOOD

OR WASHED RRYTHROCYTES

Trypsin Time

in 1 M

@g/ml) -

Erythrocytes

(hr)

* Diluted

activity

after meal *

Whole

blood *

24

7.8

8.9

70

8.8

9.2

92

6.5

8.8

120

8.6

10.5

sucrose

solution

and made

up to 80%.

Test for pepsin-like enzyme in the black-jly, Prosimulium

fuscum Syme and Davies

A pepsin-like enzyme, which is a protein-cleaving enzyme like trypsin but active at acidic pH, is regarded as uncommon in adult insects (WIGGLESWORTH,

218

Y. J. YANG

TABLE 7-TRYPSIN

AND

D. M. DAVIES

ACTIVITY IN S. venustum FEMALESAFTERFEEDINGON cow WHOLE BLOOD OR WASHEDERYTHROCYTES Trypsin activity &g/ml)

Time after meal (hr)

Erythrocytes *

Whole blood *

24 36 48 72 96 120 168 194

7.0

-

7.2 6.8

6.8 -

8.2 -

9.6 7.8 7.4 7.8 8.7

* Diluted in 1 M sucrose solution and made up to 70%.

1965). Although no such enzyme has been reported in adult bloodsucking insects, for the sake of completeness in this study, a black-fly species was assayed for this enzyme, adapting the methods of CHAMPLAIN and FISK (1956) and LAMBREMONT et al. (1959). Homogenates were prepared from unfed adult males and females of P. j&urn, a mammalophilic species autogenous for the first oijgenetic cycle, to consist of 12 flies/ml of glycine-HCl buffer at pH 2.5. Azoalbumin (Sigma Chemical Co., St. Louis) was used as substrate. Analysis for the pepsin-like enzyme was essentially the same as that of LAMBREMONT et al. except that the activity readings were made at 440 pm in the Spectronic 20 calorimeter. There was no increase in O.D.‘s in four homogenate mixtures of each sex in comparison with the values obtained from boiled homogenate mixtures or with residual colour of the substrate alone. To check the method used, crystallized pepsin (General Biochemicals, Ohio) in measured amounts was added to female homogenate mixtures, resulting in a proportional increase of O.D. with the amount of added pepsin in experimental tubes when compared with boiled homogenate mixtures. DISCUSSION

In many species of Ontario simuliids, the females require a blood meal for the second and subsequent ovarian cycles, and often for the first (DAVIES et al., 1962). In the present study a proteolytic enzyme, presumably trypsin, was demonstrated in several species of simuliids. The enzyme activity was located mainly in the midgut when various tissues were tested. Similarly in Aedes aegypti the proteinase was located mostly in the midgut (WAGNER et al., 1961). Absence of trypsin activity in black-fly salivary glands agrees with reports of no proteinase in the salivary glands of other blood-sucking insects, e.g. Glossina and Chrysops (WIGGLESWORTH, 1929, 1931), A. aegypti (METCALF, 1945), and Stomoxys

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(ROSTOMand GAMAL-EDDIN, 1962). Nor was trypsin activity found in the crop of S. venusturn even when it was filled with blood-sucrose mixture. The trypsin activity of the midgut alone differed little from that of whole-fly homogenates, suggesting that there were no proteins which inhibited trypsin activity, as has been found in other animals (KARLSON,1963), or that the presence of such proteins had little effect on the trypsin determination. The pH optimum for trypsin activity in S. venustum and S. rzlgglesi females was 5.4, similar to that found by WANSON(1950) in S. dumnosum. The level of trypsin activity determined in sugar-fed black-flies at intervals after they had emerged from pupae, differed with the species, but the level remained almost constant for the 24 hr test period. This suggests that trypsin was present before or at least at the time of emergence, and that sugar feeding caused no change. Fairly high trypsin activity in the non-bloodsucking C. dacotensis may support DOWNES’S(1958) belief that originally Diptera were bloodsucking, and that non-bloodsucking is a secondary development. This belief may also be supported by male simuliids having levels of trypsin activity similar to sugar-fed females, but failing to increase the enzyme activity after feeding on a blood-sucrose mixture. A search has been made to establish biochemical differences between species of mosquitoes and other insects (MICKS and GIBSON, 1957; MICKS et al., 1959). The differences in trypsin activity demonstrated between various black-fly species, with further extension, might prove of use in taxonomy. The possibility of enzymes having a role in animal taxonomy was reviewed by WILSON and KAPLAN (1964). In two of the black-fly species studied, high trypsin activity was sustained beyond a 24 hr period, e.g. in P. decemarticulatum the enzyme activity was much greater 4 days after a blood meal than 1 day after, In addition histological observations revealed a fair amount of undigested blood in this species and in 5’. ruggZesi even 5 days after a meal. FALLIS (1964) observed that in 5’. rugglesi blood digestion was completed in 4-6 days at 19 to 20°C. The time after a blood meal at which maximum proteinase activity occurred differs in various insects. For Aedes aegypti a peak was reported after 18 hr (FISK and SHAMBAUGH, 1952) and 24 or 36 hr (GOODING, 1966b), in Culexfutigans after 36 hr (GOODING, 1966b), and in Stomoxys after about 13 hr (CHAI\IIPLAINand FISK, 1956). In black-flies temperature influenced the rate of trypsin production and probably the time for complete digestion of blood, as is true in mosquitoes (SHLENOVA,1938). A blood-sucrose mixture, which first went to the crop, apparently stimulated trypsin activity in S. venusturn females. The amount of this enzyme increase seemed independent of the concentration of blood in the mixture, but was related to the amount of mixture ingested. However, sucrose alone produced no increase in the enzyme activity, Thus in this respect, at least, the control of enzyme production in the midgut of black-fly appears to be similar to that in GZossina (LANGLEY,

