regulation of transpiratio in n th clovee r...

jf. Exp. Biol. (1965), 43, 257-269 2 5 7With 4 text-figures

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REGULATION OF TRANSPIRATION IN THE CLOVER MITEBRYOBIA PRAETIOSA KOCH (ACARINA: TETRANYCHIDAE)

BY PAUL W. WINSTON AND V. EUGENE NELSON*

University of Colorado, U.S.A.

(Received 22 February 1965)

INTRODUCTION

The rigorous limitation of evaporative water loss is essential for the survival ofsmall arthropods normally exposed to low humidities. Such animals, particularly theAcarina, contain limited amounts of water that can be lost and a relatively largesurface area from which to lose it. Evaporation is limited primarily by physicalbarriers in the exoskeleton, the chitin-protein complex of the endo- and exocuticles(Beament, 1961) and a thin surface layer of oriented lipid molecules (Wigglesworth,1945; Beament, 1945). The intact lipid layer provides a high degree of waterproofingbut is characterized by loss of effectiveness above critical temperatures or upontreatment with solvents (Beament, 1959).

In addition, active mechanisms in the respiratory system and within the livingportions of the cuticle may further reduce and control water loss (Edney, 1957). Ofthese, control of spiracular opening has been shown by many workers to be highlyeffective for water conservation in insects. Mellanby (1935) greatly increased waterloss in Tenebrio by preventing spiracular closure with excess CO2, while it has recentlybeen shown that the degree of opening of these organs is influenced directly byhumidity in tsetse flies (Bursell, 1957) and by water balance in dragon flies (Miller,1964). Browning (1954) and McEnroe (1961) showed the importance of the mechanismto ixodid ticks and to the spider mite Tetranychus telarius.

Another active principle, a restrictive mechanism in the general body cuticle, seemsto be of secondary significance in most insects (Wigglesworth, 1945), but it was shownin ticks (Lees, 1946; Browning, 1954) to be equally as important as respiratory control.It has been called 'active retention' by Edney (1957) and others; and Lees' (1947)hypothesis that the epidermal cells are able to secrete water inward seems to offer thebest explanation, although it has not yet been experimentally confirmed. As the resultof this proposal, Edney (1957) and Beament (1961) suggested that the mechanismmight be one which would result in the uptake of atmospheric water at high humidities.It would remove the need for a different mechanism to explain this puzzling phe-nomenon which is known to occur in a limited number of arthropods, e.g. T. molitor(Buxton, 1930) and many ticks (Lees, 1946).

These active mechanisms in the cuticle and in the respiratory system may alsocontribute to at least one of the two forms of regulation that are apparent in the waterbalance of some hardy insects and arachnids. Certain insects are able to regulatetheir percentage of body water, maintaining it at essentially constant levels both with

• Present address: Department of Entomology, University of Kansas, Lawerance, Kansas, U.S.A.17 Exp. Biol. 43, a

258 PAUL W. WINSTON AND V. EUGENE NELSON

time and over a wide range of humidities, e.g. T. molitor (Buxton, 1930). In additiona very few animals, e.g. nymphs of the grasshopper Chortaphaga viridifasciata(Ludwig, 1937) and a locust Oedipha coeruliens (Jakovlev & Kriiger, 1953), have beenshown to regulate their water loss, keeping it at nearly constant rates over a substantialportion of the humidity range. The only specific studies on the mechanisms by whichthese regulatory functions are accomplished have been on the first type in which it hasbeen shown, especially in Tribolium confusum and Dermestes vulpinus (Fraenkel &Blewett, 1944), that metabolism, and thereby the production of metabolic water, isadjusted to differences in humidity. More food is consumed in dry air than in moistand the extra water produced is enough to maintain the water level in the body. Eachof these controls is such as to keep an internal variable at an essentially constant levelin the face of changing external conditions, thus conforming to the usage of the term'regulation' by Prosser & Brown (1961).

