Excretion of proteolytic enzymes by Aedes aegypti after a blood meal

J. Insect Physiol.,

1975, Vol. 21, pp. 1681 to 1684.

EXCRETION

Pergamon

Press.

Printed

in Great Britain.

OF PROTEOLYTIC ENZYMES BY AEDES AFTER A BLOOD MEAL

AEGYPT.

HANS BRIECEL * Department

of Entomology,

University of Georgia, Athens, GA 30602, U.S.A.

(Received

27 February

1975)

Abstract-Excretion of active proteolytic enzymes during the period of blood digestion in a mosquito has been demonstrated for the first time. The rate of excretion has been determined for both proteases and uric acid; each appears in a distinct peak. During the first half of the digestion period, when protease activity in the midgut is increasing, uric acid excretion predominates. During the second half of the digestion period, after the protease has reached its maximum in the midgut, there is considerable excretion of active protease, mainly trypsin. By sealing the anus after feeding (blood enema), it has been demonstrated that secretion of the proteolytic enzymes in the midgut actually stops when maximum activity is reached. Sealing the anus did not interfere with egg development. A model for protease secretion is suggested in which the proteolytic enzymes are induced by their substrate (globular proteins), and secretion stops when 80 per cent of the protein is digested, or the inducer is removed. INTRODUCTION SEVERAL factors responsible for the secretion of the proteolytic enzymes have been investigated by BRIECEL and LEA (1975). In their experiments, females A&es aegypti digest the blood meals within 30 to 40 hr. Protease activity in the midgut, triggered by the presence of blood, reached a maximum after about 20 to 24 hr. They also observed a rapid decrease in protease activity once the maximum was reached. I have now investigated this dramatic decrease in activity by measuring protease activity in the excreta. Enzyme excretion was also compared with uric acid excretion, which has been observed before (TERZIAN et al., 1957; BRIEGEL, 1969).

MATERIALS

AND

METHODS

The experiments were made with 3 to 4 day old females of Aedes aegypti, strain Segemaganga (BRIEGEL and KAISER, 1973). Adults were held at 27°C and 80”/0 r.h. Except for 1 day before the blood meal, a 10% sucrose solution was available ad lib. The experimental blood meals were given by enema at 3 ~1 of heparinized rat blood per female as described by BRIEGEL and LEA (1975). Blood fed females were kept singly in glass vials (2 cm dia.) or in groups in plastic cups. Faeces were collected from these containers by rinsing the dry material with phosphate buffer (l/15 M, pH 7.9) or lithium carbonate (1 y’). * Permanent address: Institute of Zoology, sity of Zurich, Kiinstlergasse 16, CH-8006 Switzerland.

UniverZurich,

Sealing the anus of females was achieved by administering a drop of paraffin with an electrically heated loop of nichrome wire. The females were lightly anaesthetized with ether and excessive heat was avoided. The biochemical methods were those given by an earlier paper (BRIEGEL and LEA, 1975). However, for chymotrypsin an improved assay was adopted (KANG and FUCHS, 1973). For the determination of uric acid a method developed by VAN HANDEL (1975) was used. RESULTS Sequence

of protease

and uric acid excretion

After an enema of 3 ~1 blood the maximal protease activity in the midgut measured about 70 (O.D. x 100) per female (BRIEGEL and LEA, 1975). The excreta of blood fed females were collected from the time of blood feeding until digestion was complete, and then analysed for proteases and uric acid. Protease activity was more than half the amount at maximum (45 O.D. x 100) and the uric acid excretion measured 45 pg/female. Next, I investigated the time course of protease and uric acid excretion. Immediately after an enema (3 /*l of rat blood) two groups of 10 to 15 females each were tethered in such a way that their excreta were discharged into a small beaker. The beaker for one group contained phosphate buffer for protease assay, while the beaker for the other group contained lithium carbonate for uric acid determination. These solutions were changed every 3 hr until digestion was completed in all females, i.e. 48 hr.

1681

HANSBRIEGEL

1682

Active proteases were excreted along with uric acid throughout the whole period of blood digestion (Fig. lA, B). By comparing the rate of excretion of

A

RATE

OF

HOURS

AMOUNT

EXCRETION

AFTER

OF PROTEASE

HOURS AFTER

ENEMA

EXCRETION

ENEMA

Fig. 1. Excretion of proteases and of uric acid in A. aegypti females after equal enemas of 3 rl of rat blood. (A) The rate of protease and uric acid excretion per female per hr. (B) Comparison of proteolytic activity as measured in the midgut (*, from BRIEGELand LEA, 1975) with proteolytic activity in the excrete.

