J. exp. Biol. (1982), 96, 251-262 2CIWith 2 figures

Printed in Great Britain

WATER VAPOUR ABSORPTION BY THE DESERTBURROWING COCKROACH, ARENIVAGA INVESTIGATA:

EVIDENCE AGAINST A SOLUTE DEPENDENTMECHANISM

BY M. J. O'DONNELL*Department of Zoology, University of Toronto

(Received 1 April 1981)

SUMMARY

1. Solute concentrations were measured in the frontal bodies and hypo-pharyngeal bladder surfaces, which form the absorption system of Arenivaga.Measured values were compared with the concentrations necessary to lowerrelative humidity to the absorption threshold, 81% R.H.

2. The predominant inorganic solutes, measured by instrumental neutronactivation analysis, were Na, K, and Cl. Their concentrations were 2-3orders of magnitude below those necessary for absorption.

3. Quantities of a variety of hydrophilic organic molecules were deter-mined with fluorometric reagents. Concentrations of free amino acids,peptides, reducing sugars and polyhydroxyl alcohols were negligible infrontal bodies and on bladder surfaces.

4. Frontal bodies did not appear to be active sites of protein or poly-saccharide synthesis, because they did not accumulate significant amounts ofradio-labelled amino acids and glucose injected into the hemolymph.

5. The results of this and previous studies indicate strongly that absorp-tion in Arenivaga differs markedly from the solute-dependent schemes whichhave been proposed for other arthropods.

INTRODUCTION

The desert cockroach is the only insect in which buccal structures are known to beinvolved in water vapour absorption (O'Donnell 1977a, 1980). In relative humidities(RH) above a threshold value of 81 %, vapour condenses onto a fluid layer covering twoprotrusible bladders, which are lateral diverticula of the hypopharynx (Figure 1). Thefluid is held in the interstices of fine cuticular hairs which densely cover the bladdersurface. It is composed of condensate, and the product of a pair of structures calledfrontal bodies which are situated beneath the frons and connected to the bladders by agroove in the epipharynx (O'Donnell 19776, 1981). Interrupting this connectioncauses the associated bladder to become dry. The fluid is necessary for absorptionbecause vapour does not condense onto dry bladders (O'Donnell, 1977a).

• Present address: A.R.C. Unit of Invertebrate Chemistry and Physiology, Department of Zoology,yniversity of Cambridge.

252 M. J. O'DONNELL

Frons

Frontal muscles Frontal body Labrum

Hypopharyngeal bladder

Mandible

Labium

Oesophagus Salivary duct

Fig. i. A schematic saggital section through the head of a desert cockroach with the hypo-pharyngeal bladders protruded. The dashed line indicates the path of fluid movementsthrough an epipharyngeal groove (not shown) joining the frontal body to the associatedbladder.

Recent studies in tenebrionids and acarines have suggested that the necessaryreduction in the thermodynamic activity of water is established by high soluteconcentrations at, or near, the absorption site. Ramsay (1964) measured potassiumchloride concentrations in excess of 2 M in the Malpighian tubular fluid of Tenebriomolitor. The freezing point depression of the perirectal fluid was 8 °C (Ramsay, 1964;Machin, 19796), and gradients of osmotic pressure were present, increasing posteriorlyalong the rectum and radially towards the Malpighian tubules. On the absorbingsurfaces of dried animals, crystalline deposits near the salivary glands of ticks containsignificant amounts of potassium (Rudolph & Knulle, 1974). Large quantities ofpotassium chloride are associated with the pre-coxal glands of dried mites (Wharton &Furumizo, 1977).

The predominance of solute-dependent models of absorption and epithelial trans-port (Diamond and Bossert, 1967) in other species suggested that structures associatedwith absorption in Arenivaga should be analysed to determine if high solute concen-trations exist. Many suggestions have been made concerning the properties of soluteswhich could actively be transported to lower water's thermodynamic activity. Inor-ganic ions, low molecular weight organic molecules and macromolecules have all beenproposed (Machin, 1979a; Noble-Nesbitt, 1977, 1978). Madrell (1971) suggested thatsuch substances must be highly soluble in water and possess many sites at whichhydrogen bonding can occur so as to reduce water activity. For absorption in Ther-mobia, at humidities as low as 45 % RH, he considered it doubtful that any non-toxicinorganic molecule could be used. Even for absorption by T. molitor, against muchsmaller gradients, active transport of KC1 by the leptophragmata must be achieved.

Vapour absorption by the desert cockroach 253exclusive of water move-ment from other tissues which would dilute the establishedgradient. Cells would also have to resist membrane depolarization in such a highextracellular potassium environment (Machin, 1979a).