The clover mite Bryobia praetiosa is active on warm walls and tree trunks from lateSeptember to early June in Colorado, and the ability of these very small animals(< 1 mm.) to thrive at the low humidities commonly measured on such exposedsurfaces prompted studies on their humidity tolerances and behaviour (Winston,1963 a). These have been followed in the present work by the measurement of waterloss in both living and dead mites over a wide range of humidities. Because anactive mechanism was indicated by the earlier studies, it could be expected that deadmites would show a higher rate of loss than living ones throughout the humidity range,Furthermore, it seemed likely that a comparison of weight changes in living mites atvarious humidities would reveal the existence of mechanisms for the control andrestriction of water loss and for the uptake of atmospheric water in different portionsof the humidity range. Since preliminary studies on water loss in living mites(Winston, 1964) indicated the ability to regulate over a substantial part of the range,the nature and location of the underlying mechanisms was made the primary objectiveof this investigation.

MATERIALS AND METHODS

The mite used in this study is the Bryobia praetiosa Koch of Anderson & Morgan(1958), and a brief description of its life-cycle in Colorado appears in Winston (19636).For most of the winter and spring, a plentiful supply of these parthenogenetic animalsfrom a natural population was maintained on bean plants growing on flats in thewindows of the Laboratory. A 'conditioning' or pre-experimental treatment wasadopted to assure a more uniform experimental animal than could be obtained directlyfrom a wild population. Adult mites were taken from the leaves rather than from thewindows, ensuring that the majority had fed for some time. They were then heldwithout food or water for 48 hr. at 29% R.H. and 25° C. This allowed ample time fordefecation of the copious, watery faeces and for most of the egg-laying. No faeceswere found after tests that had been preceded by this conditioning and there wereusually fewer than two eggs per mite.

Groups of sixteen mites were lightly anaesthetized, weighed as a group, and placedin screen-topped vials for exposure to air of a specified humidity. This treatment wascarried out in a water-bath at 25 ±0-5° C. in small chambers in which the humiditywas controlled by saturated salts as described by Winston & Bates (i960). The mites

Regulation of transpiration in clover mite 259

were exposed to humidities of o, 12, 29, 43, 52, 62, 76, 85, and 93%; none higher wasused because the mites easily drown in condensed droplets and because their activityis greatly reduced at very high humidities. Because all the work was done at onetemperature, relative humidity (R.H.) and saturation deficit are equivalent, and theformer term will be used throughout this paper. After 24 hr., later 18, the mites wereagain anaesthetized, weighed, dried for 24 hr. at 900 C , and weighed again. Thesetreatment periods were long enough to result in measurable weight-changes at highhumidities, but short enough to reduce mortality to a minimum at the low. Nodifferences in the calculated rates of loss were noted between the two testing periods.

The water and dry-matter content of both feeding and conditioned mites weremeasured at intervals to check for any seasonal effect on these variables and none wasfound. Feeding animals were removed from the plants, anaesthetized, weighed anddried to constant weight. The conditioned animals were treated similarly except for the48 hr. starvation period at 29% R.H. and 250 C. prior to weighing.

A correction factor of 1-5 /jg. each was obtained for the few eggs laid during thetests by calculating the volume of an egg from the diameter and assuming the densityto be about 1-02. Measurements of many eggs with an ocular micrometer showedthem to be of nearly uniform size, so a reasonably accurate correction could be madefor the loss of weight by counting the number of eggs laid. For the dry weights acorrection of 0-4 fig. per egg was used. These factors were multiplied by the numberof eggs laid per mite in each group and the result added to the average weight. Anywater loss resulting from the process of oviposition was so slight as to be undetectablewith the methods employed.

Ether was used instead of other standard anaesthetics throughout these experimentsbecause its ill-effects were much less than any others tested. The possible effect of thislipid solvent on the water-proofing layer was of some concern to us, but tests indicatedthat stronger doses of the anaesthetic than that used in practice did not increasemortality even in dry air.

Water loss in dead mites was measured after they had been killed by exposure for2 hr. to hydrogen cyanide fumes in a water-saturated atmosphere. The greatly increasedrate of loss at death made it necessary to expose these animals for only 4 hr., to preventlow water content and decay from influencing the results.

