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Exchange of water between a mite, Laelaps echidnina, and the surrounding air under equilibrium conditions
J. Insect Physiol., 1968, Vol. 14,
pp. 1303 to 1318. Pergamon Press. Printed in Great Britain
EXCHANGE OF WATER BETWEEN A MITE, LAELAPS ECHIDMNA, AND THE SURROUNDING AIR UNDER EQUILIBRIUM CONDITIONS* G. W. Acarology
Laboratory,
WHARTON College
and
T.
L. DEVINE
of Biological Sciences, Columbus, Ohio
The
Ohio State University
(Received 25 March 1968) Abstrac:t-The
spiny rat mite has been found to have a mean half-life of water exchange with vapour in the surrounding air of 18.6 hr at 25°C when the activity of the water vapour of the air a, = 0.925. Tritiated water was used in making this determination. It was found that the rate constant for transpiration (KT) could be determined by measuring changes in the tritium content (T*) of the mite with time and that the rate constant for sorption (k,) could be similarly calculated from determinations of the specific radioactivity (S). The minimum rate constant (Kp) for the pump responsible for the concentration of water from unsaturated air by the spiny rat mite can be calculated from KT, a, of the haemolymph, :md the critical equilibrium activity. These studies were done under equilibrium conditions so that no significant changes in mass occurred. The mean values of the rate constants were: k, = k, = 0.0373 hr-l
and
kp = O-0036 hr-l.
INTRODUCTION
AT AMBIENT terrestrial temperatures and pressures water in bulk loses molecules to the surrounding air, while at the same time molecules of water vapour in the air will enter the condensed phase. The loss of water to the air is evaporation or, if from an organism, transpiration. The gain of water from the air is condensation In an enclosed isobaric and isothermal system the or, if to an organism, sorption. number of molecules escaping from a water surface in a given period of time and those conde:nsing on the surface from the air will become equal and the system Under these equilibrium conditions no net change in the will be in equilibrium. amount of bulk water or water vapour will occur and in the case of an organism the rate at which water is transpired is equal to the rate at which it is sorbed. In any one such isobaric, isothermal, closed system the actual rates at equilibrium will be determined by the activity of the water (a,) in the liquid phase. The activity of a solvent is a ratio that compares its chemical potential to that of the material, in this case water, in the pure state with that of the material containing one or more solutes. Activities of solvents in ideal solutions are equal
* This work was supported Grant
NsG
by the U.S.
National
652. 1303
Aeronautics
and Space Administration:
1304
G. W. WHARTONANDT. L. DEVINE
to the concentration of the solvent expressed as the mole fraction. For non-ideal solutions the activity is equal to the mole fraction multiplied by the appropriate activity coefficient. Furthermore, under equilibrium conditions, the activity of the water in the liquid phase will be equal to the activity of the water (a,) in the vapour phase such that: a, = a,.
(1)
It follows from equation (1) that if the activity of the water in the liquid phase is increased that of the vapour will be increased by a net movement of On the other hand, if the activity of water in water from the liquid to the vapour. the liquid phase is lowered the activity of water in the vapour phase will be lowered by a net movement of water into the liquid phase. Thus, net movement of water will occur from regions of high a to regions of low a. One way to determine the activity of water in a solution is to measure the relative humidit] of air in equilibrium with the solution in question since: r.h.% %=m’ In studies on movement of water between organisms and their environments the tendency of water to move along a gradient is expressed in many ways and sometimes the units or ratios used for measurements in the environment are different from those used for the organism (EDNEY, 1966). Since the tendency of water to move from one region to another is related to its colligative properties; freezing-point depression, dew-point depression, osmolality, osmotic pressure, vapour pressure, vapour-pressure deficit, wet-bulb depression, suction pressure, diffusion-pressure deficit, and water potential as well as activity and r.h. have all been used to express the tendency for net movements of water to take place from one compartment or phase to another. In preparing this paper it has been found most convenient to use activity for this purpose (Table 1). This has proven to be the case because activity can be used with gases, liquids, and solids. Use of activity has been found most helpful in many calculations. Studies on transpiration and sorption of water by arthropods are relatively few compared to such studies on other biological material. A useful resume of many such studies on cells along with a consideration of the physical chemistry of water transport phenomena has been written by DICK (1966). In this report much information on permeability of cells to water is presented in terms of a permeability coefficient. In arthropods, studies on the net movements of water between the organism and its environment have been made. An excellent summary of such work was published ten years ago (EDNEY, 1957). Two more recent reviews (BURSELL, 1964; BAPOPORT and TSCHAPEK, 1967) cover later reports and provide excellent bibliographies. In contrast to the many studies on net transpiration and net sorption, few papers reporting on the movement of water between air and arthropods using isotope labelled water have been found in a fairly comprehensive search of the
EXCHANGE
OFWATERBETWEBN
AMITEAND
SURROUNDING
AIR
1305
literature (MARCUZZI and SANTORO, 1959; GOVAERTS and LECLERCQ, 1946). By using heavy water as a tracer the earlier authors determined that the concentration of heavy water in the body water of a number of insects became the same TABLE ~-RELATIONSHIP OF a, AT 1 atm PRESSURE AND 25°C TO SOME UNITS FREQUENTLY USED TO MEASURE THE TENDENCY OF WATER TO MOVE FROM ONE COMPARTMENT TO ANOTHER
a,
r.h. %
1.000 100 0.999 99.9 0.990 99.0 98-O 0.980 0.970 97.0 0.960 96.0 0.950 95.0 0.900 90.0 0.800 80.0 70.0 O-700 0.600 60.0 0.500 50.0 0.400 40.0 30.0 0.300 0.200 20.0 0.100 10.0 0.000 0.0
Osmotic pressure (an-n) 0 1.34 13.60 27.37 41.25 55.25 69.48 142.70 302.20 480.30 696.85 938.75 1279.2 1630.65 2179.80 3020.10 cc
Osmotic pressure (osmols kg-l)
Vapour pressure (mm Hg)
Vapour pressure deficit (mm Hg)
23.756 23,732 23.518 23.281 23,043 22.806 22.568 21.380 19.005 16.629 14.254 11.878 9.502 7.127 4.751 2.376 0
0.024 0.136 0,475 0.713 0.950 1.188 1.376 4.751 7.127 9.502 11.876 14.254 16.629 19.005 21.380 23.756
0 0.0556 0.5607 l-1329 1.7168 2.3129 2.9216 6.1679 13.8814 23.7960 37.0070 55.5160 83.2658 129.5247 222.0425 499.5950 og
0
r.h. % Column 1: given; Column 2 : a, = ~ ; Column 3 : Modified from SHULL, 1939 ; 100
Column 4: a, =
55.5106 moles kg-r 55.5106 moles kg-‘+
osmols kg-r’
Column 5 : a, = P/P, ; Column 6 : a, =
P, - VPD p
0
.
as the concentration of heavy water in the air to which the insects were exposed. The time required for the concentrations to become equal was reported to vary between 5 and 13 days depending upon the kind and stage of the insect. For example, Tenebrio molitor adults reached equilibrium in 9 days, whereas larvae did so in 13 days. A reasonable interpretation that can explain these results is that the insects exchanged their water content with the water in the surrounding air in the time required for the concentration of heavy water in their water-pools to come into equilibrium with the concentration of heavy water in the atmosphere. The latter :authors report a half-life of exchange for larvae of T. molitor of 198+21 hr. Females of the spiny rat mite, Laelaps echidnina Berlese, have been used extensively in studies on net movements of water between the mite and the
1306
G.