Certain organic molecules, on the other hand, would not lead to toxic effects andionic imbalance, and may also be tolerated by living cells at sufficiently high concen-trations (Machin, 1979a). Glycerol was suggested as a possibility by J. Diamond(personal communication to House, 1974) and T. Weis-Fogh (personal communicationto Noble-Nesbitt, 1977).

Water activity could also be lowered by high concentrations of macromolecules,especially those which have large osmotic coefficients and show strongly non-idealsolute behaviour (Machin, 1979a). Water activity could be reduced either directly bythe macromolecules, or indirectly by their enzymatic breakdown to produce a highconcentration of smaller molecules. High concentrations of amino acids and sac-charides might be produced by de-polymerization in the cuticle (Locke, 1964). It hasalso been suggested that the sub-cuticular mucopolysaccharide in the rectum ofThermobia might be a component of the absorption mechanism (Noble-Nesbitt, 1978).Reabsorption would facilitate removal of the entrained water.

Solute concentrations sufficient to lower water activity to the levels necessary forabsorption are extremely high. For example, 11700 mM of an ideal solute will equili-brate with 81% RH, the threshold for absorption, by Arenivaga. For comparison,saturated KC1 (4600 mM) equilibrates with 85 % RH, and saturated NaCl (6250 HIM)equilibrates with 75-5% RH (Weast, 1968). It therefore seemed feasible to test asolute-coupled hypothesis of water vapour absorption in Arenivaga through measure-ment of organic and inorganic solute concentrations in the frontal bodies and bladderfluid.

MATERIALS AND METHODS

Inorganic solutes were analysed by multi-element instrumental neutron activationanalysis (INAA), and by atomic absorption spectrophotometry (AAS). Hydrophilicorganic molecules were detected by fluorescent labelling techniques for amino acids,peptides, reducing sugars and polyhydroxyl alcohols. Macromolecules were detectedthrough fluorescent labelling of active groups, and through incorporation of radio-labelled precursors.

Sample preparation

Frontal bodies and hypopharyngeal bladders were excised from absorbing animalswhich had been fast-frozen during absorption and lyophilized (O'Donnell, 1980,1981).

For comparing levels of inorganic elements in bladder fluid and in bladder tissue,bladders were everted by squeezing the abdomen and washed, before freezing, with5 ml of water forcibly ejected from a syringe through a 26 gauge needle. Fluid wasalso collected on Millipore strips (o-i fim pore size) which had been moistened andapplied to the surface of protruded bladders.

Samples were weighed in platinum boats over silica gel to the nearest o-i fig using anelectronic microbalance (Mettler ME22).

9 E X B 96

254 M. J. O'DONNELL

Instrumental neutron activation analysis

Lyophilized samples, or Millipore strips used in collecting bladder fluid, wereplaced in polythene vials and irradiated in the low flux SLOWPOKE-2 (Safe Low PowerCritical Experiment) nuclear reactor (Atomic Energy Canada Ltd., CommercialProducts, Ottawa) at the University of Toronto. Details of the reactor are described byKay et al. (1973) and its use in multi-element analysis by Hancock (1976). Irradiationat 20 kW at the number 1 site in the reactor was equivalent to a neutron flux of io12

neutrons/cm2 s"1. For common biological elements 20 kW irradiation for 10 minuteswas sufficient. For greater sensitivity, and especially for potassium, samples were alsoirradiated at 5 kW for 16 h.

Elemental weights were determined from the sample's gamma irradiation. Gammaray peak intensities were evaluated by automatic analyzer integration of suitablemanually selected spectral regions of interest at, and near, each peak. Weights werecalculated using the formula:

elemental weight (jig) = (P-B)/F&t,

where P = peak intensity, B = baseline intensity, F&t = a constant, specific for eachgamma ray spectral line in the irradiating and counting conditions described. Theseconstants were determined, using standards, by R. Hancock of the SLOWPOKE

Reactor Facility. Counting efficiencies were determined according to the formula% error = (P+B) 0S/(P-B)x 100.

Estimates of the maximum volume of fluid held on the bladder surface wereobtained using a 150 mM solution of MnCl2.4H2O applied by syringe to forciblyeverted bladders. Preliminary INAA results had shown manganese was present innegligible quantities in the tissues examined, and was therefore suitable as a markersubstance. Before freezing, solution in excess of that held by the mat of cuticular hairswas removed by a jet of compressed air directed at the head of the animal for 1 s.

