Permeability of the peritrophic membrane in the larvae of Aedes aegypti

J. Insect Physiol., 1970, Vol. 16, pp. 1193 to 1202. Pergamon Press. Printed in Great Britain

PERMEABILITY OF THE PERITROPHIC MEMBRANE THE LARVAE OF AEDES AEGYPTI

IN

D. P. ZHUZHIKOV Department of Entomology, Moscow State University (Received 19 July 1969 ; revised 15 September 1969)

Abstract-Selective ionic permeability, grade of permeability, coefficient of filtration, and reflective coefficient were studied on isolated peritrophic membrane and the effective radius of the pore membrane was calculated. On the basis of the criteria obtained experimentally, conclusions have been drawn about the nature of the permeability of the peritrophic membrane for different substances. Moreover, the size of the colloidal gold particles permeating through the intact membrane of live larvae has also been determined. The results obtained show that the peritrophic membrane, by means of complex interaction with the substances permeating through it, acts as a unique ultra-filter which holds back within the trophic cavity the complex food substances while allowing the smaller molecules resulting from hydrolysis an easy passage towards the epithelium.

INTRODUCTION THE PHENOMENON of permeability

ranks among the major problems of presentday biology. The selective permeability of living cell membranes has been the subject of many intensive studies. Physiologists and physicians are also interested in the permeability of different tissues that control the directional mixture of various substances in the bodies of plants and animals. The peritrophic membrane in mosquito larvae consists of a thin-walled tube made up of hardened secretions of the epithelial cells. At its anterior end it is attached to the sclerotized ring of the oesophageal invagination. Growing from this point it gradually moves along the length of the midgut. Its hind end passes through the pyloric valve and reaches the hind gut, where it breaks down. Along the midgut the peritrophic membrane separates the food from the epithelial lining and functions not only as a mechanical but also as a physiological barrier. A general account of the permeability of the peritrophic membrane in Diptera was published a few years back (ZHUZHIKOV, 1964). In adult flies the membrane seemed to be easily permeable to ionic solutions of salts and also to the final products of digestion, i.e. the monosaccharides and the amino acids. Proteins and polysaccharides did not pass through it in any appreciable amount. But, at the same time, the digestive enzymes which are produced by the midgut epithelium and contain a protein base undoubtedly pass through it to reach the trophic cavity-the main site of the digestive process. 37

1193

1194

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ZHUZHIKOV

The present paper is the result of an attempt to study the permeability of the peritrophic membrane in more detail so as to be able to determine the fundamental principles of the passage of different substances through it. As a model object the peritrophic membrane of Aedes aegypti larvae was selected. Since the peritrophic membrane represents the hardened secretions of the epithelial cells and is not capable of self-renewal or active exchange, it may, to a great extent, be compared with the artificial, non-living membranes for which the theory of permeability has been worked out in great detail during the last few years. It should be noted that this theory accepts the possibility of certain changes in the properties, e.g. the permeability of the membrane, under the influence of the physico-chemical conditions in the solutions separated by it. Membranes of animal or plant origin similar to the peritrophic membrane structurally are unknown to us. The insect trachea and the cuticular layers of the ectodermal parts of the alimentary canal come closest to it. Recently, some Canadian workers showed that the cuticle of the grasshopper rectum is comparable in permeability to cellulose membranes. The passage of aqueous solutions through it is guided by the law of Poise11 (PHILLIPS and DOCKRILL, 1968). In studies of cellular or tissue permeability, where active transfer of ions and molecules takes place, the same physico-chemical principles as in artificial membranes are usually accepted as the basis (RUBENSTEIN,1947; HODGKIN, 1958; KEDEM and KATCHALSKY,1958; GOLDSTEINand SOLOMON,1960). For determining the characteristics of permeability a number of properties are studied, viz. selective ionic permeability, grade of permeability, coefficient of filtration, reflective coefficient, and the effective radius of the membrane pores. In the present study all these indices were determined for isolated peritrophic membranes. Moreover, the limiting size of colloidal particles passing through the intact peritrophic membrane of live larvae was also determined.