W. WHARTON ANDT. L. DEVINE
surrounding air. It has been found that dehydrated individuals can restore their lost water by extracting water from unsaturated air if the r.h. is above 90 per cent (WHARTON and KANUNGO, 1962). The lowest r.h. at which organisms can maintain their water-balance, that is an equilibrium weight, has been designated as the critical equilibrium humidity (KN~~LLE and WHARTON, 1964). In the present paper the term ‘critical equilibrium activity’ will be used in place of the critical equilibrium humidity. Thus the reported critical equilibrium humidity for Laelaps echidnina of 90 per cent will, according to equation (2), become the critical equilibrium activity of O-90. The activity of the water in the haemolymph of Laelaps echidnina has not been precisely determined but it is known to be greater than a, = 0.99. This figure reported by WHARTON and KANUNGO (1962) is verified by the observation that hydrated mites submerged in solutions where activity was a, = 0.99 rapidly lost volume and became flattened. Since at room temperatures a solution whose aqueous activity is 0.99 has an osmotic pressure of 13.6 atm and one whose activity is 0.90 has an osmotic pressure of 142.7 atm (Table l), this mite is capable of extracting water from unsaturated air against an activity difference of 0.09 or an osmotic pressure difference of 129.1 atm. The rates of the net movement of water in and out of Laelaps echidnina under various conditions are known (WHARTON and KANUNGO, 1962). Dead mites exposed to a, = 0.80 lose water at about 1.2 pg/hr whereas live mites exposed to a, = 0.99 can gain water at the rate of 0.8 pglhr. Since mites weigh about 150 ,ug these rates of net water movement represent significant shifts in total water content. The amount of energy required for net uptake of water has not been determined but it is known that it represents only a small fraction of the metabolic activity of the mite (KANUNGO, 1965). In fact, daily dehydration followed by daily rehydration of this mite has been found to have little influence on its longevity under starving conditions (KN~~LLE, 1967a). In his general conclusions on transpiration of water from arthropods EDNEY (1957) states, ‘It is also possible to eliminate transpiration from the integument to a large extent by waterproofing with wax. Here again, insects and arachnomorphs are conspicuously successful’. In a study of the fine structure of the cuticle of Laelaps echidnina (WHARTON et al., 1968) all of the essential waterproofing layers were seen, and it can be concluded that this mite has a wellwaterproofed cuticle. In most terrestrial arthropods the site of maximum transpiration is the respiratory surface. Despite the excellent waterproofing of the external cuticle and the high transpiration rates from respiratory surfaces, it is important to emphasize that transpiration through a well-waterproofed cuticle is significant to the water-balance of arthropods. This conclusion is reinforced by the fact that gain of water by arthropods from unsaturated air takes place through the cuticle (LEES, 1946). Another consideration that reinforces the importance of a well-waterproofed cuticle as a pathway for the exchange of water is that the total or gross movement of water across the cuticle under most circumstances will be much greater than the net movement.
EXCHANGE OF WATER BETWEEN A MITE AND SURROUNDING AIR
1307
studies on the gross movement of water between female Laelaps air at a, = O-925 and T = 25°C will be reported. Finally the significance of the findings on the ability of the spiny rat mite to gain water from the air against an activity difference of 0.09 or an osmotic pressure difference of over 100 atm will be discussed. In this paper
echidnina and
MATERIALS
AND METHODS
Mites used in these studies were obtained from cultures at the Institute of Acarology of the Ohio Agricultural Research and Development Center. Mites were placed in an arena formed from an enamel tray bordered by hot nichrome wire that served as a barrier to the mites. From this arena female mites were aspirated into glass tubes. The tubes were closed at each end with nylon net so that free flow of air through the tubes was possible. Such tubes of mites were then placed in a dry atmosphere in desiccators containing dehydrated silica gel. After exposure to the low a, of the desiccators for 6 hr the tubes of mites were removed. A meal of rat blood was then offered to the mites (CROSS, 1954). Females that engorged on the blood were aspirated into clean tubes and held in moist air (a, > O-95) over moist activated charcoal and plaster of Paris (HUBER, 1958) for at least a week. This procedure provided standardized mites that had completed digestion of their previous meal (KANUNGO, 1964) and that produced neither faecal pellets nor eggs. By using only standardized mites in experiments, time since the last meal was uniform for each mite. An important variable that was not controlled by this procedure was the age of the mites (LEES, 1964). A plastic exposure chamber in which the air was in equilibrium with a saturated solution of KNO, and thus had an activity (a,) of 0.925 at 25°C (WINSTON :and BATES, 1960) was used in these experiments. Atmospheres containing tritiated water were obtained by adding tritiated water at about 2 PC of tritium/ml of saturated salt solution. The temperature of 25°C was maintained within 1°C by keeping the chamber in a BOD incubator. Standardized mites were exposed in glass tubes that were closed at either end by nylon bolting cloth. Studies on sorption of tritiated water involved placing mites in an atmosphere containing tritiated water vapour and determining the radioactivity of the mites at various intervals of time of exposure to the tritiated atmosphere. This technique was found to introduce a systematic error if a series of readings was made. The error resulted from the fact that removal of a sample from the exposure chamber introduced air from the room which then lowered the tritium concentration of the water vapour in the exposure chamber. This introduction of air from the room resulted in lowered values of tritium in the mites for a given period of time and thus lowered observed sorption. This error was avoided by using multiple exposures at different times instead of taking a series of samples at different intervals from the same exposure. Studies on transpiration of tritiated water were made in the same manner as those on sorption except that the standardized mites were exposed to an
1308
G. W. WHARTON AND T. L. DEVINE
atmosphere containing tritiated water vapour until they had come into virtual equilibrium with the vapour. They were then exposed to an atmosphere that Studies on transpiration of tritiated water contained no tritiated water vapour. were more convenient to do because they could be done without using atmospheres containing tritiated water vapour except for the single equilibration chamber in which large numbers of mites could be exposed at the same time. Since all exposures were made at a, = 0.925, an activity above the critical equilibrium activity of L. echidnina, no significant net movement of water occurred in mites All mites that died during an exposure or that maintained normal weights. weighed less than 100 pg were discarded. Under these conditions sorption and transpiration are equal and the measurement of one will provide the value for the other. Three data were obtained for each exposure interval: time of exposure in the test atmosphere, weight of each mite in pg and the radioactivity or tritium content Mites were weighed on a Cahn electrobalance (T*) of each mite in counts/min. to the nearest pg. The radioactivity of the mites was determined by placing a single intact weighed mite in 10 ml phosphor solution and determining the counts/min in a liquid scintillation counter. The phosphor solution consisted of 4 g of Packard BBOT scintillator dissolved in 1 1. of a 1 : 1 mixture of toluene The background count rate was subtracted from the observed -methanol. counts/mm to provide the counts/min attributable to the tritium from the mite. The mite had no significant effect on background or counting efficiency and virtually all of the tritium content of the mite was determined by this technique. Because the methods employed in this investigation resulted in the destruction of the specimen, it was impossible to obtain a series of observations on a single specimen. For this reason mean values are reported. One important value needed for an interpretation of the results is the mean water content of the mites. Another important consideration is the fate of tritiated water in the mites. If the tritiated water remains in the water-pool of the mite then the counts/min recorded for mites in equilibrium with a given concentration of tritiated water vapour should be proportional to the amount of water in the mite. If this be the case, then a plot of tritium content vs. weight should result in a straight line that crosses the weight axis at the mean dry weight. Fifty mites were dried over vacuum and P,O,. The mean dry weight for these mites was 37.54 pg, the heaviest individual weighed 43.7 pg and the lightest 27.8 pg. Fifteen mites, after they had come into equilibrium, were weighed and their radioactivity determined. The values for each mite were plotted on linear co-ordinate paper ruled off in counts/min along the abscissa and pg on the ordinate. A straight line originating at 37.54 pg (the previously determined mean dry weight) and terminating at the highest countsjmin recorded runs through the array of data points representing mites, (Fig. 1). It is on the basis of the agreement between weight and tritium content illustrated in Fig. 1 that the hypothesis, stating that all of the observed tritium remains in the waterpool of the mite, is accepted. Furthermore, when the hypothesis that all of the
EXCHANGE OF WATER BETWEEN A MITE AND SURROUNDING AIR
observed tritmm remains in the water-pool is accepted, as an independent determination of dry weight and obtained by (dehydration over vacuum and PsO,.
1309
Fig. 1 can be considered then confirms the value
160 140 g
120
i? .; 100 E c .-
80
ii r"
60 40 20
200
400
600
800
1000
1200
1400
CPM FIG.
1.
The relationship between weight (pg) and radioactivity (counts/min) of mites in equilibrium with water vapour containing tritiated water
The analysis of the data is based on a theoretical model derived from CRANK I or organism initially in equilibrium with a given a, will gain or (1956). A so I’d lose weight when placed in an atmosphere at a different a,. The relations for evaporation or condensation by a small surface-limited solid which had been in equilibrium with one vapour pressure and then plunged into another at some constant vapour pressure reduce to first order kinetics: m = m,exp(-K,t)
or
lnGs=
-K,t,
where m is the mass yet to be transferred and m, is the total mass that evaporated Ior sorbed. The rate riz of net mass change is the derivative above equation with respect to time (t): riz = -k,m,exp(-k,t)
= -k,m.
will be of the
(4)
G. W. WHARTON AND T. L. DEVINE
1310
This net mass change rate must equal the difference and the rate of transpiration (rizr):
in the rate of sorption
riz = r&-r&. Dividing
equation
(5) by the water
(rizs)
(5)
mass gives the rate constants:
k, = k,-k, where k, = 2,
k, = 2,
and
k, = 2.
(6)
An organism containing an amount of tritium (T*) when placed in a constant untritiated vapour will, in an increment of time (dt), lose an increment of tritium (dT*) equal to the increment of water transpired (-~$dt) multiplied by the specific radioactivity (S) where S = T*lm, and using the relation for k, above: dT*=-Sh,dt=-;ria,dt=-T*k,dt.
With
an initial
tritium
content
(7)
(T*) this integrates
T* = T*,exp(-k,t)
or
ln$
to :
0
= -k,t.
