In vitro properties of proteinases in the midgut of adult Aedes aegypti L. and Culex fatigans (Wiedemann)

Comp. Biochem. Physiol., 1966, Vol. 17, pp. 115 to 127. PergamonPressLtd. Printedin Great Britain

I N V I T R O PROPERTIES OF PROTEINASES IN T H E M I D G U T OF A D U L T A E D E S A E G Y P T I L. AND C U L E X F A T I G A N S (WIEDEMANN) R. H. G O O D I N G * Laboratories of Medical Entomology, Department of Pathobiology, The Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland (Received 28 May 1965)

Abstract--1. The midgut proteinases from Aedes aegypti and Culex fatigans appear to have similar properties. These proteinases show a small peak of activity near pH 5.5, but the greatest activity was found in a broad peak in the alkaline region with a temperature optimum near 46-50°C. 2. Hemoglobin was a more suitable substrate than albumin or y-globulin. 3. The hydrolysis of N-benzoyl-L-arginine ethyl ester and N-benzoyl-Ltyrosine ethyl ester was demonstrated. 4. The alkaline proteinases were inhibited by serum from normal and malarious chicks, and to a limited extent by disodium ethylenediaminetetraacetate, and Mg 2+, Ca 2+ and Mn z+. INTRODUCTION MOST of the parasites transmitted by mosquitoes spend part of their life cycle in the midgut of these insects. At this time, when the female mosquito is digesting the blood meal, an initial host parasite interaction may take place. Comparative studies of digestive enzymes of different species may reveal whether or not digestive processes affect the intensity of an infection or the parasite-host specificity. Such studies may also uncover systems applicable to biochemical taxonomy. Proteolytic enzymes from the midgut of adult Aedes aegypti have been reported by Fisk (1950), Fisk & Shambaugh (1952), and Shambaugh (1954), and in extracts of whole mosquitoes (Wagner et al., 1961). T h e object of this investigation was to determine some properties of the proteinases in the midguts of Aedes aegypti (L.) Culex fatigans (Wiedemann). These species were selected especially for two reasons. First, the former is highly susceptible to Plasmodium gallinaceum but is refractory to P. cathemerium, while the latter is a host for P. cathemerium but not for P. gallinaceum. Secondly, these two mosquitoes are widely separated by the usual morphologic taxonomic characters, and if proteolytic enzymatic digestive processes have any taxonomic significance, some difference should be detectable by a comparative study of these enzymes in the two mosquitoes. * Present address: Department of Microbiology, School of Medicine, Vanderbilt University, Nashville, Tennessee 37203. 115

116

R.H. GOODING MATERIALS AND METHODS

Experimental animals The A. aegypti used in this study were from a laboratory colony started about 8 years ago with material from Lagos, Nigeria, but perhaps contaminated with various other strains that have been held in this laboratory. Mosquitoes from this colony are considered to be representative of the "wild type" of A. aegypti. The C. fatigans used in these experiments were from a colony established from specimens collected near Manila and Angeles in the Philippines. The colony has been maintained at Johns Hopkins School of Hygiene since 1956. Both species were reared and maintained in the insectary at 75-80°F. The larvae were fed guinea-pig chow, and the adults 5-10 per cent sucrose as well as blood. Leghorn chicks obtained when they were approximately 10 days old were used for feeding mosquitoes until they were about 2 months old. When malaria-infected chickens were needed, the infections were induced by intramuscular injections of blood from birds infected with Plasmodium gallinaceum. Chicks used as sources of serum were not used to feed mosquitoes, but were held in separate cages until old enough to provide sufficient serum. The serum was obtained under sterile conditions and stored frozen until used.