MATERIALS AND METHODS Actively feeding fourth instar larvae from a laboratory culture were used throughout this work. Peritrophic membranes were dissected out and thoroughly washed in water just before use. The membrane potential that arose when the membrane is placed between two solutions of different make-up or concentrations was measured in order to determine its selective ionic permeability. A right-angled glass vial, divided into two equal parts by a cover-glass with a 1 mm dia. opening in the centre, served as the measuring chamber. Quadrangular pieces cut from the peritrophic membrane were placed over the opening with a drop of water. When the water on the glass surface dried out, the membrane was firmly attached to the glass. The opening in the cover-glass was filled with water all the time to prevent the membrane from drying out. The boundaries of the membrane around the opening were gummed to the glass by applying pesein. The cover-glass thus prepared was lowered into the glass vial, and the two halves

PERMEABILITY

OF PERITROPHIC MEMBRANE IN AEDES

LARVAE

1195

of the vial were filled with the required solutions. Thus a system of two solutions separated by the peritrophic membrane was obtained. Potentials were measured with the help of non-polar calomel electrodes. The first series of experiments were carried out with 0.01 M and O*OOlM solutions of KCl. Measurements were recorded first by one position of the electrodes and then the positions of the two electrodes were reversed. This gave rise to a potential with reverse signs. Control measurements were done under similar conditions but without the peritrophic membrane cover on the cover-glass opening. In another series of experiments the solutions were prepared by adding potassium chloride to 0.01 M solutions of HCl, NaCl, and NaOH. A tapering 5 mm high glass cone was used as the measuring chamber for determining the grade of permeability of the peritrophic membrane. From the top of the chamber two platinum electrodes 0.3 mm apart were introduced 3 mm deep into the broader part of the chamber. The electrodes were platinized beforehand( RABOTNOVA, 1957). Measurements of the electroconduction of the solution in the chamber were recorded with a Watson’s bridge. The bridge was fed with 1 kc/set and 0.5 V impulses from a sound generator. A signal from the bridge after increase was registered by an oscillograph and auto-recording ampere-voltmeter. The bridge was brought into equilibrium by comparison with the solution of corresponding salts at a concentration of O-1 g-equiv./l. All the solutions were prepared in redistilled water. In such a system the grade of permeability (W) may be determined as a ratio of the time taken to attain a certain concentration of the substance at the point of measurement in the absence (control--tg) and presence (experiment-r,) of the membrane. For this, the time from the beginning of the experiment until attainment of equilibrium of the bridge by diffusion of the solution through the free lower opening of the chamber and the time taken through the same opening covered with peritrophic membrane were measured. In a series of experiments the grade of permeability of the peritrophic membrane for a number of chlorides was studied. In a second series, the grade of permeability of potassium sulphate and calcium chloride in media containing various concentrations of H- ions were studied. Water was either acidulated with HCl to pH 4, or alkalinated with KOH to pH 10, or left at neutral but with a little NaCl added for equalizing the basic electroconduction of all the three media. These solutions were placed in the measuring chamber. Solutions of the experimental salts were prepared in the measuring chamber. All measurements were carried out in the thermostat at a temperature of 25 + 0*5”C. In all about 200 experimental and 90 control measurements in the first series and 80 experimental and 50 control measurements in the second series were recorded.’ When there is water on both sides of the membrane, its volume (V) permeating through the membrane in unit time is determined solely by the difference in hydrostatic pressure (AP) on the two sides of membrane and the hydraulic permeability (L,) of the membrane, i.e. I’ = L,AP. The value L,, also known as the

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ZHUZHIKOV

coejicient of jiltration, is determined, accordingly as Lp = VIAP and is constant for each membrane. For determining the coefficient of filtration one end of the peritrophic membrane was tied with thread and the other end was put over the narrow end of a graduated glass capillary tube and fixed with thread. The height of the water column in the tube determined the extra pressure on the membrane. The movement of the water meniscus in the tube was observed under a microscope with an ocular micrometer. The time required for the passage of a small quantity of water through the membrane was recorded. The area of each membrane was calculated from its diameter and length. If on one side of the membrane there is water and on the other a solution of a partially permeating substance, then, in the condition of equilibrium when V = 0, AP< AT or AP = Ava = SAT, where Ta is the observed or apparent osmotic pressure, the value of which is always lesser than the actual osmotic pressure m, and 6 is the reflective coefficient, the value of which varies between 0 and 1 for different substances. For such a system V = LJAP- SA?T)/~,where +jis the amount of solution held within the membrane itself. From this equation 6 may be determined as 6 = L,AP- I? (1) L,AP *