(8)
Thus the rate constant for tritium loss is identical to that for the transpiration of water and is independent of sorption. If k, is determined from mass data with equation (4) and k, is determined from tritium content data with equation (8) then k, is determined by equation (6). When mass is constant k, = 0 and k, = k,. Sorption of untritiated water vapour by an initially tritiated organism results in a change of the specific activity of the body fluids which is determined by and substituting relations from differentiating (S) with respect to time equations (6) and (7): 1 dT” =-_-----_~ m dt With
the initial
specific
S
-T*
T*ldm m m dt
radioactivity
s, = exp(-kst)
T* -,(k,-k,)
m
(So) this integrates or
lnS,
S
= -Sk,.
(9)
to:
= -k,t.
Thus the rate constant for the decrease of specific radioactivity is identical rate constant for sorption of water and is independent of transpiration.
to the
EXCHANGE
OF WATER
BETWEEN
A MITE
AND
SURROUNDING
An initially untritiated organism gains both tritium radioactivity when plunged into a vapour of tritiated water. to the above., results in the equations:
T* = l-exp(-&t) KC
-
or
In
(
1-g
m)
1311
AIR
content and A derivation,
specific similar
(11)
=-krt,
-k,t, where the radioactivity respectively.
and specific
radioactivity
(12)
at equilibrium
are T, and S,
RESULTS
Water was found to move in and out of the mites during exposures at an easily measurable rate as was indicated by changes with time (Table 2). TABLE
~---N[EAN
WEIGHTS, EXPOSED
TRITIUM
CONTENTS,
TO EXPERIMENTAL
AND
TRITIUM
ATMOSPHERES
CONCENTRATIONS
FOR VARIOUS
experimental in T* and S
FOR
MITES
TIMES
Mean S Time (hr)
Mean weight (ELg)
Mean T* (counts/min)
(counts/min per Fg)
+1s Equilibrium -18.5 -27 -44
144f9 153k5 149k6 149*7 139kl3
552f43 1303 f 57 559 + 63 488 f 90 23lk 102
5.2 11.3 5.4 4.4 2.3
No. of mites 23 11 15 15 6
Positive and equilibrium values represent periods of exposure to a tritiated atmosphere; negative values represent time in an untritiated atmosphere since attaining equilibrium in a tritiated atmcsphere.
In order to calculate the amount of water transpired and sorbed from the data obtained it is necessary to make three assumptions. That the tritium observed re-mains in the water-pool of the mite is an assumption the validity of which has Eleen demonstrated (Fig. 1). The second essential assumption is that the observe’d movement of the tritium is indicative of the movement of water. This assumption is justified in that tritium is introduced into the system only in the form of water, and is recovered from the system only in the aqueous component. The third assumption that no significant net movement of water occurred during the course of the experiments is justified in that the experiments were all run at an a, above the critical equilibrium level and that the mean weights for the various times are not significantly different (Table 2).
G. W. WHARTONAND T. L. DEVINE
1312
In making calculations of gross transpiration and sorption of water between the mites and the air, only the mean values have been used. It is recognized that values for mites will vary from individual to individual and from time to time in the case of the same mite. An estimate of the amount of variation to be expected is reflected in the 5 per cent confidence intervals given for the mean weights and mean tritium contents (Table 2). As can be predicted from equations (8) and (10) the relationship between time and relative specific radioactivity is that of a first-order kinetic equation. When time (t) is plotted on a linear scale on the abscissa and S/S,, or 1 -(S/S,) 1.0
.9 .8 .7 .6 .5 a cl??
.4
6
12
18 Time
24
30 in
36
42
48
j,
SO
Hours
FIG. 2. The relationship between time of exposure and relative specific radioactivity of mites under equilibrium conditions.
is plotted on a logarithmic ordinate a straight line of slope -k can be seen to describe the relationship (Fig. 2). Using the data in Table 2 and equations (8) and (11) it is possible to calculate k for each exposure period. The results of
EXCHANGE
these calculations
OF WATER
BETWEEN
A MITE
AND
SURROUNDING
AIR
1313
are : k = 0.0342 hr
for
+ 18 hr,
K = 0.0397 ~ hr
for
- 18.5 hr,
k: = 0.0385 hr
for
-27
k: = 0.0366 hr
for
- 44 hr.
hr,
The mean value for K taking into account the number of observations represented by the means for each exposure period is 0_0373/hr. Another way of expressing k = 0*0373/hr is to say that transpiration and sorption each proceeds at a rate of 3.73 per cent of the total water mass/hr. The half.-life of a first order kinetic reaction can be determined by taking advantage of the fact that the time taken to complete a definite fraction of the process is independent of the initial amount of the material under study. Thus when one half of the process is completed, that is when T*/T*,, is equal to 0.5, substitution of this value in equation (8) gives: ln0.5
= -k,t,
or
tg =p
ln2 T
where tt is equal to the time required for T*/T, to become equal to 0.5 or another way of expressing it is that tt represents the half-life of the process. Inserting the mean value of k in equation (8a) the mean half-life is calculated to be 18.6 hr (Fig. 2). The rate constant KS = 0*0342/hr calculated on the basis of 18 hr of sorption and the rate constant k, = 0*0397/hr calculated on the basis of 18.5 hr of’ transpiration are well within the limits of the biological diversity and experimental error of these observations and are in good agreement with the assumption that no significant net movement of water occurred during the experiments. It has been determined that the rate constants k, and k, determined for the movement of tritium are equal to the rate constants for the fraction of the water labelled with1 tritium and thus for all of the water. In the case of tritiated mites exposed to an untritiated atmosphere containing a large tritium sink, loss in tritium content is associated solely with transpiration (equation (8)). On the other hand changes in the concentration of tritium are brought about solely by the sorption of water (equation (10)). Th e validity of the mathematical argument can be appreciated intuitively if one considers a separator-y funnel containing a dye dissolved in some solvent. The total amount of the dye in the funnel can be changed only by opening the stopcock and removing some of the solution. This action will have no effect on the concentration of the dye or the intensity of the
1314
G. W. WHARTONAND T. L. DEVINE
colour of the solution. Removal of material is analogous to transpiration and its only effect is the reduction in the quantity of the diluent, dye in the case of this analogy or tritium in the case of the mite. If solvent is added to the funnel, the concentration of the dye or the intensity of colour of the solution will be changed. Addition of solvent is comparable to sorption. Thus in the experiment reported here, measure of the change of concentration of tritium is not a measure of the movement of tritiated water but is in fact a measure of the movement of water itself. Since K gives the per cent of the total reaction taking place in unit time, the amount of water transpired in unit time is equal to k, times the amount present. In the case of a spiny rat mite containing 100 pg of water under equilibrium conditions this means that transpiration amounts to 3.73 pg/hr and the same amount of water will be sorbed when equilibrium weight is maintained. Thus the total movement of water amounts to 7.46 pg/hr under equilibrium conditions. When compared to rates of 1.2 and 0.8 pg of water/hr reported as net movement (WHARTON and KANUNGO, 1962) it can be seen that concepts concerning the permeability of the arthropod cuticle based on studies of the net movement of water are likely to give an exaggerated impression of its impermeability. DISCUSSION
Exchange rates Although a half-life of 18.6 hr for the exchange of water between an organism and the water vapour in the surrounding air is significant and will appear to many entomologists as surprisingly short, it is actually quite slow when compared to exchange of water through cell membranes. DICK (1966) reviews values of permeability of various animal cells to water. He uses as a measure a permeability coefficient (k*) whose formula in tracer studies is: k*
=
IlnCo-Ce At
C-Ce’
(13)
where V is volume; A, area; t, time; Co, initial concentration of the tracer in the cell; Ce, concentration of the tracer in the environment; and C, concentration of the tracer in the cell at time t. Values of 12” reported for various free living cells range from 400 psec-1 for canine erythrocytes to 0.37 psec-l for certain protozoa. While some of the difference is probably caused by differences in permeability of the cell membranes, a more significant factor is the surface to volume ratio, k* being roughly proportional to this ratio. The surface to volume ratio reported for the canine erythrocytes is 1.82 p-l and that for the protozoan O-017 p--l. In the case of the smaller k* values, it is probable that diffusion within the cell is so slow that concentration differences across the cell membrane remain slight, thus slowing the net movement across the membrane. Substituting the mean values for S at tt obtained for the spiny rat mite in equation (13) and assuming a volume of l-2757 x 108 p3 and an area of
EXCHANGE
OF WATER
BETWEEN
A MITE
AND
SURROUNDING
AIR
1315
9.25 x lo5 pz its k* value is 0.0014 psec-I. Since this value is so much smaller than any of the values reported for cells, even those with larger volumes than that of the mite, it is obvious that the internal mixing of the water is rapid enough to maintain internal concentrations at a uniform level throughout the system. Under these conditions the low K* value must be interpreted as revealing a highly imperlmeable barrier to the passage of water. Such an interpretation is consistent with the generally held view that the arthropod cuticle is well waterproofed. In studies of cells reported by DICK (1966), it is pointed out that k* values obtained from tracer studies, and those from studies on net movement of water, are not the same for the same cell system. In general, k* values calculated on the basis of the net movement of water were found to be about twice as great as those determined from tracer techniques. It should be pointed out that in the calculation of k* only sorption is taken into account by Dick’s formula, and, since tracer studies are usually made under conditions where no net movement occurs, sorption and transpiration must be equal and the movement of water across the membrane will be twice that obtained by his method, The assumption that rate constants for exchange of HTO, which were determined, are approximateky equal to rate constants for exchange of Hz0 can be challenged on the basis of the difference of the two molecules in weight and configuration. Since in these studies actual water movement as well as tracer movement have been determined and found to correspond, the challenge that artefacts produced by isotopic eRects may contaminate the results is groundless. Site of exchange Three main avenues of water uptake are available to most arthropods. These are the general body surface, the respiratory surface, and the digestive tract. In the experiments reported here the digestive tract as a portal of entry for liquid water can be ruled out because no liquid water was available to the mites. It is possible that water from the air could be obtained via the digestive tract. If the mites were to secrete a saliva whose a, was 0.90 or below, a net gain in water from air wh’ose a, was greater than 0.90 would result and ingestion of this vapour-enriclhed saliva could account for water uptake. On the other hand, failure to have observed such salivary secretions on countless microscopic examinations suggests that this is not the mechanism involved. It is also probable that the portal of entry is not the respiratory system. Two considerations are important here. The first is the fact that excessive water loss even at a,‘s well above the crucial equilibrium level has been observed whenever the respiratory rate of the spiny rat mite is elevated much above its basic level (KANUNGO, 1965). The second observation is that acari with no special respiratory system such as cheese mites and ixodid larvae have the capacity to extract water from unThere remains as the most probable saturated air (KN~~LLE, 1965, 1966). exchange surface the general body cuticle or some portion of it. All of the studies on the phenomenon of water uptake from unsaturated air by arthropods support
1316
G. W. WHARTONAND
the view that the cuticle or some portion water from unsaturated air.