Proteinase determinations Concentrated protein solutions were dialyzed against 1% NaC1 for 24 hr, except for that of denatured hemoglobin which was continued for 72 hr. At the end of the dialysis, the protein solutions were centrifuged (1400g for 20 min at 2°C). The protein concentration in the supernatant was adjusted to the desired level by the addition of 1°/o NaC1. Preparation of a large pool of enzyme material was usually accomplished by using mosquitoes that were 1-5-2 weeks old. In most cases the mosquitoes were deprived of sugar for 12 hr and then allowed to take their first blood meal on a normal chick during a 2 hr period. No attempt was made to separate partially gorged from gorged mosquitoes. The mosquitoes were kept in the holding cages at 75-80°F for periods of 24-36 hr, and then immobilized by chilling. The midguts were dissected from fed mosquitoes, placed in labeled vials and stored in a deep freeze until they were to be homogenized. The assays, run in duplicate, followed a modification of the method of Kunitz (1947), which measures the hydrolysis of the protein by the release of trichloroacetic acid (TCA) soluble material absorbing light at 280 m~. Controls were run to correct for endogenous substrate (EAOD) and spontaneous breakdown of the substrate (SAOD). The net change in optical density (NAOD) was calculated as NAOD = TAOD--(EAOD+SAOD) and is reported in the results. Unless otherwise stated, the conditions for assay were the following: The substrate was 0.25 ml of 10% denatured hemoglobin (wt. protein/vol, in 1% NaC1 (wt/vol.). For reactions carried out at 46°C, the buffer was 0-50 ml of 0-2 M bicarbonate; and for assays run at 49°C, 0.50 ml of 0.2 M phosphate buffer was used. Sodium salts were employed in all cases. The reaction was started by the

117

M I D G U T PROTEINASES OF A E D E S A E G Y P T I ( L . ) AND C U L E X F A T I G A N S

0.30

0"20

D E

0'25

X

.~ 0.2C

O'15 AEGYPTI

FATIGANS

E

~

!

o.,o

O.IC

L8

/

<3 0.05

Z 0.10

Z

0 -~O2

L

e, I I

I 2

I 3

I 4

[ 5

l 6

I 7

I 8

I 9

I I I0 II

I 12

. .

Fed nfed

0

-0"02

pH

', ; ; " ~ ~ ' ~ ;,~,',,'~ pH

FIG. 1. T h e effect of pH upon the proteinase activities of the A. aegypti and o£ C. fatigans midgut homogenates. Buffers and symbols: + = citric acid/HPO4; © = H=POJHPO4; • = HPO4/PO4; x = HCOs/COs.

O'3C 0.30

x

/\

0.25

x

0"25 3 o~ 0"20 E

0.2C

E --Io0

AEGYPTI

X FATIGANS

Oq5

18 0.,5 <~

Z

Z

~10

0.10 0"05 I 20

I

30

I

40

Temperature,

I

50 °C

I

60

°:I

X

-0.021-

/

I

20

I

30

I

40

Temperature,

f

I

50 60 °C

FIG. 2. T h e effect of temperature upon the proteinase activities of the A. aegypti and of C. fatigans midgut homogenates. Same buffers used as in Fig. 1.

118

R . H . GOODING

addition of 0.25 ml of a midgut homogenate prepared in cold distilled water which had been neutralized with dilute sodium hydroxide and/or hydrochloric acid. T h e homogenate was centrifuged (1400g, at 2°C for 20 rain) and the supernatant adjusted to contain an extract of four midguts/ml for A. aegypti or of two midguts/ml for C. fatigans. Incubation was carried out for 6 min, and the reaction stopped by the addition of 1 ml 6% (wt/vol.) trichloroacetic acid. T h e protein content of the midgut homogenates was determined by the method of L o w r y et al. (1951). These concentrations (mg protein/reaction mixture) were as follows: fed A. aegypti, from 0-155 to 0-269; unfed A. aegypti, 0.044± 0.001 ; fed C.fatigans, from 0.188 to 0-277; unfed C. fatigans, 0.042± 0.001.

T A B L E 1 - - E S T I M A T I O N S OF THE M I C H A E L I S - - M E N T O N CONSTANT CONFIDENCE INTERVAL FOR Kr,~*

A. aegypti

Assay conditioni

1

Protein

Denatured hemoglobin Twice cryst. hemoglobin Denatured hemoglobin Twice cryst. hemoglobin BSA V v-Globulin II

C. fatigans

1

Denatured hemoglobin Twice cryst. hemoglobin Denatured hemoglobin Twice cryst. hemoglobin BSA V 7-Globulin II

AND THE 95 PER CENT

K,~

95 per cent confidence interval

Conc (mg/tt)

(mg/1 ml reaction mixture)

(mg/1 ml reaction mixture)