In practice, for determining the reflective coefficient in the presence of hydrostatic pressure the tube was filled with a O-1 M solution of raffinose or a 0.01 M solution of inulin. The membrane was constantly flushed externally with water. For each solution, the quantity passing through a unit area of the membrane in 1 set was determined; 6 was calculated from equation (1). The system takes a different form if on one side of the membrane is placed a solution of a substance (i) not permeable through the membrane, and on the other side a solution of a substance (p) which will permeate through it. The concentrations of these two solutions are so chosen that in the absence of hydrostatic pressure (AP = 0) the system comes to equilibrium (V = 0). Then 6,~~ = Qrt since, for substances permeating through the membrane, ai = 1, 6, = 7r&r~. (2) The experiments were conducted in the following manner. Washed peritrophic membranes were filled with protargol solution of known concentration and the ends were ligatured. The sac thus obtained was placed in a sucrose solution. By choosing the concentrations of solutions inside and outside the membrane it was possible to reach a condition of equilibrium when the volume of the solution in the The osmotic pressure of protargol was determined sac remained unchanged. experimentally with the help of an osmometer built according to the scheme of Dogadkin (DOGADKIN et al., 1949). The reflective coefficient was then calculated according to equation (2). Colloidal gold was used to determine the limiting size of the colloidal particles capable of permeating through the peritrophic membrane of live larvae. A highly

PERMEABILITY

OF PRRITROPHIC

MEMBRANE

IN AEDES LARVAE

1197

dispersive solution of gold was prepared by the reduction of hydrochloro-auric acid by white phosphor. All the reagents were carefully purified. The larvae, after being kept in clear water, were transferred to colloidal gold solution where powdered Mipor was added as an inert solid substance. When the intestine was filled with gold and colour visible in the caeca, the larvae were dissected and a portion of the caecal content drawn out with a micropipette and placed directly on a slide under an electron microscope with an angular base. These slides were photographed at 100,000 x and the size of the particles were measured directly from the photoplates with a microscope oculometer. Besides this, drops of the colloidal gold solution from the dish and the trophic cavity of the larvae were also examined from time to time. RESULTS

Determination of the selective ionic permeability of the peritrophk membrane A comparison of the potential during the free passage of a solution through an open pore in a glass separating two units and the potential when the pore is covered with peritrophic membrane did not show much difference (experimental average 4.5 mV; control average 4.3 mV). It follows, therefore, that with a tenfold difference of KC1 concentration on the two sides of the membrane no appreciable amount of membrane potential is created in the system. The second series of experiments where media with different pH values were used also gave analogous results. Addition of acid to the medium changes the potential inversely, but this is not related to the peritrophic membrane, since the membrane did not generate membrane potential in acid, alkaline, or neutral media. This means that solutions of different ions permeate through the membrane at practically equal speed. The speed, it seems, is determined by the laws of free diffusion and depends upon the gradient of concentration and the coefficient of diffusion of the substance concerned. The level of permeability of the peritrophic membrane for solutions of electrolytes confirmed this idea. Experiments showed that all the substances used permeate through the membrane in both directions at equal speed. Therefore, the average time required to pass through the membrane was calculated without consideration of the direction of movement. Table 1 shows that the level of permeability (W) of the peritrophic membrane for all the substances used is expressed by values close to each other. In general, it may be said that the level for mono-monovalent electrolytes is 0.3 and for mono-bivalent electrolytes it is 0.2, but there are exceptions in both the cases. Thus, the level of permeability for chloride of lithium was found to be lower (O-25) than for other mono-monovalent electrolytes, whereas for chloride of magnesium it is higher (O-3) than for the rest of the substances with a similar valency. A comparison of the speed of permeation of substances through the peritrophic membrane in acidic, neutral and alkaline media also did not show any appreciable difference. This further confirmed the idea that the peritrophic membrane of

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D. P. ZHUZHIKOV

mosquito larvae does not possess any selective permeability for simple anions and cations. The values of the level of permeability of the peritrophic membrane that we obtained quite clearly show the high speed of permeation of ionic solutions through it. In fact, the speed of ionic permeation through an opening covered with the peritrophic membrane is only three to five times slower than the speed of passage through the unhindered opening. TABLE I-LEVEL

OF PERMEABILITYOF THE PERITROPHIC MEMBRANE OF Aedes aegyfiti LARVAE FOR SOLUTIONS OF ELECTROLYTES Electrolytes

Hydrochloric acid Potassium chloride Ammonium chloride Sodium chloride Lithium chloride Barium chloride Calcium chloride Magnesium chloride Manganese chloride Potassium sulphate

tK

tE

13.2 16.5 20.3 20.3 17.3 18.0 14.9 23.0 17.7 14.8

45.2 56.0 63.9 68.5 68.4 84.7 69.4 72.4 79.5 68.5

w = t&E 0.3 0.3 0.3 0.3 0.25 0.2 0.2 0.3 0.2 0.2

Equal speed of ionic permeation through the membrane in either direction shows that, for the ions, there is no polarity on the outer or inner surfaces of the membrane. The coefficient of permeation of the peritrophic membrane is the same in both directions. Coefficient of$Ztrutif3n (LJ