T. L. DEVINE
of it is responsible
for the uptake
of
Gain of water from unsaturated air The most recent comments concerning the mechanism by which water is gained by arthropods from unsaturated air are those of KN~~LLE (196713). His brief discussion points out the activity of water molecules at the absorbing site must be lower than the a, of the air from which water is extracted and that an active mechanism or pump is required to move the water from the site of sorption to the haemolymph. KN~~LLE (1967a) re f rains from speculating on the mechanics of the pump but does refer to papers that do (BEAMENT, 1964; LOCKE, 1964). Recognizing that it is the difference between the a, of the site of sorption and the a, of the surrounding air that is responsible for net gain or loss of water it follows that at equilibrium where no net movement of water occurs k, = ks. Now k, is independent of k, and is proportional to the activity of the water at the surface from which transpiration takes place such that: k,,
=
zr a,
for transpiration where k, equals the rate constant rate constant for transpiration at a particular a,. for ks, that is: k,
=
2.
(14) at azr = 1 and k, equals the The same relationship holds
(15)
”
It follows from equations (14) and (15) that at equilibrium the effective a, of the exchange surface between the organism and the air must equal a,. If the a, at the exchange surface and the a, of the haemolymph are the same no active transport or pump is required for the maintenance of equilibrium. If, on the other hand, the a, of the haemolymph is O-99 or greater, as is usual, then a pump is required to maintain lower a, at the exchange surface of those arthropods that can extract water from air whose a” is below 0.99. Whether or not water or some carrier molecule is involved in the molecular mechanisms of the pump measurable quantities of water are moved. This flow of water should have a rate constant (kp) such that sufficient water can be removed from the exchange surface to be able to maintain an a, at the surface as low as the a, at the critical equilibrium activity. From equation (14) it is possible to calculate k, for any a,. When no pump is in operation, k, will be equal to k, x a, (haemolymph). When the pump is operating at the critical equilibrium level, k, will be equal to k,, x a, (critical equilibrium activity). It follows therefore that the rate constant for the pump (kp) for net movement of water when operating at the critical equilibrium level must be the difference between k, (haemolymph) and k, (critical equilibrium
EXCHANGEOF WATERBETWEENA MITE ANDSURROUNDING AIR
1317
activity). Using the values of k, determined in these studies for the spiny rat mite k, becomes 0*0373/0*925 or 0.0403, and the rate constant for the pump (kp) is found to be 0*0036/hr or about one-tenth the value of k,. The amount of water that must be pumped for a mite containing 100 pg of water is 0.36 pg/hr. This is of course the net movement. Knowledge of the net movement of water required of the pump opens the way to the development of more realistic models of possible mechanisms of pump action. For example, if the pump cycle is 1 set in duration, the amount of water involved/cycle need be only 0.0001 ,ug or 1 ppm of the total water content of the mite.
REFERENCES BEAMENTJ. W. L. (1964) The active transport and passive movement of water in insects. Adv. Insect Physiol. 2, 67-129. BURSELL E. (1964) Environmental aspects: Humidity. In The Physiology of Insectu (Ed. by ROCKSTEIN, M.), 1, 323-361. Academic Press, New York. CRANKJ. (1956) The Mathematics of Diffusion. Oxford University Press, London. CROSS H. F. (1954) Feeding tests with blood sucking mites on heparinized blood. J. econ. Ent. 47, 1155. DICK D. A. T. (1966) Cell Wuter. Butterworths, Washington. EDNEY E. B. (7.957) The Wuter Relations of Terrestrial Arthropods. Cambridge University Press, London. EDNEY E. B. (1966) Absorption of water vapour from unsaturated air by Areniwaga sp. (Polyphagidae, Dictyoptera). Comp. Biochem. Physiol. 19, 387-408. GOVAERTSJ. and LECLERCQ J. (1946) Water exchange between insects and air moisture. Nature, Lond. 157, 483. HUBER I. (1958) Color as an index to the relative humidity of plaster of Paris culture jars. Proc. ent. 6’0~. Wash. 60, 289-291. I(ANUNG0 K. (1964) Disappearance of blood from the gut of engorged Echinolaelaps echidninus (Acarina: Laelaptidae). Ann. ent. Sot. Am. 57,427-428. KANUNCOK. (1965) Oxygen uptake in relation to water balance of a mite (EchinoZueZups e&id&us) in unsaturated air. J. Insect Physiol. 11, 557-568. KN~~LLE W. (1965) Die Sorption und Transpiration des Wasserdampfes bei der Mehlmilbe (Acarus sire L.). 2. vergl. Physiol. 49, 586604. KN~~LLE W. (1966) Equilibrium humidities and survival of some tick larvae. J. med. Ent. 2, 335-33s. KN~ILLE W. (1967a) The significance of fluctuating humidities and frequency of blood meals on the survival of the spiny rat mite, Echinolaelaps echidninus (Berlese). J. med. Ent. 4, 322-325. KN~LLE W. (1!)67b) Physiological properties and biological implications of the water vapour sorption mechanism in’larvae of the oriental rat flea, XenopsyZZu cheopis (Roths.). r. Insect Physiol. 13, 333-357. KN~LLE W. and WHARTONG. W. (1964) Equilibrium humidities in arthropods and their ecological significance. Acarologia 6, 299-306. LEESA. D. (1946) The water balance in Ixodes ricks L. and certain other species of ticks. Parasitology 37, I-20. LEES A. D. (1964) The effect of ageing and locomotor activity on the water transport mechanism of ticks. AcaroZogia 6, 315-323. LOCKE M. (1964) The structure and formation of the integument in insects. In The PhysioZogy of Insecta (Ed. by ROCKSTEINM.), 3, 379470. Academic Press, New York. 84
1318
G. W. WHARTONANDT. L. DEVINE
MARCUZZIG. and SANTOROV. (1959) Indagini sul ricambio idrico de1 Tenebrio molitor mediante acqua tritiata. Ric. sci. 29, 2576-2581. RAPOPORTE. H. and TSCHAPEKM. (1967) Soil water and soil fauna. Rev. Ecol. Biol. Sol. 4, l-58. SHULL C. A. (1939) Atmospheric humidity and temperature in relation to the water system of plants and soils. Plant Physiol. 14, 401-422. WHARTONG. W. and KANUNGOK. (1962) Some effects of temperature and relative humidity on water-balance in females of the spiny rat mite, Echinolaelaps echidninus (Acarina: Laelaptidae). Ann. ent. Sot. Am. 55, 483-492. WHARTONG. W., PARRISHW., and JOHNSTOND. E .(1968) Observations on the fine structure of the cuticle of the spiny rat mite, Laelaps echidnina (Atari-Mesostigmata). Acarologia. In press. WINSTONP. W. and BATESD. H. (1960) Saturated solutions for the control of humidity in biological research. Ecology 41, 232-237.
pp. 1303 to 1318. Pergamon Press. Printed in Great Britain
EXCHANGE OF WATER BETWEEN A MITE, LAELAPS ECHIDMNA, AND THE SURROUNDING AIR UNDER EQUILIBRIUM CONDITIONS* G. W. Acarology
Laboratory,
WHARTON College
and
T.