Atomic absorption spectrophotometry

Measurements of potassium levels by INAA were corroborated by atomic absorp-tion spectrophotometry. Frontal bodies were dissected from fast frozen animals,macerated with tungsten needles and centrifuged in flame-sealed micropipettescontaining immersion oil. Fluid volumes were calculated from drop diameters in oil,prior to addition of 1 ml distilled water. The spectrophotometer, a Varian TechtronAA6 (Springvale, Australia), was calibrated with solutions of Analytical Reagent gradepotassium chloride.

Analysis of primary amines

Amino acids in frontal bodies and bladder fluid were detected as the highly fluores-cent compounds produced upon reaction with O-pthalaldehyde (OPA; Roth, 1971). Astock solution of OPA was prepared as a 15 mg % solution (w/v) in glass distilledwater with a drop of 1 N NaOH added for every 200 ml. The reagent solution,prepared just before use, consisted of 20 ml stock OPA and o-oi ml 2-mercaptoethanol

Vapour absorption by the desert cockroach 255

(Stephens et al., 1978). For peptides, sensitivity was maximized by adding o-oi ml^riethanolamine to 15 mg % OP A stock. Standard solutions were applied to a strip ofMillipore filter (1 cm x 0-5-1-0 mm) and allowed to dry. OPA reagent (0-5-1-0 /A) wasthen pipetted onto the same region of the strip. For peptides, a 10% aqueous solutionof triethanolamine was applied after the OPA solution. For determination of aminoacids in bladder fluid, OPA reagent was applied directly to the surface of protrudedbladders of animals maintained in humid conditions in transparent chambers.Alternatively, OPA was applied to o-i fim. pore size Millipore strips which had beenmoistened and applied one or more times to the bladder surface (O'Donnell, 1980).Filter strips were examined under a long wave ultraviolet lamp. The limit of detectionwas determined for several solutions of amino acids and peptides applied to the strips.

Analysis of reducing sugars

DANSYL hydrazine (i-naptholenesulfonyl 5-dimethylamino hydrazine; Sigma,St Louis, Missouri) was used as a sensitive fluorometric reagent for detection ofreducing sugars. Over 90 % of a glucose standard is converted to glucose DANSYLhydrazone (Avigad, 1977). Because DANSYL hydrazine is itself fluorescent, reactionproducts were separated by thin layer chromatography (TLC).

As a standard, glucose (0-05-2-0 /rniol in 100 /il water) was added to 100 /i\ of o-iM/1 acetic acid and 200 fi\ of DANSYL hydrazine in a 2 ml plastic vial, heated to80 CC for 10 min, then cooled to room temperature and spotted on the plate.

Reducing sugars in lyophilized frontal bodies were extracted in 5-10 fi\ of water.Tissues were either macerated in Reacti-Vials (Pierce Chemical Co., Rockford,Illinois) using a glass rod ground to fit the conical vial, or were ultrasonicated, frozen,then thawed, three times. Volumes were reduced to 0-5-1-o/d by evaporation underpartial vacuum, then mixed with 2 fi\ acetic acid (0-05 M) and 2 fi\ DANSYL hydra-zine (1 % in ethanol). Samples were heated, and in some cases volumes reduced to lessthan 1 fi\ by evaporation. Proteins were first precipitated from hemolymph samples,either by heating (70 °C for 5 min) or by addition of an equal volume of o-i M aceticacid. Samples (1 fi\) were spotted onto high performance thin layer chromatographicplates (HPTLC) under a stream of warm air. Detection limit of glucose by HPTCLplates developed in chloroform-ethyl acetate- 1% boric acid (3:5:2; Avigad, 1977)was 30 pmol. 1 fd aliquots of the reagent mixture were spotted on the HPTLC plateunder a stream of water air. The detection limit was extended by applying the super-natant collected from the centrifuged homogenate of as many as ten frontal bodies toone spot on the plate.

Analysis of polyhydroxy alcohols

DANSYL hydrazine has also been used to detect aldehydes produced by periodateoxidation of glycoproteins in polyacrylamide gels (Eckhardt, Hayer & Goldstein,1976). A method was developed to produce polyhydroxy alcohols by periodateoxidation. Frontal bodies were homogenized and added to an equal volume ofaqueous periodate (0-5%), then heated at 75 °C for 30 min. A small volume of 0-5%sodium metabisulfite in 5 % aqueous acetic acid was added to destroy excess periodate.The mixture was then treated as for reducing sugars. Mannitol and glycerol were used

9-2

256 M. J. O'DONNELL

Table 1. Frontal bodies and hypopharyngeal bladders: elemental analysis byINAA

Tissue*

Frontal bodies

Bladders (absorbing)

Bladders (washed)

SampleWeight

(MS)

420

801

S83

Irradiation

kW Time (h)

S

2 0

5

2 0

5

2 0

16

0-17

16

0 1 7

16

0 1 7

Element

NaKNaClNaKNaClNaKNaCl

Weight

i '33 91-3

3 37-64 18-i

I2-O

6-83'56-S

io-6

% error

1

67S0-5

633

o-S1 0

33

% of sampleby weight

0-30 90-3o-80-90-50 9

i'S

1-2

o-61-2

i-8

• Frontal bodies or bladders from 20-24 animals were pooled for each sample.

as standards. Visual detection limits on HPTLC plates were 1 and 10 nmol formannitol and glycerol, respectively.