In a series of experiments it was found that for every peritrophic membrane the coefficient of filtration remains the same when hydrostatic pressure varies from 20 to 500 mm of water column. This coefficient varies slightly for the membranes from different larvae. For later calculations the average value of Lp = 3.36.10-s cms/sec through an area of 1 cm2 at a pressure of 1 bar. was used. RejZective coeficient

(6)

In experiments with raffinose and inulin in the presence of hydrostatic pressure, the following average values of 8 were found: for rafIinose 0.001 and for inulin O-0007 cm3. The osmotic pressure of O-1 M solutions of raffinose and 0.01 M solutions of inulin, calculated according to the equation of Vant-Goff, are equal to 2.35. lo6 and 2.35. lo5 bar. respectively. Hydrostatic pressure was kept constant at 39,220 bar. in all the experiments. By using these values the reflective coefficient

PERMEABILITY

OF PERITROPHIC MEMBRANE IN AEDES

1199

LARVAE

can be calculated from equation (1). orsti =

3.36.10~*.39220-0.001 3.36.10-s.2.35.106

= o oo4

%ml =

3.36. 1O-s. 39220 - 0.0007 = o 078 ’ ’ 3.36.10-s. 2.35. lo5

’

-

In experiments where the difference of hydrostatic pressure is zero, i.e. P = 0, it was found that a O~015°/osolution of protargol neutralizes a 10% solution of sucrose. The values of the reflective coefficient for sucrose in these experiments were calculated from equation (2) : Asno= 1~?/7000 = OGlO2. The value of the reflective coefficient is related to the effective radius (u) of the permeating substance and the effective radius (r) of the membrane pore by a rather complex equation (GOLDSTEIN and SOLOMON, 1960): I _ 6 = [2( 1- a/r)” - (1 - a/~)~] [ 1- 2*104a/r + 2.09(# - 0.95(u/~)s] [2(1 -b/r)2- (1- b/~)~] [l - 2.104b/r+2.09(b/~)~- 0.95@/~)~] ’

(31

where b is the crystallographic radius of the water molecule and is equal to 1.5 A. The effective radius of molecules of sucrose is 5.3 8, for raflinose 6-l A, and for inulin 12 A. The effective radius of the peritrophic membrane pore for all the three substances examined was determined by substituting these values in equation (3).

rA

sugars

40,000 3,000 250

1. Sucrose 2. Raffinose 3. Inulin

Permeability of the intact membrane for particles of colloidal gold

The results obtained in a series of measurements are recorded in Table 2. In this table only those particles with a completely round form are included-irregular particles formed by adhesion of several smaller ones not being considered. The size of such (round) particles did not exceed 100 A. TABLE ~--DISTRIBUTION OF COLLOIDALGOLD PARTICLESACCORDINGTO SIZE

Size (A) Place from which the probes were drawn Outer solution Trophic cavity Caeca

10

20

30

40

50

60

70

80

90

100

3 5 15

5 8 19

10 12 58

27 17 84

35 25 39

27 15 16

15 11 4

2 3 1

3

1: 4

_’

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D.P.

ZHUZHIKOV

Statistical analysis of the results showed that the size of particles in the trophic cavity coincides wholly with the size of those in the outer solution. In both the cases a normal distribution without any material difference between the two was observed. It follows, therefore, that the larvae swallow all the colloidal particles without filtering the biggest or the smallest ones. Analysis of the size of particles in the caeca showed that the size distribution of these particles may be regarded as normal only with a level of probability of 0.95. A graph of this distribution does not possess any reliable asymmetry (t, = 2.49) or axis (t, = 2.81). The maximum particle size, calculated according to the law of normal distribution, does not exceed 81.42 A. Since we are concerned here with a not very strict normality of distribution, the maximum particle size may be regarded as 90 A. This distribution differs distinctly from the first two and shows that the smaller particles permeate through the peritrophic membrane in larger numbers and the bigger particles find their way into the caecal cavity only very rarely. DISCUSSION