L. DEVINE
of Biological Sciences, Columbus, Ohio
The
Ohio State University
(Received 25 March 1968) Abstrac:t-The
spiny rat mite has been found to have a mean half-life of water exchange with vapour in the surrounding air of 18.6 hr at 25°C when the activity of the water vapour of the air a, = 0.925. Tritiated water was used in making this determination. It was found that the rate constant for transpiration (KT) could be determined by measuring changes in the tritium content (T*) of the mite with time and that the rate constant for sorption (k,) could be similarly calculated from determinations of the specific radioactivity (S). The minimum rate constant (Kp) for the pump responsible for the concentration of water from unsaturated air by the spiny rat mite can be calculated from KT, a, of the haemolymph, :md the critical equilibrium activity. These studies were done under equilibrium conditions so that no significant changes in mass occurred. The mean values of the rate constants were: k, = k, = 0.0373 hr-l
and
kp = O-0036 hr-l.
INTRODUCTION
AT AMBIENT terrestrial temperatures and pressures water in bulk loses molecules to the surrounding air, while at the same time molecules of water vapour in the air will enter the condensed phase. The loss of water to the air is evaporation or, if from an organism, transpiration. The gain of water from the air is condensation In an enclosed isobaric and isothermal system the or, if to an organism, sorption. number of molecules escaping from a water surface in a given period of time and those conde:nsing on the surface from the air will become equal and the system Under these equilibrium conditions no net change in the will be in equilibrium. amount of bulk water or water vapour will occur and in the case of an organism the rate at which water is transpired is equal to the rate at which it is sorbed. In any one such isobaric, isothermal, closed system the actual rates at equilibrium will be determined by the activity of the water (a,) in the liquid phase. The activity of a solvent is a ratio that compares its chemical potential to that of the material, in this case water, in the pure state with that of the material containing one or more solutes. Activities of solvents in ideal solutions are equal
* This work was supported Grant
NsG
by the U.S.
National
652. 1303
Aeronautics
and Space Administration:
1304
G. W. WHARTONANDT. L. DEVINE
to the concentration of the solvent expressed as the mole fraction. For non-ideal solutions the activity is equal to the mole fraction multiplied by the appropriate activity coefficient. Furthermore, under equilibrium conditions, the activity of the water in the liquid phase will be equal to the activity of the water (a,) in the vapour phase such that: a, = a,.
(1)
It follows from equation (1) that if the activity of the water in the liquid phase is increased that of the vapour will be increased by a net movement of On the other hand, if the activity of water in water from the liquid to the vapour. the liquid phase is lowered the activity of water in the vapour phase will be lowered by a net movement of water into the liquid phase. Thus, net movement of water will occur from regions of high a to regions of low a. One way to determine the activity of water in a solution is to measure the relative humidit] of air in equilibrium with the solution in question since: r.h.% %=m’ In studies on movement of water between organisms and their environments the tendency of water to move along a gradient is expressed in many ways and sometimes the units or ratios used for measurements in the environment are different from those used for the organism (EDNEY, 1966). Since the tendency of water to move from one region to another is related to its colligative properties; freezing-point depression, dew-point depression, osmolality, osmotic pressure, vapour pressure, vapour-pressure deficit, wet-bulb depression, suction pressure, diffusion-pressure deficit, and water potential as well as activity and r.h. have all been used to express the tendency for net movements of water to take place from one compartment or phase to another. In preparing this paper it has been found most convenient to use activity for this purpose (Table 1). This has proven to be the case because activity can be used with gases, liquids, and solids. Use of activity has been found most helpful in many calculations. Studies on transpiration and sorption of water by arthropods are relatively few compared to such studies on other biological material. A useful resume of many such studies on cells along with a consideration of the physical chemistry of water transport phenomena has been written by DICK (1966). In this report much information on permeability of cells to water is presented in terms of a permeability coefficient. In arthropods, studies on the net movements of water between the organism and its environment have been made. An excellent summary of such work was published ten years ago (EDNEY, 1957). Two more recent reviews (BURSELL, 1964; BAPOPORT and TSCHAPEK, 1967) cover later reports and provide excellent bibliographies. In contrast to the many studies on net transpiration and net sorption, few papers reporting on the movement of water between air and arthropods using isotope labelled water have been found in a fairly comprehensive search of the
EXCHANGE
OFWATERBETWEBN
AMITEAND
SURROUNDING
AIR
1305
literature (MARCUZZI and SANTORO, 1959; GOVAERTS and LECLERCQ, 1946). By using heavy water as a tracer the earlier authors determined that the concentration of heavy water in the body water of a number of insects became the same TABLE ~-RELATIONSHIP OF a, AT 1 atm PRESSURE AND 25°C TO SOME UNITS FREQUENTLY USED TO MEASURE THE TENDENCY OF WATER TO MOVE FROM ONE COMPARTMENT TO ANOTHER
a,
r.h. %
1.000 100 0.999 99.9 0.990 99.0 98-O 0.980 0.970 97.0 0.960 96.0 0.950 95.0 0.900 90.0 0.800 80.0 70.0 O-700 0.600 60.0 0.500 50.0 0.400 40.0 30.0 0.300 0.200 20.0 0.100 10.0 0.000 0.0
Osmotic pressure (an-n) 0 1.34 13.60 27.37 41.25 55.25 69.48 142.70 302.20 480.30 696.85 938.75 1279.2 1630.65 2179.80 3020.10 cc
Osmotic pressure (osmols kg-l)
Vapour pressure (mm Hg)
Vapour pressure deficit (mm Hg)
23.756 23,732 23.518 23.281 23,043 22.806 22.568 21.380 19.005 16.629 14.254 11.878 9.502 7.127 4.751 2.376 0
0.024 0.136 0,475 0.713 0.950 1.188 1.376 4.751 7.127 9.502 11.876 14.254 16.629 19.005 21.380 23.756
0 0.0556 0.5607 l-1329 1.7168 2.3129 2.9216 6.1679 13.8814 23.7960 37.0070 55.5160 83.2658 129.5247 222.0425 499.5950 og
0
r.h. % Column 1: given; Column 2 : a, = ~ ; Column 3 : Modified from SHULL, 1939 ; 100
Column 4: a, =
55.5106 moles kg-r 55.5106 moles kg-‘+
osmols kg-r’
Column 5 : a, = P/P, ; Column 6 : a, =
P, - VPD p
0
.
as the concentration of heavy water in the air to which the insects were exposed. The time required for the concentrations to become equal was reported to vary between 5 and 13 days depending upon the kind and stage of the insect. For example, Tenebrio molitor adults reached equilibrium in 9 days, whereas larvae did so in 13 days. A reasonable interpretation that can explain these results is that the insects exchanged their water content with the water in the surrounding air in the time required for the concentration of heavy water in their water-pools to come into equilibrium with the concentration of heavy water in the atmosphere. The latter :authors report a half-life of exchange for larvae of T. molitor of 198+21 hr. Females of the spiny rat mite, Laelaps echidnina Berlese, have been used extensively in studies on net movements of water between the mite and the
1306
G.