0-625-25

2'75

2'45

3.15

0-625-25

4.08

2.63

7.25

0.625-10

1'51

1 '07

2'18

0"625-25 0"625-25 2"5-25

1.84 19.31 374'15

0"69 17.37

6.79 36.70

0.625-25

2"10

1"72

2-60

0"625-20

2'17

1"74

2-74

0-625-10

1-32

1"02

1"72

0-625-10 0"625-15 2'5-25

3"15 8"51 6"22

2"23

4"79

2.92

14-07

Substrate Species

(K,n)

* When Fieller's theorem failed to yield meaningful confidence intervals (i.e. the intervals were infinite), they were omitted from the table. t Assay condition 1--bicarbonate buffer (pH 9"5) at 46°C; condition 2--phosphate buffer (pH 7"9) at 49°C.

MIDGUT PROTEINASES OF A E D E S A E G Y P T I (L.) AND C U L E X F A T 1 G A N S

EXPERIMENTAL

119

RESULTS

1. The effects of pH, temperatures and substrates Figures 1 and 2 show that the effects of pH and temperature upon the in vitro activity of the proteinases from the midguts were similar in A. aegypti and C. fatigans. The effects of the protein substrates and their concentrations are reproduced in Figs. 3 and 4. From the data obtained from substrate concentrations 0'30

A

0'55

8 o

o

0"25

0"30

0'20

0-25 0.20

~

°~°~-

0-15

Z

I~ "

~/

<1

z °'.°

gl

0"10

&05

005 C -0"02~

' +..+_ I 5

.t~i~llt~ I io

I 15

Substrate concentration,

ll 1 20

I 25

I 5

0

rng/test tube

Subsfrate

I iO

I 15

I 20

concentration,

I 25

r n g / t e s t tube

FIG. 3. T h e effect of substrate concentration u p o n the proteinase activities of

A. aegypti m i d g u t homogenates at p H 9.5 (bicarbonate buffer) and 46°C (a) and at p H 7"9 (phosphate buffer) and 49°C (b). Substrates and symbols: © = denatured h e m o g l o b i n ; • = twice crystallized hemoglobin; • = 7-globulin I I ; x = bovine sentrn albumin (BSA V); + = twice crystallized BSA. 0.25

A

B 0"20

o,5 :.

~ y

_~*

~ 0.,5 " ~."

"

o.,ot.Ai 0.05~o L q B ~ J ~ : F ~ + r - - - - - -t 0 5 10 15 Substrofe concentration,

: i x T ~ r 20 25 rng/tesf tube

p ,'<" . , _ . . . . . i Z _ ? _ ~ i ~ 5 l0 15 Substrote concentration,

20 25 rag/test tube

FIG. 4. The effect of substrate concentration upon the proteinase activities of C. fatigans m i d g u t homogenates at p H 9"5 (bicarbonate buffer) and 46°C (a) and at p H 7"9 (phosphate buffer) and 49°C (b). Substrates and symbols: O = denatured h e m o g l o b i n ; • = twice crystallized hemoglobin ; • = 7-globulin I I ; x = B S A V ; + = twice crystallized BSA.

120

R.H.

GOODING

below the point at which the enzyme is saturated, the values of the MichealisMenton constant (Kin) were calculated. The reciprocals of the activity and substrate concentrations were fitted with a straight line by the method of least squares (Moroney, 1956). Estimates of Km and the 95 per cent confidence interval for Km (obtained by the method of Fieller, 1954, Williams, 1959) are given in Table 1. 0"70

0.60

0-20

<1

18

z OqO

Z

AEGYPTI/

I~ ol5

<1

J

0'05 I

0-50 040

FATIGANS~

/-

0.30

0'20

I

I

I

O.lO

I

0"05 0"10 0"15 0"20 0"25 mg protein in 2 5 m l

e

I

o

homogenate

I

I

I

I

005 OdO 0'15 0"20 0'25 mg protein in 25 ml homogenale

FIG. 5. T h e effect o f the concentrations o f extracts u p o n the proteinase activities o f .4. aegypti and o f C. fatigans. © = p H 9"5 (bicarbonate buffer) and 46°C; • = p H 7"9 (phosphate buffer) and 49°C. 0.30