For measurement of the effects of high concentrations of CO2 on weight loss themites were exposed to atmospheres containing 10% of the gas at various humidities.An approximation to the lowest concentration which would produce a maximum effectwas obtained by determining the water loss in 5, 10, and 15% mixtures of the gas inair at one humidity. The two higher concentrations produced essentially the same losswhile at 5% it was significantly lower; the 10% mixture was therefore used throughoutthe tests. No anaesthetic effect was observed at these concentrations. Humidity inthis series of experiments was controlled by appropriate concentrations of sulphuricacid (Solomon, 1951) because, unlike many of the saturated salts, these solutionsabsorb very little CO2.

A Cahn Electrobalance, model Mio, was modified to obtain a sensitivity of lessthan 1 fig. with a range of 1 mg. Changes in weight of individual mites were quitesmall, and to obtain greater accuracy we weighed them in groups of sixteen andcalculated the average loss per mite. Standard weights approximating the weight of

17-2


a group of mites were weighed frequently to check for drift in the balance circuit.Single adult mites averaged 36±n/<g. in weight; and, to counteract the widevariations found, groups were made up of animals as close to the same size as possible.

Standard deviations and standard errors of the means of all values were calculatedby the methods of Arkin & Colton (1955) and the significance of the difference betweenmeans of adjacent values was tested by the use of the t-test of Fisher (1950).

In these experiments weight loss was determined on 560 groups of 16 mites, about9000 individuals. Each group was weighed at least twice, and most three times, toobtain an initial weight, a final weight, and a final dry weight.

RESULTS

Water content

The water content of the mites was calculated as a percentage of the final totalweight and plotted against R.H. in Fig. 1. The freshly fed animals have a much higherwater content than that of conditioned ones, a result to be expected from the largeamount of water in the plant cells on which they feed. Much of this water is lostrapidly soon after feeding, but the remaining water is only slightly reduced during

78 r

74 Feeding mites

20 40 60Relative humidity

80 100

Fig. 1. The water content of untreated mites and of mites treated with 10% CO^ expressedas a percentage of the final weight, was plotted against the various humidities to which theywere exposed. As freshly fed mites were not exposed to any specific humidities their averagewater content is indicated by the dotted lino. Vertical lines represent the standard errors of themeans.


the subsequent treatment periods of 18 or 24 hr., indicating a form of regulation inrelation to time. It can also be seen that there is very little effect of R.H. on the watercontent of control mites throughout the humidity range. They are thus able to regulatethe percentage of body water despite different rates of water loss over a wide range ofhumidities.

The water content of CO2-treated mites falls below that of the controls (Fig. 1) andis essentially proportional to the R.H. AS the dry weights are the same in both groups,the reduction in water is probably due to loss through the opened spiracles, whichwould indicate that the mechanism for regulation is based in the control of thesestructures. In addition, the differences in rate of loss of solids in control mites wereso slight (Fig. 4) that it does not appear possible that maintenance of a constant watercontent could be the result of adjustments in metabolic rate in response to water loss.This problem is really tangential to the present one of regulation of water loss, however,and further study must be deferred for the present.

Water loss

Though the object of this study was to determine water loss in groups of mites, itwas obviously possible to obtain only the loss in total weight. The final weight waseasily resolved into solids and water by obtaining the dry weight and then subtractingthis value from the total to get the water. For the initial weight, however, these hadto be calculated by using the percentage of water and of solids in the conditionedmites. Water and dry-weight losses were then obtained by subtracting the final fromthe initial values. The results of these calculations are plotted in Fig, 4 and show thatwater loss follows total weight loss quite faithfully, and that the latter is not affectedby the slight changes in dry weight. We will, therefore, use both terms interchangeablyin the remainder of this paper even though the values presented will actually be oftotal weight loss. These animals vary so much in size that only relativecomparisons arepossible, and because of this we have expressed weight changes as percentages of theinitial total weights, in percentage per hour per mite.

There are four avenues for the escape of water in Bryobia: urine and faeces,oviposition, cuticular transpiration, and spiracular loss. In our work the 48 hr.conditioning period eliminated urine, faeces, and most of oviposition as factors; andonly water loss through the cuticle and through the spiracles remained to account forthat which was measured.