W. WHARTON ANDT. L. DEVINE
surrounding air. It has been found that dehydrated individuals can restore their lost water by extracting water from unsaturated air if the r.h. is above 90 per cent (WHARTON and KANUNGO, 1962). The lowest r.h. at which organisms can maintain their water-balance, that is an equilibrium weight, has been designated as the critical equilibrium humidity (KN~~LLE and WHARTON, 1964). In the present paper the term ‘critical equilibrium activity’ will be used in place of the critical equilibrium humidity. Thus the reported critical equilibrium humidity for Laelaps echidnina of 90 per cent will, according to equation (2), become the critical equilibrium activity of O-90. The activity of the water in the haemolymph of Laelaps echidnina has not been precisely determined but it is known to be greater than a, = 0.99. This figure reported by WHARTON and KANUNGO (1962) is verified by the observation that hydrated mites submerged in solutions where activity was a, = 0.99 rapidly lost volume and became flattened. Since at room temperatures a solution whose aqueous activity is 0.99 has an osmotic pressure of 13.6 atm and one whose activity is 0.90 has an osmotic pressure of 142.7 atm (Table l), this mite is capable of extracting water from unsaturated air against an activity difference of 0.09 or an osmotic pressure difference of 129.1 atm. The rates of the net movement of water in and out of Laelaps echidnina under various conditions are known (WHARTON and KANUNGO, 1962). Dead mites exposed to a, = 0.80 lose water at about 1.2 pg/hr whereas live mites exposed to a, = 0.99 can gain water at the rate of 0.8 pglhr. Since mites weigh about 150 ,ug these rates of net water movement represent significant shifts in total water content. The amount of energy required for net uptake of water has not been determined but it is known that it represents only a small fraction of the metabolic activity of the mite (KANUNGO, 1965). In fact, daily dehydration followed by daily rehydration of this mite has been found to have little influence on its longevity under starving conditions (KN~~LLE, 1967a). In his general conclusions on transpiration of water from arthropods EDNEY (1957) states, ‘It is also possible to eliminate transpiration from the integument to a large extent by waterproofing with wax. Here again, insects and arachnomorphs are conspicuously successful’. In a study of the fine structure of the cuticle of Laelaps echidnina (WHARTON et al., 1968) all of the essential waterproofing layers were seen, and it can be concluded that this mite has a wellwaterproofed cuticle. In most terrestrial arthropods the site of maximum transpiration is the respiratory surface. Despite the excellent waterproofing of the external cuticle and the high transpiration rates from respiratory surfaces, it is important to emphasize that transpiration through a well-waterproofed cuticle is significant to the water-balance of arthropods. This conclusion is reinforced by the fact that gain of water by arthropods from unsaturated air takes place through the cuticle (LEES, 1946). Another consideration that reinforces the importance of a well-waterproofed cuticle as a pathway for the exchange of water is that the total or gross movement of water across the cuticle under most circumstances will be much greater than the net movement.
EXCHANGE OF WATER BETWEEN A MITE AND SURROUNDING AIR
1307
studies on the gross movement of water between female Laelaps air at a, = O-925 and T = 25°C will be reported. Finally the significance of the findings on the ability of the spiny rat mite to gain water from the air against an activity difference of 0.09 or an osmotic pressure difference of over 100 atm will be discussed. In this paper
echidnina and
MATERIALS
AND METHODS
Mites used in these studies were obtained from cultures at the Institute of Acarology of the Ohio Agricultural Research and Development Center. Mites were placed in an arena formed from an enamel tray bordered by hot nichrome wire that served as a barrier to the mites. From this arena female mites were aspirated into glass tubes. The tubes were closed at each end with nylon net so that free flow of air through the tubes was possible. Such tubes of mites were then placed in a dry atmosphere in desiccators containing dehydrated silica gel. After exposure to the low a, of the desiccators for 6 hr the tubes of mites were removed. A meal of rat blood was then offered to the mites (CROSS, 1954). Females that engorged on the blood were aspirated into clean tubes and held in moist air (a, > O-95) over moist activated charcoal and plaster of Paris (HUBER, 1958) for at least a week. This procedure provided standardized mites that had completed digestion of their previous meal (KANUNGO, 1964) and that produced neither faecal pellets nor eggs. By using only standardized mites in experiments, time since the last meal was uniform for each mite. An important variable that was not controlled by this procedure was the age of the mites (LEES, 1964). A plastic exposure chamber in which the air was in equilibrium with a saturated solution of KNO, and thus had an activity (a,) of 0.925 at 25°C (WINSTON :and BATES, 1960) was used in these experiments. Atmospheres containing tritiated water were obtained by adding tritiated water at about 2 PC of tritium/ml of saturated salt solution. The temperature of 25°C was maintained within 1°C by keeping the chamber in a BOD incubator. Standardized mites were exposed in glass tubes that were closed at either end by nylon bolting cloth. Studies on sorption of tritiated water involved placing mites in an atmosphere containing tritiated water vapour and determining the radioactivity of the mites at various intervals of time of exposure to the tritiated atmosphere. This technique was found to introduce a systematic error if a series of readings was made. The error resulted from the fact that removal of a sample from the exposure chamber introduced air from the room which then lowered the tritium concentration of the water vapour in the exposure chamber. This introduction of air from the room resulted in lowered values of tritium in the mites for a given period of time and thus lowered observed sorption. This error was avoided by using multiple exposures at different times instead of taking a series of samples at different intervals from the same exposure. Studies on transpiration of tritiated water were made in the same manner as those on sorption except that the standardized mites were exposed to an
1308
G. W. WHARTON AND T. L. DEVINE
atmosphere containing tritiated water vapour until they had come into virtual equilibrium with the vapour. They were then exposed to an atmosphere that Studies on transpiration of tritiated water contained no tritiated water vapour. were more convenient to do because they could be done without using atmospheres containing tritiated water vapour except for the single equilibration chamber in which large numbers of mites could be exposed at the same time. Since all exposures were made at a, = 0.925, an activity above the critical equilibrium activity of L. echidnina, no significant net movement of water occurred in mites All mites that died during an exposure or that maintained normal weights. weighed less than 100 pg were discarded. Under these conditions sorption and transpiration are equal and the measurement of one will provide the value for the other. Three data were obtained for each exposure interval: time of exposure in the test atmosphere, weight of each mite in pg and the radioactivity or tritium content Mites were weighed on a Cahn electrobalance (T*) of each mite in counts/min. to the nearest pg. The radioactivity of the mites was determined by placing a single intact weighed mite in 10 ml phosphor solution and determining the counts/min in a liquid scintillation counter. The phosphor solution consisted of 4 g of Packard BBOT scintillator dissolved in 1 1. of a 1 : 1 mixture of toluene The background count rate was subtracted from the observed -methanol. counts/mm to provide the counts/min attributable to the tritium from the mite. The mite had no significant effect on background or counting efficiency and virtually all of the tritium content of the mite was determined by this technique. Because the methods employed in this investigation resulted in the destruction of the specimen, it was impossible to obtain a series of observations on a single specimen. For this reason mean values are reported. One important value needed for an interpretation of the results is the mean water content of the mites. Another important consideration is the fate of tritiated water in the mites. If the tritiated water remains in the water-pool of the mite then the counts/min recorded for mites in equilibrium with a given concentration of tritiated water vapour should be proportional to the amount of water in the mite. If this be the case, then a plot of tritium content vs. weight should result in a straight line that crosses the weight axis at the mean dry weight. Fifty mites were dried over vacuum and P,O,. The mean dry weight for these mites was 37.54 pg, the heaviest individual weighed 43.7 pg and the lightest 27.8 pg. Fifteen mites, after they had come into equilibrium, were weighed and their radioactivity determined. The values for each mite were plotted on linear co-ordinate paper ruled off in counts/min along the abscissa and pg on the ordinate. A straight line originating at 37.54 pg (the previously determined mean dry weight) and terminating at the highest countsjmin recorded runs through the array of data points representing mites, (Fig. 1). It is on the basis of the agreement between weight and tritium content illustrated in Fig. 1 that the hypothesis, stating that all of the observed tritium remains in the waterpool of the mite, is accepted. Furthermore, when the hypothesis that all of the
EXCHANGE OF WATER BETWEEN A MITE AND SURROUNDING AIR
observed tritmm remains in the water-pool is accepted, as an independent determination of dry weight and obtained by (dehydration over vacuum and PsO,.
1309
Fig. 1 can be considered then confirms the value
160 140 g
120
i? .; 100 E c .-
80
ii r"
60 40 20
200
400
600
800
1000
1200
1400
CPM FIG.
1.
The relationship between weight (pg) and radioactivity (counts/min) of mites in equilibrium with water vapour containing tritiated water
The analysis of the data is based on a theoretical model derived from CRANK I or organism initially in equilibrium with a given a, will gain or (1956). A so I’d lose weight when placed in an atmosphere at a different a,. The relations for evaporation or condensation by a small surface-limited solid which had been in equilibrium with one vapour pressure and then plunged into another at some constant vapour pressure reduce to first order kinetics: m = m,exp(-K,t)
or
lnGs=
-K,t,
where m is the mass yet to be transferred and m, is the total mass that evaporated Ior sorbed. The rate riz of net mass change is the derivative above equation with respect to time (t): riz = -k,m,exp(-k,t)
= -k,m.
will be of the
(4)
G. W. WHARTON AND T. L. DEVINE
1310
This net mass change rate must equal the difference and the rate of transpiration (rizr):
in the rate of sorption
riz = r&-r&. Dividing
equation
(5) by the water
(rizs)
(5)
mass gives the rate constants:
k, = k,-k, where k, = 2,
k, = 2,
and
k, = 2.
(6)
An organism containing an amount of tritium (T*) when placed in a constant untritiated vapour will, in an increment of time (dt), lose an increment of tritium (dT*) equal to the increment of water transpired (-~$dt) multiplied by the specific radioactivity (S) where S = T*lm, and using the relation for k, above: dT*=-Sh,dt=-;ria,dt=-T*k,dt.
With
an initial
tritium
content
(7)
(T*) this integrates
T* = T*,exp(-k,t)
or
ln$
to :
0
= -k,t.