0"70 0.25

0"60 AEG

18 YPT/

~

0.50

I~

0'40

<3

Z

o.15

~ . ,// .o // °"

0"30

o.2o

-IN

//

<1 ~ o.,o

0.20 0.IO

/

/ o/ //I

6

0.05

I

12

Reaction

I

18 time,

I

24 min

I

30

0

/ ,/ I

.j.J" • /

I

t

I

6 12 18 24 Reaction time, rnin

I

30

FIG. 6. T h e effect o f time on proteinase activities o f h o m o g e n a t e s of A. aegypti and o f C. fatigans. O = assays run at p H 9"5 (bicarbonate buffer) at 46°C; • = assays run at p H 7"9 (phosphate buffer) at 49°C.

2. The effect of enzyme concentration and of time Figure 5 shows an approximately linear relationship between proteinase activity and enzyme concentration for both species of mosquitoes. In preliminary experi-

MIDGUT PROTEINASES OF A E D E S A E G Y P T I (L.) AND C U L E X F A T I G A N S

|21

ments, it was found that the proportionality does not hold at values above an enzyme concentration contained in one midgut per test-tube. Figure 6 illustrates the necessity of using a short reaction time in assaying proteinase activity in A. aegypti and C. fatiga~, particularly at pH 9.5.

3. The hydrolysis of synthetic substrates An attempt was m a d e to determine, by spectrophotometric methods, the substrate specificity of the proteinases at pH 5.5, 7.9 and 9.5. Both A. aegypti and C. fatigans homogenates catalyzed the hydrolysis of N-benzoyl-L-arginine ethyl ester and that of a-p-toluenesulphonyl-L-arginine methyl ester at pH 5.5 and 7.9, as determined by the methods of Schwert & Takenaka (1955) and Hummel (1959) respectively. The extracts of both species hydrolyzed only slowly N-benzoylL-tyrosine ethyl ester at pH 7.9 and 9.5. No evidence was found to indicate hydrolysis of L-tyrosine ethyl ester (method of Schwert & Takenaka, 1955), leucinamide (method of Binkley & Torres, 1960) or hippuryl-L-arginine (method of Folk et al., 1960) at pH 5-5, 7.9 or 9.5.

4. lnhibitors of proteinase activities E D T A had little or no effect during an incubation period of 6 min, with denatured hemoglobin (25 mg/tt) as the substrate, and with either a phosphate buffer (pH 7.9) assayed at 49°C, or a bicarbonate buffer (pH 9.5) assayed at 46°C. With bovine serum albumin (fraction V, BSA V) and an incubation period of 15 min, the activities of extracts of both A. aegypti and C. fatigans were inhibited to an extent of 2 5 4 1 per cent by E D T A (0.01 M) at pH 7.9 (phosphate or tris buffers), while no inhibition was observed at pH 9.5. An attempt was made to overcome the E D T A inhibition of A. aegypti midgut proteinase by adding calcium, magnesium or manganese chloride to the reaction mixture. The E D T A and the salt were mixed with the midgut homogenate at 0°C before the homogenate was added to the substrate. In all cases, the presence of the cations in the reaction mixture increased the inhibition of the ,4. aegypti proteinase. Various concentrations of the salts were mixed with the A. aegypti midgut homogenate at 0°C, and then proteinase activity was assayed in tris buffer at pH 9.5 with BSA V as the substrate and an incubation period of 15 min at 46°C. The results indicated that the increased inhibition of the proteinase was due to the presence of the salts. These inhibitory effects were observed when either denatured hemoglobin or BSA was used as the substrate (Table 2). There was no difference in activity between the two mosquito species. Inhibition of the BSA V digestion was greater than that of denatured hemoglobin. The effect of chick serum upon the in vitro activity of the mosquito proteinases with denatured hemoglobin as the substrate was studied by addition of 0.1 ml of various dilutions of chick serum to the reaction mixture and reducing the volume of buffer to 0-4 ml. Two experiments were performed; in the first, there were four normal and four infected chicks; in the second, there were three normal and one infected chicks. The concentrations of serum necessary for

122

R . H . GOODING TABLE 2--THE

EFFECT OF CATIONS U P O N THE PROTEINASE A C T I V I T Y OF

A. aegypti

AND

C. fatigans MIDGUT HOMOGENATES* Substrate

Species

Assay conditiont

A. aegypti

C. fatigans

Cation (0"01 M)