The nature of the barriers to transpiration from these two areas was studied bythe measurement of water loss in living mites, in those killed by HCN gas, and inothers killed in chloroform vapour (Fig. 2). As the lowest rates of loss were fromliving mites, rates above these can be considered due to the removal or disruption ofone or more of these barriers. By far the highest rates of loss were from the chloroform-treated animals, demonstrating changes in the lipid waterproofing layer. Gibbs &Morrison (1959) recently showed the same kind of layer for another tetranychid, thespider mite Tetranyckus telarius. In both this species and Bryobia the cuticle under-lying the wax layer is so thin that it is difficult to see how it could be much of abarrier, but Beament (1961) has shown that, though very permeable, such a layerwould reduce evaporation considerably below that from a free-water surface. It canbe seen that the passive barriers provide the major reduction of evaporation, but the


substantially higher rates of loss in dead mites over those in living ones suggest thepresence of an active mechanism which further restricts water loss.

The respiratory system is considered to be a primary site of evaporative water lossin many terrestrial arthropods (Wigglesworth, 1953), and the relatively simple tech-nique needed to demonstrate this offered an obvious starting-point for the problem

70 r

Dead mites-chloroform treated

10 -

20 •40 60 80 100

Relative humidity

Fig. 2. The average loss in weight in percentage per hour per mite was measured at varioushumidities for mites killed in chloroform vapour and in cyanide fumes (lower line). Verticallines represent the standard error of the means.

in Bryobia. Since CO2 at concentrations of 5-10% is known to maintain the spiraclesin an open position in most arthropods (Wigglesworth, 1953) including ticks (Browning,1954), the rates of loss of CO8-treated and untreated mites were compared (Fig, 3).Tests at five humidities indicated that a CO2-sensitive mechanism, presumably controlof the respiratory openings, would account for about half the difference in the rate ofwater loss between live, untreated animals and dead ones; thus about half the loss isconsidered to be through the spiracles and half through the cuticle. Lees (1946) andBrowning (1954) found a similar relationship in ticks.

There is no doubt of the existence of organs acting as spiracles in clover mites andmany other Acarina, but little is known of their behaviour and controls (Winston,


1964). Bryobia is usually very active in dry air and one would expect this to result inhigh rates of water loss through necessarily opened spiracles. There is higher loss at0% R.H. than in more humid air, but it is so small that these animals can survive forrelatively long periods under dry conditions. It must be that they, like the tsetse fly

24-r

20 40 60

Relative humidity

100

Fig. 3. The average loss in weight in percentage per hour per mite was measured at varioushumidities for those killed in cyanide fumes, for living mites treated with 10% CO,, and foruntreated animals. Vertical lines represent the standard errors of the means.

{Bursell, 1957), can obtain enough oxygen for activity through nearly closed spiraclesand thus make gas exchange subordinate to control of water loss in these organs. Ithas been reported that the respiratory organs of spider mites are exposed to the air toa greater or lesser extent in response to changing levels of activity (Blauvelt, 1945),humidity, and water content (McEnroe, 1961). Observations in this laboratory havefailed, however, to show any visible positional changes of these organs in Bryobia thatwould indicate a control function. They have been seen to be exposed only when themites were actively feeding.

Since the differences between living and killed mites cannot be explained whollyon the basis of a CO2-sensitive respiratory mechanism an additional one is indicated.


This is most likely some form of active retention of water by the general body cuticle,though Beament's (1961) suggestion of a rapid change in the cuticle at death stillremains a remote possibility.

The better to show important deflexions in the curves for water loss, the rates forliving mites are given on a larger scale in Fig. 4. The general tendency of the curve,

Total weight loss

\

20 40 60 80 100

Relative humidity

Fig. 4. The average loss in weight in percentage per hour per mite was measured at varioushumidities for untreated (control) mites (solid line). Calculated values for water loss and forloss of solids are shown by the dotted lines. Vertical lines represent the standard errors of themeans.

as shown by the broken line between 0% and 85% R.H., indicates an overall inverserelationship between water loss and R.H. The deviation from this straight line between53% and 85%, however, indicates that the mites are able to modify the relationshipin this part of the range. Between these humidities, loss is maintained at a nearlyconstant rate, showing that the mites can regulate water loss in the face of considerabledifferences in the evaporating power of the air.