(8)
Thus the rate constant for tritium loss is identical to that for the transpiration of water and is independent of sorption. If k, is determined from mass data with equation (4) and k, is determined from tritium content data with equation (8) then k, is determined by equation (6). When mass is constant k, = 0 and k, = k,. Sorption of untritiated water vapour by an initially tritiated organism results in a change of the specific activity of the body fluids which is determined by and substituting relations from differentiating (S) with respect to time equations (6) and (7): 1 dT” =-_-----_~ m dt With
the initial
specific
S
-T*
T*ldm m m dt
radioactivity
s, = exp(-kst)
T* -,(k,-k,)
m
(So) this integrates or
lnS,
S
= -Sk,.
(9)
to:
= -k,t.
Thus the rate constant for the decrease of specific radioactivity is identical rate constant for sorption of water and is independent of transpiration.
to the
EXCHANGE
OF WATER
BETWEEN
A MITE
AND
SURROUNDING
An initially untritiated organism gains both tritium radioactivity when plunged into a vapour of tritiated water. to the above., results in the equations:
T* = l-exp(-&t) KC
-
or
In
(
1-g
m)
1311
AIR
content and A derivation,
specific similar
(11)
=-krt,
-k,t, where the radioactivity respectively.
and specific
radioactivity
(12)
at equilibrium
are T, and S,
RESULTS
Water was found to move in and out of the mites during exposures at an easily measurable rate as was indicated by changes with time (Table 2). TABLE
~---N[EAN
WEIGHTS, EXPOSED
TRITIUM
CONTENTS,
TO EXPERIMENTAL
AND
TRITIUM
ATMOSPHERES
CONCENTRATIONS
FOR VARIOUS
experimental in T* and S
FOR
MITES
TIMES
Mean S Time (hr)
Mean weight (ELg)
Mean T* (counts/min)
(counts/min per Fg)
+1s Equilibrium -18.5 -27 -44
144f9 153k5 149k6 149*7 139kl3
552f43 1303 f 57 559 + 63 488 f 90 23lk 102
5.2 11.3 5.4 4.4 2.3
No. of mites 23 11 15 15 6
Positive and equilibrium values represent periods of exposure to a tritiated atmosphere; negative values represent time in an untritiated atmosphere since attaining equilibrium in a tritiated atmcsphere.
In order to calculate the amount of water transpired and sorbed from the data obtained it is necessary to make three assumptions. That the tritium observed re-mains in the water-pool of the mite is an assumption the validity of which has Eleen demonstrated (Fig. 1). The second essential assumption is that the observe’d movement of the tritium is indicative of the movement of water. This assumption is justified in that tritium is introduced into the system only in the form of water, and is recovered from the system only in the aqueous component. The third assumption that no significant net movement of water occurred during the course of the experiments is justified in that the experiments were all run at an a, above the critical equilibrium level and that the mean weights for the various times are not significantly different (Table 2).
G. W. WHARTONAND T. L. DEVINE
1312
In making calculations of gross transpiration and sorption of water between the mites and the air, only the mean values have been used. It is recognized that values for mites will vary from individual to individual and from time to time in the case of the same mite. An estimate of the amount of variation to be expected is reflected in the 5 per cent confidence intervals given for the mean weights and mean tritium contents (Table 2). As can be predicted from equations (8) and (10) the relationship between time and relative specific radioactivity is that of a first-order kinetic equation. When time (t) is plotted on a linear scale on the abscissa and S/S,, or 1 -(S/S,) 1.0
.9 .8 .7 .6 .5 a cl??
.4
6
12
18 Time
24
30 in
36
42
48
j,
SO
Hours
FIG. 2. The relationship between time of exposure and relative specific radioactivity of mites under equilibrium conditions.
is plotted on a logarithmic ordinate a straight line of slope -k can be seen to describe the relationship (Fig. 2). Using the data in Table 2 and equations (8) and (11) it is possible to calculate k for each exposure period. The results of
EXCHANGE
these calculations
OF WATER
BETWEEN
A MITE
AND
SURROUNDING
AIR
1313
are : k = 0.0342 hr
for
+ 18 hr,
K = 0.0397 ~ hr
for
- 18.5 hr,
k: = 0.0385 hr
for
-27
k: = 0.0366 hr
for
- 44 hr.
hr,
The mean value for K taking into account the number of observations represented by the means for each exposure period is 0_0373/hr. Another way of expressing k = 0*0373/hr is to say that transpiration and sorption each proceeds at a rate of 3.73 per cent of the total water mass/hr. The half.-life of a first order kinetic reaction can be determined by taking advantage of the fact that the time taken to complete a definite fraction of the process is independent of the initial amount of the material under study. Thus when one half of the process is completed, that is when T*/T*,, is equal to 0.5, substitution of this value in equation (8) gives: ln0.5
= -k,t,
or
tg =p
ln2 T
where tt is equal to the time required for T*/T, to become equal to 0.5 or another way of expressing it is that tt represents the half-life of the process. Inserting the mean value of k in equation (8a) the mean half-life is calculated to be 18.6 hr (Fig. 2). The rate constant KS = 0*0342/hr calculated on the basis of 18 hr of sorption and the rate constant k, = 0*0397/hr calculated on the basis of 18.5 hr of’ transpiration are well within the limits of the biological diversity and experimental error of these observations and are in good agreement with the assumption that no significant net movement of water occurred during the experiments. It has been determined that the rate constants k, and k, determined for the movement of tritium are equal to the rate constants for the fraction of the water labelled with1 tritium and thus for all of the water. In the case of tritiated mites exposed to an untritiated atmosphere containing a large tritium sink, loss in tritium content is associated solely with transpiration (equation (8)). On the other hand changes in the concentration of tritium are brought about solely by the sorption of water (equation (10)). Th e validity of the mathematical argument can be appreciated intuitively if one considers a separator-y funnel containing a dye dissolved in some solvent. The total amount of the dye in the funnel can be changed only by opening the stopcock and removing some of the solution. This action will have no effect on the concentration of the dye or the intensity of the
1314
G. W. WHARTONAND T. L. DEVINE
colour of the solution. Removal of material is analogous to transpiration and its only effect is the reduction in the quantity of the diluent, dye in the case of this analogy or tritium in the case of the mite. If solvent is added to the funnel, the concentration of the dye or the intensity of colour of the solution will be changed. Addition of solvent is comparable to sorption. Thus in the experiment reported here, measure of the change of concentration of tritium is not a measure of the movement of tritiated water but is in fact a measure of the movement of water itself. Since K gives the per cent of the total reaction taking place in unit time, the amount of water transpired in unit time is equal to k, times the amount present. In the case of a spiny rat mite containing 100 pg of water under equilibrium conditions this means that transpiration amounts to 3.73 pg/hr and the same amount of water will be sorbed when equilibrium weight is maintained. Thus the total movement of water amounts to 7.46 pg/hr under equilibrium conditions. When compared to rates of 1.2 and 0.8 pg of water/hr reported as net movement (WHARTON and KANUNGO, 1962) it can be seen that concepts concerning the permeability of the arthropod cuticle based on studies of the net movement of water are likely to give an exaggerated impression of its impermeability. DISCUSSION
Exchange rates Although a half-life of 18.6 hr for the exchange of water between an organism and the water vapour in the surrounding air is significant and will appear to many entomologists as surprisingly short, it is actually quite slow when compared to exchange of water through cell membranes. DICK (1966) reviews values of permeability of various animal cells to water. He uses as a measure a permeability coefficient (k*) whose formula in tracer studies is: k*
=
IlnCo-Ce At
C-Ce’
(13)
where V is volume; A, area; t, time; Co, initial concentration of the tracer in the cell; Ce, concentration of the tracer in the environment; and C, concentration of the tracer in the cell at time t. Values of 12” reported for various free living cells range from 400 psec-1 for canine erythrocytes to 0.37 psec-l for certain protozoa. While some of the difference is probably caused by differences in permeability of the cell membranes, a more significant factor is the surface to volume ratio, k* being roughly proportional to this ratio. The surface to volume ratio reported for the canine erythrocytes is 1.82 p-l and that for the protozoan O-017 p--l. In the case of the smaller k* values, it is probable that diffusion within the cell is so slow that concentration differences across the cell membrane remain slight, thus slowing the net movement across the membrane. Substituting the mean values for S at tt obtained for the spiny rat mite in equation (13) and assuming a volume of l-2757 x 108 p3 and an area of
EXCHANGE
OF WATER
BETWEEN
A MITE
AND
SURROUNDING
AIR
1315
9.25 x lo5 pz its k* value is 0.0014 psec-I. Since this value is so much smaller than any of the values reported for cells, even those with larger volumes than that of the mite, it is obvious that the internal mixing of the water is rapid enough to maintain internal concentrations at a uniform level throughout the system. Under these conditions the low K* value must be interpreted as revealing a highly imperlmeable barrier to the passage of water. Such an interpretation is consistent with the generally held view that the arthropod cuticle is well waterproofed. In studies of cells reported by DICK (1966), it is pointed out that k* values obtained from tracer studies, and those from studies on net movement of water, are not the same for the same cell system. In general, k* values calculated on the basis of the net movement of water were found to be about twice as great as those determined from tracer techniques. It should be pointed out that in the calculation of k* only sorption is taken into account by Dick’s formula, and, since tracer studies are usually made under conditions where no net movement occurs, sorption and transpiration must be equal and the movement of water across the membrane will be twice that obtained by his method, The assumption that rate constants for exchange of HTO, which were determined, are approximateky equal to rate constants for exchange of Hz0 can be challenged on the basis of the difference of the two molecules in weight and configuration. Since in these studies actual water movement as well as tracer movement have been determined and found to correspond, the challenge that artefacts produced by isotopic eRects may contaminate the results is groundless. Site of exchange Three main avenues of water uptake are available to most arthropods. These are the general body surface, the respiratory surface, and the digestive tract. In the experiments reported here the digestive tract as a portal of entry for liquid water can be ruled out because no liquid water was available to the mites. It is possible that water from the air could be obtained via the digestive tract. If the mites were to secrete a saliva whose a, was 0.90 or below, a net gain in water from air wh’ose a, was greater than 0.90 would result and ingestion of this vapour-enriclhed saliva could account for water uptake. On the other hand, failure to have observed such salivary secretions on countless microscopic examinations suggests that this is not the mechanism involved. It is also probable that the portal of entry is not the respiratory system. Two considerations are important here. The first is the fact that excessive water loss even at a,‘s well above the crucial equilibrium level has been observed whenever the respiratory rate of the spiny rat mite is elevated much above its basic level (KANUNGO, 1965). The second observation is that acari with no special respiratory system such as cheese mites and ixodid larvae have the capacity to extract water from unThere remains as the most probable saturated air (KN~~LLE, 1965, 1966). exchange surface the general body cuticle or some portion of it. All of the studies on the phenomenon of water uptake from unsaturated air by arthropods support
1316
G. W. WHARTONAND
the view that the cuticle or some portion water from unsaturated air.