1966).

activity was almost identical whether the females of S. venustum fed on human, cow, or duck whole blood or blood cells suspended in sucrose solution. Trypsin

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M. DAVIES

The proteinases of Aedes and Culex mosquitoes hydrolysed a haemoglobin preparation much more rapidly than they did the serum protein in vitro (GOODING, 1966a). The haemoglobin, which is the most abundant protein in the blood, is normally found in the erythrocytes, from which it may be released by haemolysis of the cells. In Aedes mosquitoes (SHAMBAUGH,1954) and in Glossina (LANGLEY, 1966), proteinase activity was less stimulated by blood cells suspended in saline than by serum or whole blood. The discrepancy between these findings may be because of species differences or because in the simuliids the erythrocytes were suspended in sucrose solution rather than isotonic saline. No pepsin-like enzyme was found in sugar-fed adults of the black-fly, P. fuscum which agrees with the findings for blood-fed adults of Stomoxys (CHAMPLAINand FISK, 1956) and of Gloss&a (LANGLEY, 1966), but Calliphora females contain a pepsin-like enzyme (FRASERet al., 1961). The distribution of blood-sucrose mixture in the alimentary canal of S. wenustum females resulted in part from the difference in concentration of the two components in the mixture. When the sucrose level in the blood was more than O-4 M, most of the mixture went to the crop, but when it was less than 0.2 M, more of the mixture went to the midgut. WENK (1965) observed similar results with another species of black-fly, Boophthora erythrocephala. The passage of blood-sugar mixtures into crop, midgut or both in black-flies also resembles that in mosquitoes (DAY, 1954; Hosor, 1959). I n mosquitoes the switching mechanism of the food passage may be controlled by various sensilla in the labrum and cibarial pump (HOSOI, 1959; VON GERNET and BUERGER,1966). Investigations are under way on the sensory organs in the black-fly which may regulate the food distribution. AcknowZedgements-The authors wish to thank Mrs. H. GYORKOSfor technical assistance and Mr. V. GOLINI for collecting and processing blood-fed S. ndgglesi in the field. We are grateful for facilities provided by the Ontario Department of Lands and Forests at the Wildlife Research Station in Algonquin Park. The senior author gratefully acknowledges scholarships from the Department of Biology, McMaster University, and an Ontario Government fellowship. The research was supported by grants from the National Research Council of Canada and from the Ontario Department of University Affairs. REFERENCES BENNETT G. F. (1960) On some ornithophilic blood-sucking Diptera in Algonquin Park, Ontario, Canada. Can. r. Zoo& 38, 377-389. BRUESC. T. (1946) Insect Dietary-An Account ofFoodHabits ofInsects. Harvard University Press, Cambridge, Mass. CHAMPLAINR. A. and FISK F. W. (1956) The digestive enzymes of the stable fly, Stomoxys calcitrans L. OhioJ. Sci. 56, 52-62. Cox J. A. (1938) Morphology of the digestive tract of the blackfly (Simulium nigroparvum). J. agric. Res. 57, 443-448. DADD R. H. (1961) Evidence for humoral regulation of digestive secretion in the beetle, Tenebrio molitor. J. exp. Biol. 38, 259-266 DAVIES D. M. and PETERSONB. V. (1956) Observations on the mating, feeding, ovarian development, and oviposition of adult black flies (Simuliidae, Diptera). Can. J. Zool. 34, 615-655.