Despite the slight influence of humidity in the zone of regulation, the differencesbetween adjacent means were found to be not significant to the 0-05 level, though thatbetween the two extremes was close to it. At humidities above and below this region


the differences between adjacent means were found to be highly significant, at least tothe 001 level. It is to be noted that cyanide-treated mites do not exhibit this plateau,and so it can be assumed to be the result of an active process. The sharp breaks ateither end of this zone of regulation are indications that some distinct changes in theactive process take place between 85 and 93% and between 43 and 53% R.H.

In contrast, the greater slope of the line between 43 and 0% shows that loss dependsmore on the humidity, but this slope is still much less than was found in dead mites.Thus the sudden increase in rate of loss from 53 to 43% is not to a condition which iscompletely dependent on the evaporating power of the air. Instead, there is evidenceof an active mechanism functioning at low humidities that restricts, but does notregulate, water loss.

The apparently insignificant effect of R.H. on dry-weight losses (Fig. 4) does notsupport the hypothesis that energy is expended for the retention of water, as anychange in respiratory activity should produce a corresponding change in the loss ofsolids. Other evidence for an active principle is too strong to be ignored, however.Furthermore, it is quite probable that the amount of energy used is too small to beshown by measuring the very slight changes in solid matter in these animals.

The mechanism for this regulation could be based either in the respiratory systemor in an active component of the cuticle. If it were in the cuticle the regulatory patternshould still be evident after CO2 treatment, but if it were in the spiracles, whichpresumably were wide open, the line should show inverse proportionality to thehumidity over its entire length. It is evident that the line for average rates of loss inthe treated mites (Fig. 3) shows the same deviation as that for the control animals,indicating a zone of regulation. This is good evidence that the mechanism is indeed apart of the cuticular activity rather than being based in the respiratory system. Treatedanimals showed a much greater water loss than did the controls, but this is to beexpected when the spiracles were kept open. Moreover, if this were a spiracularmechanism, one would not anticipate the sharp breaks at either end of the zone ofregulation found in untreated mites. Spiracular control would probably requirehygroreceptors of some sort, and such abrupt changes are not characteristic of receptorsin general.

DISCUSSION

The restriction of water loss by active work of some mechanism in the cuticle,possibly the epidermal cells, has been shown to be an important factor in only a fewother animals, notably in several species of both ixodid and argasid ticks (Lees, 1946).This mechanism is thought to be of only slight importance in most insects (Wiggle-worth, 1945), and it may be that it is more significant in the Acarina because of therelatively greater surface area for water loss in these small animals. Little has beendone on the very small hardy insects, however, and this approach might lead to otherideas on the subject.

It would be convenient to assume that the active cuticular restriction of loss below50% R.H. and the regulation above it are part of the same mechanism, if for no otherreason than simplicity. There is little or no evidence for either one mechanism or twoat this time, and a second ought not to be assumed if it is not necessary. This can bebetter justified by postulating that above 50% R.H. the mechanism can regulate water


loss, causing the deviation from proportionality to humidity that we have shown. Inall humidities below this zone of regulation, though, it would be working at the samerate, its full capacity. Hence, differences would be due to variations in the dryingpower of the air. The mechanism would only be restricting transpiration in the lowerhumidities.

The mechanism by which regulation and restriction of water loss are accomplishedis unknown, but Edney's (1957) and Beament's (1961) suggestions, that it might be thesame as the one that produces active uptake of atmospheric water at high humiditiesin some arthropods, should be considered. Beament (1954) hypothesized that uptakeis based on the active transport of water; and, to follow this same line of thought,regulation and restriction would then be based on active transport. If this were so, onewould expect a sharp change such as that between 43% and 53% R.H., indicating abreakdown of the mechanism at lower vapour pressures. It would mean that theregulation of transpiration at humidities below 50% R.H. requires more energy thanis available to the system, and the rate of loss rises at 43 % to become proportional tothe evaporating power of the air. Such an abrupt change between these two humiditiesis also typical of several other aspects of the humidity relations of B. praetiosa,especially of survival (Winston, 1963 a); and Edney (1945) and Beament, Noble-Nesbitt & Watson (1964) found that the lower limits for uptake were near 50% R.H. inprepupae of rat fleas and in the common firebrat. It is possible that these changes arethe result of the breakdown of a mechanism common to many species.