protease and uric acid it becomes evident that each shows a distinct peak. The majority of uric acid (about 80 per cent of the total) appears during the first 24 hr and decreases thereafter. On the other hand, very little protease was excreted for the first 24 hr ; the major output of active enzyme occurred between 30 and 40 hr after the blood meal. These two phases can also be recognized visually in a mosquito cage : during the first 24 hr the dry excreta have a white or faintly yellow colour, due to the predominance of uric acid. During the next 24 hr brown or black colours prevail, indicating excretion of haematin and possibly other haemoglobin derivatives. This ‘dark fraction’ contains most of the active proteases. Dry excreta were eluted and then tested for trypsin and chymotrypsin activities. Using a specific trypsin inhibitor (BRIEGEL and LEA, 1975) I found that trypsin accounts for 75 per cent of the total proteolytic activity in the excreta. This is very close to the proportion found in the midgut homogenates (BRIEGEL and LEA, 1975). Chymotrypsin was not found in faeces. Its absence was not

surprising in view of the very small amounts in the midgut (BRIEGEL and LEA, 1975). Thus, the mosquito trypsin obviously remains active in the dry as well as in frozen excreta for days. Tests for an alternative

protease loss in the midgut

To determine whether beside excretion proteases are subject to inactivation (e.g. inhibition) or autodigestion within the midgut, excretion was prevented by sealing the anus and measuring the enzyme activity in the midgut. Sealing the anus immediately after an enema caused an unacceptable level of mortality and delayed secretion of the enzymes for 30 hr in the survivors. Therefore, females were sealed at 12 or 24 hr after the enema. The protease activity in the midgut homogenates and protein digestion was measured. Egg maturation was measured as an additional control. When the anus was sealed at 12 hr, the increase in protease activity was comparable to the unsealed controls (Fig. 2A); after the maximum at 24 hr, the activity remained nearly unchanged until 72 to 96 hr, when the females finally died. Basically the same results were obtained by sealing at 24 hr after an enema; having reached maximal activity, proteases remained at this level until death occurred. Likewise, protein digestion and egg maturation followed the same rates in sealed as in the control females (Fig. 2B,C). These results demonstrate that release and production of proteolytic enzymes actually ends once maximum activity has been reached. Digestive potential

of the proteases

Since the secretion of proteases was discontinued after a certain time I undertook to estimate their proteolytic potential after secretion before they were discarded. In other words, how much protein can be digested by the enzymes secreted up to a certain time when their supply was interrupted. First, the stability of the enzymes in a cell-free homogenate had to be determined. Midguts were homogenized at 8, 16, or 24 hr after a blood meal. The homogenates were then preincubated at 27°C (rearing temperature) with phosphate buffer. At regular intervals, aliquots were withdrawn and incubated for the protease assay. The activity measured at the time of homogenization is taken as 100 per cent and the data in Fig. 3(A) show the decrease in activity during preincubation. At all times tested, protease activity did not drop significantly below an 80 per cent level, where it remained stable for 2 days. Thus, homogenization of midguts did interrupt the supply of new enzyme and most of the activity present at that time was retained. To show the digestive potential of the enzymes which had been secreted by a certain time after a

Excretion

enzymes by Aedes aegyfiri

of proteolytic

o+

HOURS AFTER

.

.

0 24 (3 ~1 OF BLOOD )

ENEMA

.

1683

.

*

40

*

72

Fig. 2. Effect of sealing the anus 12 hr after enema in female A. oegypti. (A) On protease activity. (B) On protein digestion. (C) On yolk deposition. 0, Anus sealed; 0, anus open (controls). blood meal, the protein content was determined in the same homogenates (Fig. 3B). At 8 hr after blood meal only 40 to 50 per cent of the protein can

be digested during 20 hr of preincubation, but at 24 hr it takes about 10 hr to digest 90 per cent of the intact protein present. In other words, at 8 hr after a blood meal the enzymes already secreted, allowed digestion of about half of the intact protein, whereas at 24 hr when only 20 to 30 per cent of the protein was left (BRIEGEL and LEA, 1975), the high titre of enzyme secreted by this time was enough to complete digestion rapidly.

A

B

100% PRYTEASE ACTMY

100% PROTEIN

IN MIDGUT !iOMOGENATES

IN MIDGUT

HOMOGENATES

AT

AT

DISCUSSION The demonstration of a discontinuation in protease secretion after its maximal activity in the midgut and the absence of any inactivation mechanism within the intestinal tract of the mosquito contribute to an explanation of the mechanism of protease regulation. As reported by BRIEGEL and LEA (1975), maximal protease activity occurs at the time when about 70 to 80 per cent of the initial protein has been digested. Generally speaking, the process of secretion can now be described as follows : the proteins in the blood meal stimulate the secretion of trypsin; its activity rises and at the maximum secretion stops, when very little of the protein is left intact. In a few hours, all material still present in the midgut is excreted, i.e. enzymes, uric acid, and certain haemoglobin derivatives.