Denatured hemoglobin

BSA V

NAOD

Per cent inhibition

NAOD

Per cent inhibition

1

None M g 2 '~ Ca 2 ~ M n 2+

0"210 0"171 0-172 0-108

0 18"6 18.9 48-6

0"076 0"067 0"052 0'011

0 11"8 31-6 85'5

2

None M g z+ Ca 2+ M n 2+

0"228 0"205 0-179 0" 166

0 10.1 24"1 27 '2

0"115 0"086 0-078 0"018

0 25.2 32'2 84" 3

1

None M g 2 ~Ca ~ M n 2+

0"189 0'171 0"174 0'091

0 9'5 7'9 51 "9

0'075 0'072 0"050 0"001

0 4'0 33'3 98 -7

2

None M g 2+ Ca 2 ~ M n 2+

0'195 0.170 0"160 0"121

0 12-8 17"9 37"9

0'132 0.087 0.077 0"024

0 34.1 41 "7 81 "8

* T h e p r o t e i n c o n t e n t of the A. aegypti h o m o g e n a t e was 0-197 __+_0"003 mg/0"25 m l and of the C. fatigans h o m o g e n a t e was 0.183 _+0"001 mg/0"25 ml. t A tris buffer was u s e d in all assays. C o n d i t i o n 1 was p H 9-5 at 46°C and condition 2 was p H 7"9 at 49°C. T h e reactions w e r e r u n for 6 rain w h e n h e m o g l o b i n was the substrate and 15 rain w h e n BSA V was t h e substrate. 'FABLE 3--ESTIMATES

OF C O N C E N T R A T I O N OF SERUM G I V I N G A 5 0 PER CENT I N H I B I T I O N OF

A. aegypti

AND

C. fat~ans

M I D G U T PROTEINASES

IC~0 (ml serum/1 ml reaction m i x t u r e )

Species

A. aegypti C. fatigans A. aegypti C. fatigans

Exp. No.

Assay condition*

Normal chick serum

Malarious chick serum

MCS/NCSt

1

1 2

0"0043 0'0027

0-00335 0"0007

0-78 0"73

2

1 2 1 2

0"0058 0"00105 0'0018 0'00115

0"0068 0.0020 0-0025 0"0015

1-17 1.90 1-39 1 '30

* C o n d i t i o n 1 - - b i c a r b o n a t e buffer ( p H 9"5) a n d 46°C; condition 2 - - p h o s p h a t e buffer ( p H 7"9) a n d 49°C. t M C S / N C S = ratio of the value for malarial chick s e r u m to n o r m a l chick serum.

MIDGUT PROTEINASES OF A E D E S A E G Y P T I (L.) AND C U L E X F A T I G A N S

123

50 per cent inhibition were estimated graphically from a plot of the log of the serum concentration against the percentage of inhibition, and are in the range of from 0.001 to 0.007 ml serum/ml reaction mixture. The results of this experiment showed that the degree of inhibition of proteinase activity was about the same in both species of mosquitoes, and that normal and malarial sera had about the same effect (Table 3). DISCUSSION The in vitro properties of the proteinases of `4. aegypti and C. fatigans were studied by the use of crude homogenates of the midguts of mosquitoes which had fed about 24 hr earlier on a presumably normal chick. The preparation assayed was a mixture of mosquito tissue, mosquito secretion, the blood of a chicken (the elements of which were in various stages of breakdown due to the action of the enzymes in the midgut) and probably a mixture of microorganisms and their products. The pH optima for proteinase activity in these crude homogenates of A. aegypti and C. fatigans midguts are very similar. This activity is much greater in fed than in unfed midguts, which indicates that the proteinases are present in the midgut as a response to the blood meal, and that their function is digestive rather than catheptic. The pH of the midgut of blood-fed mosquitoes has been reported to be in the range 7-3-7.9 (Fisk, 1950; Micks et al., 1948; Bishop & McConnachie, 1956). Thus it appears that the proteinases responsible for digestion of the blood meal must function in a slightly alkaline medium. In a phosphate buffer, the maximum activity in the crude midgut homogenate corresponds fairly closely to the pH of the gut. If the increased activity at pH 9-5 was caused by a separate proteinase, it is questionable whether this enzyme would contribute significantly to the process of digestion in guts with a normal pH of about 7"7. It may represent the retention of a digestive enzyme that was functional in the phytophagous ancestor of the mosquitoes. In general, the gut contents of the phytophagous insects are more alkaline than the carnivorous insects (Day & Waterhouse, 1953) and their enzymes would be expected to have higher pH optima. By use of synthetic substrates it has been possible to demonstrate tryptic and chymotryptic activity at pH 7'9, but only chymotryptic activity at pH 9"5. The temperature-activity curves indicate that the proteinases from the midguts of fed mosquitoes are most active at rather high temperature and that the optimal activity seems to depend more upon the buffer than upon the mosquito species from which the enzyme came. The temperatures to which the mosquitoes are exposed in nature during the digestion of the blood meal are variable and well below that found in this study to be optimal for the in vitro activity. If engorged females were exposed to a constant 30°C, digestion would be at a rate of 26-27 per cent of the maximum rate observed at optimum (in vitro) temperatures. The temperature optima (46-50°C) found here for .4. aegypti and C. fatigans are similar to those of alkaline proteinases. For mammalian trypsins it ranges from