Though regulation is apparent, we have been unable to demonstrate active uptakein the clover mite by any of the techniques used successfully on other acarines andinsects. One might expect to find it in this mite, though it is the first tetranychid to bestudied in this way, because the other Acarina which have been properly tested haveshown the phenomenon quite readily—for example, ticks of several species (Lees, 1946;Belozerov & Seravin, i960), grain mites (Kniille, 1962; Solomon, 1962), spiny ratmites (Wharton & Kanungo, 1962) and rabbit ticks (Camin, 1963). None of these isas well adapted to dry air as are clover mites, however, except for some of the argasidticks, and it may be that uptake is not present in some hardy forms such as Bryobia.Water loss is slow enough to make it possible for them to replenish their watersupply by drinking or feeding well before their water content has dropped to acutelevels, and uptake would not be of any particular survival value. Thus the functionmay have either been lost completely or masked so that the usual techniques would notreveal it.

To show that there is regulation of water loss one must expose the experimentalanimals to a wide range of humidities, and very few workers have used this approach.Of these, only Ludwig (1937) and Jakovlev & Kriiger (1953), working with twoorthopterans, were able to demonstrate regulation. It is probably only because so fewanimals have been tested in this way that the regulation of transpiration is not morecommonly known among terrestrial arthropods.

Such a regulatory system would be a major factor in the maintenance of a stableinternal environment over a major portion of the humidity range. Even though manyarthropods are apparently able to withstand wide variations in their internal medium,there must be an advantage to having as much constancy of the blood as possible. Thedevelopment of homeostatic mechanisms has usually brought advances for the


fortunate species that had them, and, in general, fluctuating humidities representmuch more of a stress factor for small animals than for large ones. This ability tocontrol and restrict evaporative water loss in all but the highest humidities, coupledwith broad temperature tolerances (Anderson & Morgan, 1958), may be the primaryfactors that make it possible for these mites to be almost the only arthropods activein numbers above the soil surface during the winter months when competition andpredation are essentially nil. They carry on their life-cycle in normal fashion duringa long period of the year in which daytime humidities in this region may range downto 5% or below and in which temperatures commonly vary between 250 and — io° C.They are to be found on sun-warmed walls all during this period, limited in theiractivity by low light intensities and extremely low temperatures.

The humidities encountered by this mite over most of its distribution in the Northand South Temperate Zones (Morgan, i960) are almost always within or above thezone of regulation. Presumably, they evolved under such conditions where life wouldseem to be easiest for them from the standpoint of humidity. There is no question,though, from the data presented in this paper, that the mites can cope without difficultywith the occasional periods below 50% R.H. which would be experienced. Drierareas, such as this one at Boulder, Colorado, where the humidity during the day isalmost always below the zone of regulation and the evaporating power of the air ishigh, represent nearly marginal habitats that must tax their control mechanismsconsiderably. Nevertheless, they are able to thrive under such conditions.

SUMMARY

1. Groups of sixteen mites were starved for 48 hr. at 29% R.H. and then exposedfor 18 or 24 hr. to one of nine humidities, from 0% to 93% R.H. They were weighedas groups before and after the treatments to determine total weight loss. Dry weightswere also obtained to find water content and for the calculation of water and dry-weightlosses. All work was done at 250 C.

2. Water loss, considered equivalent to total weight loss, was also obtained underseveral other conditions; and at all humidities it was found to be highest in miteskilled in chloroform vapour while it was considerably less in those killed in HCN gas.Mites with spiracles kept open by air with 10% CO2 lost weight at rates midwaybetween those for dead and those for living animals.

3. There is apparent regulation of body-water content as a percentage of the finalweight over the whole humidity range.

4. Water loss is restricted by a CO2-sensitive mechanism, presumably the spiracles.5. Active regulation of water loss by a cuticular mechanim was shown between

53% and 85% R.H., while at humidities below this, loss was actively restricted but notregulated.

6. It is postulated that both restriction and regulation are brought about by thesame mechanism, which might be a form of active transport.

7. Uptake of water from unsaturated air was not found with any of the methodsused.

8. Regulation such as was found here would help to maintain the internal environ-ment of these mites as nearly constant as possible in the face of fluctuating humidities.


The authors wish to acknowledge support of this research by the University ofColorado and by Research Grant no. G14517 to the senior author from the NationalScience Foundation.

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