HOURS

OF

PREiNCUBATlON

Fig. 3. (A) Thermal stability of proteases during preincubation of midgut homogenates in buffer. (B) Demonstration of the proteolytic potential of the proteases present in midgut homogenates at certain times after the blood meal. For further explanations see text.

1684

HANS BRIEGEL

In summary, for mosquitoes we suggest the following model: secretion (or synthesis) of mosquito trypsin is induced by its substrate (globular proteins) and when the substrate is digested (i.e. the inducer removed), enzyme secretion stops. The enzyme is now expelled from the midgut and excreted, clearing the way for the next blood meal. Two distinct peaks of excretion have been demonstrated. Uric acid excretion was prevalent during the period of constantly increasing protease activity in the midgut. In Culux pipiens I have shown that free histidine is also excreted during the early part of blood digestion (BRIEGEL, 1969). Uric acid and histidine are both compounds rich in nitrogen. Therefore, the coincidence between their excretion and the time of intense and increasing proteolytic activity is understandable. Excretion of the proteases themselves marked the second peak; this can be looked at simply as a mechanical discharge together with haematin and other remaining waste products. Reports on excretion of active enzyme are rather limited in insects. Among higher Diptera, larvae of Phormia (BROOKES, 1961) and of Lucilia (HOBSON, 1931) are known to release proteolytic enzymes into their breeding substrate. In this case a predigestive function has been assumed. MARTIN and MARTIN (1970) have shown that fungus-culturing ants excrete proteases in addition to several other enzymes. This is an important basis of their symbiosis with the fungus. YANGand DAVIES(1971)

found excreted chymotrypsin in the rearing water of A. aegypti larvae, but for adult mosquitoes this is the first observation of protease excretion. However, in C. pipiens acid hydrolysates of the excreta had a high content of glycine (BRIEGEL, 1969). This observation could now be explained, if mosquito trypsin had as high a glycine content as reported for human trypsin (TRAVIS and ROBERTS, 1969). The fact that mosquito trypsin retains its activity in the dry excreta for several days will allow us a new and relatively simple approach for isolation and purification of this enzyme.

Acknowledgements-This work was supported by a fellowship of the Swiss National Fund for Scientific Research and partially by a grant from the National Institute of Health to Dr. A. 0. LEA (No. AI-0954). The Department of Entomology, University of Georgia, is acknowledged for providing space and facilities. Dr. E. V.4NHANDEL (Florida Entomological Laboratory, Vero Beach, Florida) kindly allowed me to use his method prior to publication. I thank Mrs. U. BRIECEL for technical assistance.

REFERENCES BRIEGEL H. (1969) Untersuchungen zum Arninoslurenund Proteinstoffwechsel wZhrend der autogenen und anautogenen Eireifung von Culex pip&s. .r. Insect Physioi 15, 1137-1166. BRIEGEL H. and KAISERC. (1973) Life-soan ofmosauitoes (Culicidae, Diptera) under’ laboratory conditions. Gerontologia 19, 240-249. BRIEGEL H. and LEA A. 0. (1975) Relationships between protein and proteolytic activity in the midgut of mosquitoes. J. Insect Physiol. 21, 1597-1604. BROOKES V. J. (1961) Partial purification of a proteolytic enzyme from an insect, Phormia regina. Biochim. biophys. Acta 46, 13-21. HOBSON R. P. (1931) On an enzyme from blow-fly larvae (Lucilia sericata) which digests collagen in alkaline solution. Biochem. J. 25, 1458-1463. KANG S.-H. and FUCHS M. S. (1973) An improvement in the Hummel chymotrypsin assay. Analyt. Biochem. 54, 262-265. MARTIN M. M. and MARTIN J. S. (1970) The biochemical basis for the symbiosis between the ant, Atta columbica tonsipes, and its food fungus. 3. Insect Physiol. 16, 109-119. TERZIAN L. A., IRRWERRE F., and STAHLER N. (1957) A study of nitrogen and uric acid patterns in the excreta and body tissues of adult Aedes aegypti. J. Insect Physiol. 1, 221-228. TRAVIS T. and ROBERTS R. C. (1969) Human trypsin. Isolation and physical-chemical characterization. Biochemistry 8, 2884-2889. VAN HANDEL E. (1975) Direct determination of uric acid in fecal material. Biochem. Med. 12, 92-93. YANG Y. J. and DAVIES D. M. (1971) Digestive enzymes in the excreta Aedes aegypti larvae. r. Insect _ _. of_.__ Physiol. 17, 2119-2123.