124

R.H. GOODING

45 to 55°C (Buck et al., 1962); for the housefly, it is 45°C (Lin & Richards, 1956); and for the larval blowfly, it is 44°C (Evans, 1958). The proteinases of A. aegypti and C. fatigans are very similar in that both hydrolyze hemoglobin preparations much more rapidly than they do the serum proteins. If these in vitro findings are indicative of the relative susceptibility of the blood proteins in vivo, it suggests that the hemoglobin is more available for digestion than are the serum proteins. Crystallized hemoglobin was almost as good a substrate as the denatured hemoglobin. Fisk (1950) raised the question of whether the proteinases of mosquitoes are able to break down native proteins, or whether proteins are denatured by some as yet unknown mechanism. It appears that the proteinases of these mosquitoes differ from mammalian trypsins which do not readily attack native proteins (Sumner & Somers, 1947). At pH 9-5 and 46°C, the proteinase activity, particularly of A. aeg3?ti, is largely restricted to the digestion of hemoglobin. If under these conditions a different enzyme is being assayed than at pH 7.9, it would be a proteinase with a very high degree of substrate specificity. Such high degrees of substrate specificity are rare among the digestive proteinases. A proteinase from Schistosoma mansoni which attacks only hemoglobin or globin has been partially purified (Timms & Bueding, 1959) and has a pH optimum at pH 3.9. As suggested above, the proteinase functioning at pH 9-5 may be a vestige of a phytophagous ancestor. It is also possible that hemoglobin more closely resembles the substrate to which this enzyme was originally adapted than do any of the other blood proteins tested. The relatively low activity of the proteinases on the serum proteins found here is interesting in view of the findings of Shambaugh (1954) that it is the serum proteins which cause the stimulation of proteinase secretion in A. aegypti. The hydrolysis of N-benzoyl-L-arginine ethyl ester (BAEE) and N-benzoylL-tyrosine ethyl ester (BTEE) indicates the presence in the midgut homogenate of both A. aegypti and C. fatigans of enzymes having substrate specificities similar to mammalian trypsin and chymotrypsin respectively. The hydrolysis of BAEE by midgut homogenates of female A. aegypti had previously been demonstrated by Wagner et al. (1961). E D T A was observed to have at most only a slight effect upon the in vitro activity of the mosquito proteinases studied. In attempts to overcome this inhibition by the addition of divalent cations (Ca 2+, Mg ~+, Mn2+), it was found that the cations themselves inhibit the proteinases when either denatured hemoglobin or BSA V was used as the substrate. The demonstration of this inhibition confirms the finding by Wagner et al. (1961) that these cations inhibit the action of A. aegypti proteinase upon hemoglobin. In addition, it shows that they inhibit proteinase activity upon bovine serum albumin, and that they have a similar effect upon the proteinase from C.fatigans. Terzian (1958, 1963) and Terzian & Stahler (1964) have demonstrated that the digestive processes may be inhibited in A. aegypti and A. quadrimaculatus by feeding the mosquitoes solutions containing the chlorides of calcium, magnesium manganese or iron. Terzian (1958) concluded that since A. aegypti which had ingested calcium or magnesium chloride were