T. L. DEVINE
of it is responsible
for the uptake
of
Gain of water from unsaturated air The most recent comments concerning the mechanism by which water is gained by arthropods from unsaturated air are those of KN~~LLE (196713). His brief discussion points out the activity of water molecules at the absorbing site must be lower than the a, of the air from which water is extracted and that an active mechanism or pump is required to move the water from the site of sorption to the haemolymph. KN~~LLE (1967a) re f rains from speculating on the mechanics of the pump but does refer to papers that do (BEAMENT, 1964; LOCKE, 1964). Recognizing that it is the difference between the a, of the site of sorption and the a, of the surrounding air that is responsible for net gain or loss of water it follows that at equilibrium where no net movement of water occurs k, = ks. Now k, is independent of k, and is proportional to the activity of the water at the surface from which transpiration takes place such that: k,,
=
zr a,
for transpiration where k, equals the rate constant rate constant for transpiration at a particular a,. for ks, that is: k,
=
2.
(14) at azr = 1 and k, equals the The same relationship holds
(15)
”
It follows from equations (14) and (15) that at equilibrium the effective a, of the exchange surface between the organism and the air must equal a,. If the a, at the exchange surface and the a, of the haemolymph are the same no active transport or pump is required for the maintenance of equilibrium. If, on the other hand, the a, of the haemolymph is O-99 or greater, as is usual, then a pump is required to maintain lower a, at the exchange surface of those arthropods that can extract water from air whose a” is below 0.99. Whether or not water or some carrier molecule is involved in the molecular mechanisms of the pump measurable quantities of water are moved. This flow of water should have a rate constant (kp) such that sufficient water can be removed from the exchange surface to be able to maintain an a, at the surface as low as the a, at the critical equilibrium activity. From equation (14) it is possible to calculate k, for any a,. When no pump is in operation, k, will be equal to k, x a, (haemolymph). When the pump is operating at the critical equilibrium level, k, will be equal to k,, x a, (critical equilibrium activity). It follows therefore that the rate constant for the pump (kp) for net movement of water when operating at the critical equilibrium level must be the difference between k, (haemolymph) and k, (critical equilibrium
EXCHANGEOF WATERBETWEENA MITE ANDSURROUNDING AIR
1317
activity). Using the values of k, determined in these studies for the spiny rat mite k, becomes 0*0373/0*925 or 0.0403, and the rate constant for the pump (kp) is found to be 0*0036/hr or about one-tenth the value of k,. The amount of water that must be pumped for a mite containing 100 pg of water is 0.36 pg/hr. This is of course the net movement. Knowledge of the net movement of water required of the pump opens the way to the development of more realistic models of possible mechanisms of pump action. For example, if the pump cycle is 1 set in duration, the amount of water involved/cycle need be only 0.0001 ,ug or 1 ppm of the total water content of the mite.
REFERENCES BEAMENTJ. W. L. (1964) The active transport and passive movement of water in insects. Adv. Insect Physiol. 2, 67-129. BURSELL E. (1964) Environmental aspects: Humidity. In The Physiology of Insectu (Ed. by ROCKSTEIN, M.), 1, 323-361. Academic Press, New York. CRANKJ. (1956) The Mathematics of Diffusion. Oxford University Press, London. CROSS H. F. (1954) Feeding tests with blood sucking mites on heparinized blood. J. econ. Ent. 47, 1155. DICK D. A. T. (1966) Cell Wuter. Butterworths, Washington. EDNEY E. B. (7.957) The Wuter Relations of Terrestrial Arthropods. Cambridge University Press, London. EDNEY E. B. (1966) Absorption of water vapour from unsaturated air by Areniwaga sp. (Polyphagidae, Dictyoptera). Comp. Biochem. Physiol. 19, 387-408. GOVAERTSJ. and LECLERCQ J. (1946) Water exchange between insects and air moisture. Nature, Lond. 157, 483. HUBER I. (1958) Color as an index to the relative humidity of plaster of Paris culture jars. Proc. ent. 6’0~. Wash. 60, 289-291. I(ANUNG0 K. (1964) Disappearance of blood from the gut of engorged Echinolaelaps echidninus (Acarina: Laelaptidae). Ann. ent. Sot. Am. 57,427-428. KANUNCOK. (1965) Oxygen uptake in relation to water balance of a mite (EchinoZueZups e&id&us) in unsaturated air. J. Insect Physiol. 11, 557-568. KN~~LLE W. (1965) Die Sorption und Transpiration des Wasserdampfes bei der Mehlmilbe (Acarus sire L.). 2. vergl. Physiol. 49, 586604. KN~~LLE W. (1966) Equilibrium humidities and survival of some tick larvae. J. med. Ent. 2, 335-33s. KN~ILLE W. (1967a) The significance of fluctuating humidities and frequency of blood meals on the survival of the spiny rat mite, Echinolaelaps echidninus (Berlese). J. med. Ent. 4, 322-325. KN~LLE W. (1!)67b) Physiological properties and biological implications of the water vapour sorption mechanism in’larvae of the oriental rat flea, XenopsyZZu cheopis (Roths.). r. Insect Physiol. 13, 333-357. KN~LLE W. and WHARTONG. W. (1964) Equilibrium humidities in arthropods and their ecological significance. Acarologia 6, 299-306. LEESA. D. (1946) The water balance in Ixodes ricks L. and certain other species of ticks. Parasitology 37, I-20. LEES A. D. (1964) The effect of ageing and locomotor activity on the water transport mechanism of ticks. AcaroZogia 6, 315-323. LOCKE M. (1964) The structure and formation of the integument in insects. In The PhysioZogy of Insecta (Ed. by ROCKSTEINM.), 3, 379470. Academic Press, New York. 84
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G. W. WHARTONANDT. L. DEVINE
MARCUZZIG. and SANTOROV. (1959) Indagini sul ricambio idrico de1 Tenebrio molitor mediante acqua tritiata. Ric. sci. 29, 2576-2581. RAPOPORTE. H. and TSCHAPEKM. (1967) Soil water and soil fauna. Rev. Ecol. Biol. Sol. 4, l-58. SHULL C. A. (1939) Atmospheric humidity and temperature in relation to the water system of plants and soils. Plant Physiol. 14, 401-422. WHARTONG. W. and KANUNGOK. (1962) Some effects of temperature and relative humidity on water-balance in females of the spiny rat mite, Echinolaelaps echidninus (Acarina: Laelaptidae). Ann. ent. Sot. Am. 55, 483-492. WHARTONG. W., PARRISHW., and JOHNSTOND. E .(1968) Observations on the fine structure of the cuticle of the spiny rat mite, Laelaps echidnina (Atari-Mesostigmata). Acarologia. In press. WINSTONP. W. and BATESD. H. (1960) Saturated solutions for the control of humidity in biological research. Ecology 41, 232-237.