The single-layer peritrophic membrane of Aedes aegypti larvae, as has been shown in these experiments, does not possess selective ionic permeability. This confirms the idea that exchange of anions or cations in well-dissociating inorganic compounds used as stomach poisons will not vitally affect their passage through the It should, however, be taken into consideration that peritrophic membrane. absorption of such substances by the gut epithelium may be different and therefore their toxicity may also differ. It was found that the coefficient of filtration values for the peritrophic membrane is two to three times higher than the known values for cellulose membranes (DURBIN, 1960), i.e. water passes through the peritrophic membrane at a much higher rate than through different cellulose membranes. This coefficient remains constant at hydrostatic pressures between 2 and 50 cm of HsO, which points towards the absence of any considerable expansion of the peritrophic membrane at such pressures. The high level of permeability of the peritrophic membrane for ionic solutions shows that the membrane does not offer any appreciable hindrance for such solutions. Sugar solutions permeate through the membrane practically without any dissociation of solutes from the solvents (S,,, = 0*0002; araft = 0*004), i.e. the membrane is fully permeable for them. Even the comparatively larger molecules of inulin (M = 3100) p ermeate through it sufficiently freely (6 = O-078). In connexion with such easy permeability of the membrane for all these sugars, the calculated effective radius of the membrane pore was found to be very large, but its value for the three sugars differs greatly (40,000, 3000, and 250 A). It seems probable that the theoretical assumptions worked out for the inert cellulose membranes are not fully suited for the peritrophic membrane. Its permeability for the non-ionic substances depends not only on the pore size but is

PERMEABILITYOF PERITROPHIC MEMBRANJZ IN AED.?%LARVAE

1201

also regulated, to a considerable extent, by the interaction between the matrix of the membrane and the permeating substance. This interaction becomes more effective the bigger the size of the permeating molecule. Thus, the isolated membrane does not act upon the small sugar molecules, while its retarding forces become more effective for the bigger molecules of inulin and, as a result, the calculated effective pore radius becomes smaller. In this way, the peritrophic membrane causes more hindrance for high molecular complexes than is expected from the law of Poisell. The results obtained in these experiments show that the substances to which the peritrophic membrane is permeable pass through it so rapidly that the study of their speed or quantity does not seem to be worth while. This is because, in the presence of a sufficient gradient of concentration or osmotic pressure, the quantity of permeating substance will depend on these indices and not on the characteristics of the membrane. High molecular aggregates or colloidal food substances may be held back, to a great extent, in the trophic cavity and may not pass through the peritrophic membrane. At present nothing can be said about the nature of the forces obstructing the passage of these substances through the membrane. The only thing that we know for sure is that there is some mechanism holding back some of the high molecular substances which differ from the rest not only in the size of the component molecules but also in some other characteristics. In fact, molecules of digestive enzymes are not smaller (in some cases even bigger) than the molecules of proteins and polysaccharides, but the enzyme molecules permeate through the membrane freely while colloidal food substances are held back by the membrane. On the basis of these findings it may be concluded that the peritrophic membrane should not be regarded as a simple ultra-filter similar to cellulose membranes. On the other hand, it is not related to living cells and cannot, therefore, possess energy mechanisms allowing active transport of substances through it. The peritrophic membrane is, thus, a unique non-cellular membrane selectively permeable to high molecular complexes. REFERENCES DOGADKINB., SOBOLEVAI., and ARCHANGELSKAJA M. (1949) Comparative determination of molecular weight of rubber by the method of light refraction and osmometry. Reports of the 6th Conference on High Molecular Compounds, pp. 253-262. USSR Academy of Science Publications, Moscow. (In Russian.) DURBIN R. P. (1960) Osmotic flow of water across permeable cellulose membranes. J. gen. Physiol. 44, 315-326. GOLDSTEIND. and SOLOMONA. K. (1960) Determination of equivalent pore radius for human red cells by osmotic pressure measurement. J. gen. Physiol. 44, 1-17. HODGKINA. L. (1958) Ionic movements and electrical activity in giant nerve fibres. Proc. R. Sot. (B) 148, l-37. KEDEM 0. and KATCHAL~KYA. (1958) Thermodynamic analysis of the permeability of biological membranes to non-electrolytes. Biochim. biophys. Acta 27, 229-246. PHILLIPS J. E. and DOCKRILLA. A. (1968) Molecular sieving of hydrophilic molecules by the rectal intima of the desert locust (Schistocerca gregaria). J. exp. Biol. 48, 521-532.

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RABOTNOVA I. L. (1957) Role of Physico-chemical Conditions (pH and rHJ in Life Processes of Micro-organisms. USSR Academy of Science Publications, Moscow. (In Russian.) RUBENSTEIN D. L. (1947) General Physiology. Medgiz, Moscow. (In Russian.) ZHUZHIKOVD. P. (1964) Function of the peritrophic membrane in Musca domestica L. and Calliphora erythrocephala Meig. J. Insect Physiol. 10,273-278.