MIDGUT PROTEINASES OF A E D E S A E G Y P T 1 (L.) AND C U L E X F A T I G A N S

125

able to develop eggs, it was "reasonable to assume that only digestion of the hemoglobin fraction, of all the fractions contained in the original blood meal, is affected by the cations or cation antibiotic mixtures". In the present study the in vitro hydrolysis of bovine serum albumin was inhibited by the cations. However, this does not prove that in vivo digestion of serum proteins is inhibited by these cations. One question which arises at this point is what effect do the cations, although present in the blood meal in very low concentrations, have upon the mosquito's proteinases ? The possibility exists that these inhibitory cations may be removed from the midgnt and eliminated per annum during, or immediately after, the ingestion of the blood meal. There is a dearth of information concerning the inorganic content of mosqiuto feces. Fisk (1950) suggested the possibility that blood from the rabbit may contain some substances which inhibit the midgut proteinase of A. aegypti. The inhibition of A. aegypti and C. fatigans proteinases by serum from normal chicks and chicks infected with P. gallinaceum has been demonstrated in this study. The degree of inhibition by normal and malarial sera is not markedly different. The inhibition of mammalian trypsin by sera from normal and diseased mammals has been the subject of numerous investigations and the subject has been reviewed recently (Laskowski & Laskowski, 1954; Zipf et al., 1961). It was pointed out (Zipf et al., 1961) that the level of serum anti-trypsin increases during the course of diseases characterized by extensive tissue destruction. The inhibitory activity of sera from malarial chicks did not differ markedly from sera from normal chicks. The volume of blood ingested by A. aegypti has been estimated at 0.0026 ml when fed on humans (Jeffery, 1956). If one assumes that mosquitoes take the same volume of chicken blood and that half of this is serum, they must ingest about 0-0013 ml of serum. Such a volume of serum would inhibit to an extent of about 25-35 per cent of the maximum proteinase activity found in the midgut. It is not known whether the mosquitoes are capable of selectively removing the inhibitor(s) from the blood meal or whether, as suggested by Fisk (1950), some of the mosquito midgut proteinase combines with the inhibitor to neutralize it. It is known that the fluid defecated by .4. aegypti just after a blood meal contains proteins and that at least some of the fluid comes from the blood meal (Boorman, 1960). A. aegypti is a markedly susceptible host for Plasmodium gallinaceum, while C. fatigans is so refractory that the parasite is unable to become embedded in the gut wall. Destruction of the parasite evidently takes place in the lumen of the gut. From the present studies it seems unlikely that this specific action against the parasite can be attributed to a proteolytic enzyme that is present in C. fatigans, but is absent from A. aegypti. The search for biochemical differences between living organisms represents a new approach to problems in taxonomy. Attempts have been made recently to demonstrate discontinuities between mosquito species in amino acid composition (Micks & Gibson, 1957), vitamin content (Micks et al., 1959) and some other

126

R . H . GOODING

biochemical properties. H o w e v e r , R o z e b o o m (1962) has expressed d o u b t that biochemical techniques will be of practical use in ecological or epidemiological studies w h i c h require identification of mosquitoes in the field. I f proteolytic e n z y m e systems differ sufficiently even between unrelated species of m o s q u i t o e s to be capable of separation, the analytical procedures will have to be far m o r e exacting than those used in the present study. I ) o w n e s (1958) believes that bloodsucking in mosquitoes and other g r o u p s of biting N e m a t o c e r a was the original condition, and that n o n - b l o o d s u c k i n g is a m o r e recent development. T h e finding of an apparently different proteinase in the two species of mosquitoes, similar to those in p h y t o p h a g o u s insects in that it is active at a high p H , s u p p o r t s the view of those w h o consider b l o o d s u c k i n g to be a secondarily acquired habit. Acknowledgements--This work was supported in part by Research Grant No. AI-00351, The National Institute of Health, USPHS. The writer is indebted to Dr. L. E. Rozeboom and Dr. E. Bueding for suggestions and criticisms, and to Dr. J. Gart for assistance with the statistical treatment given in Table 1.

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