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DAVIESD. M., PETER.SONB. V., and WOOD D. M. (1962) The black flies (Diptera, Simuliidae) of Ontario. Part I. Adult identification and distribution with descriptions of six new species. Proc. ent. Sot. Ont. 92, 71-154 (1961). DAY M. F. (1954) The mechanism of food distribution to midgut or diverticula in the mosquito. Aust. J. biol. Sci. 7, 515-524. DOWNESJ. A. (1958) The feeding habits of biting flies and their significance in classification. A. Rev. Ent. 3, 249-266. FALLIS A. M. (1964) Feeding and related behavior of female Simuliidae (Diptera). Exp. Parasit. X,439-470. FISK F. W. and SHAMBAUGH G. H. (1952) Protease activity in adult Aedes aegypti mosquitoes as related to feeding. OhioJ. Sci. 52, 80-88. FRASER A., RING R., and STEWART R. (1961) Intestinal proteinase in an insect, Calliphora vomitoria L. Nature, Lond. 192, 999-1000. GOODING R. H. (1966a) 1n vitro properties of proteinases in the midgut of adult Aedes aegypti L. and Culexfatigans (Wiedemann). Comp. Biochem. Physiol. 17, 115-127. GOODING R. H. (1966b) Physiological aspects of digestion of the blood meal by Aedes aegypti (L.) and Cztlex fatigans Wied. r. med. Ent., Honolulu 3, 53-61. HOSOI T. (1959) Identification of blood components which induce gorging of the mosquito. J. Insect Physiol. 3, 191-218. HUMMEL C. W. (1959) A modified spectrophotometric determination of chymotrypsin and thrombin. Can. J. Biochem. Physiol. 37, 1393-l 399. &w&SON P. (1963) Introduction to Modern Biochemistry, p. 147. Academic Press, New York. LAMBREMONTE. N., FISK F. W., and ASHRAFI S. (1959) Pepsin-like enzyme in larvae of stable flies. Science, N. Y. 129, 1484-1485. LANGLEYP. A. (1966) The control of digestion in the tsetse fly, Gloss&a morsitans. Enzyme activity in relation to the size and the nature of the meal. J. Insect PhysioE. 12, 439-W. METCALF R. L. (1945) The physiology of the salivary glands of Anopheles quardrimaculatus. J. nut. Malar. Sot. 4, 271-278. MICKS D. W. and GIBSON F. J. (1957) The characterization of insects and ticks by their free amino acids patterns. Ann. ent. Sot. Am. 50, 500-505. MICKS D. W., STARR C. F., and PARTRIDGEM. H. (1959) The vitamin-B content of mosquitoes. Ann. at. Sot. Am. 52, 26-28. PATTERSONR. A. and FISK W. F. (1958) A study of trypsin-like protease of the adult stable fly, Stomoxys calcitrans (L.). Ohio J. Sci. 58, 299-309. RAO B. R. and FISK F. W. (1965) Trypsin activity associated with reproductive development in the cockroach, Nauphoeta cinereu (Blattaria). J. Insect Physiol. 11,961-971. ROSTOM 2. M. F. and GAMAL-EDDINF. M. (1962) The chemical composition of the salivary glands of some blood sucking and of some non-biting flies in Egypt (Diptera: Cyclorrhapha)-I. Enzymes. Proc. Egypt. Acad. Sci. 16, 21-28. RUBZOV I. A. (1958) Gonotrophic cycle in bloodsucking black flies (Simuliidae). Parasit. Symp. Zool. Inst. Acad. Sci. U.S.S.R. 18, 255-282 (in Russian). SCHWERT G. W. and EISENBERG M. A. (1949) The kinetics of the amidase and esterase activities of trypsin. J. biol. Chem. 179, 665. SCHWERT G. W., NEURATHH., KAUFMANS., and SNOKE J. E. (1948) The specific esterase activity of trypsin. J. biol. Chem. 172, 221. SHAMBAUGHG. F. (1954) Protease stimulation by foods in adult Aedes aegypti L. OhioJ. Sci. 54, 151-160. SHLENOVAM. F. (1938) Vitesse de la digestion du sang par la femelle de l’dnopheles maculopennis messeae aux tempkratures effectives constantes. Med. Parasit., Moscow 7, 716-735 (in Russian with French summary). SIEGELMANA. M., CARLSONA. S., and ROBERTSONT. (1962). Investigation of serum trypsin and related substances-I. The quantitative demonstration of trypsin-like activity in human blood serum by a micromethod. Archs Biochem. Biophys. 97, 1.50-163.

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