Incorporation of ̂ C-labelled compounds

5 /A each of 14C-labelled glucose, alanine and an amino acid mixture were injectedinto the haemocoel of desiccated animals through the arthrodial membrane betweenthe forelegs. Animals were placed in glass vials which were humidified by pluggingwith moistened cotton wool. They were fast-frozen at intervals after injection of thelabel, and then decapitated, lyophilized, dissected with tungsten needles, and weighed.Tissues were placed in 1 ml of Protosol (New England Nuclear Corp.) and heated to55 °C for 18-24 n m glass scintillation vials with polythene-lined aluminium screwcaps. Nine ml of scintillation fluid (6 g diphenyloxazole/litre toluene) were added andsamples analysed by LSC. Aliquots of hemolymph (usually 5 fil) were collected inmicropipettes from an incision in the arthrodial membrane between the forelegs. Thehaemolymph was dispensed into 1 ml of Protosol and the pipette broken and includedin the vial. Washed bladders were prepared as above.

Bladder fluid was also collected on Millipore strips after injection of uC-labelledglucose and amino acid mixture.

RESULTS

Inorganic solutes

Instrumental neutron activation analysis showed that inorganic material contributesonly a small part to the total dry weight of frontal bodies and bladders, the principalelements (Na, K, Cl) all constituting less than 4% dry weight (Table 1). The basisfor the apparent increase in the proportion of each of these elements in washedbladders, and estimated elemental concentrations (Table 3) will be discussed. Calcium,magnesium and bromine were present in trace amounts, less than 0-06 % of total dryweight for each. Iodine was present in significant quantities in bladders from absorb-

Vapour absorption by the desert cockroach 257

Table 2. Analysis of primary amines by ortho-pthalaldehyde (OPA): detection limitsfor samples applied to millipore filter strips

Compound

GlycinePenta-L-alanineOxytocin

BSAfTrypsinOvalbumin

Method a:

OxytocinPenta-L-alanine

Ovalbumin

Volume applied(nl)

Concentration(miu)

VxC*(pmol)

Method 1: OPA + 2-mercaptoethanol

4 6

2 5

6 9

3°2-4

1 0

2 0

asgl"1

o-ii -oi-o

OPA + a-mercaptoethanol, followed by

3-S8 0

8 0

mM

252 0

gr1

1-0

463°62

ng0 73 0

2 4

Tests detectable(%)

750

1 0 0

4060

1 0 0

10% triethanolamine

pmol

87150

ng8

801 0 0

so• VxC = volume * concentration. \ BSA: Bovine Serum Albumin.

ing animals (0-07%) and in washed bladders (0-04%) but was undetectable in frontalbodies. No elements were detectable in Millipore strips applied to the bladder surface.

Potassium concentrations in fluid from frontal bodies were measured by AAS,which gave values of 99 + 49 mequiv/1 (X±SD\ n = 5). The large deviation ofindividual values from the mean is possibly due to errors inherent in manipulatingsmall samples (5-23 nl) and in separating the frontal bodies from the surroundingmuscle tissue.

INAA was used to determine the maximum volume of fluid held in spaces betweenhairs on the bladder surface. The quantity of the manganese chloride marker solutionretained on the bladder surface was a constant proportion of the dry weight of thebladder. Calculations indicated that 6-2 + 01 nl//ig dry weight were retained on thebladder. For a typical animal weighing 364 mg, a maximum of 273 ± 6 nl wereretained on both bladders, or 137 nl/bladder.

Organic solutes

No amino acids were detectable by OPA applied either directly to the bladder ofabsorbing animals or to Millipore strips used to collect fluid from the surface. Themaximum undetectable concentrations of amino acids and peptides present in thebladder fluid were therefore estimated by determining the sensitivity of OPA toamino acids, peptides or proteins applied to the bladder surface. Application of OPAonto the bladder surface within 5 s of a preceding application of several nanoliters of10 mM glycine, o-i mg/1 bovine serum albumin, or 1 mg/ml trypsin by micropipetteshowed clear, visible fluorescence. OPA was an extremely sensitive detector of aminoacids, peptides or proteins on Millipore strips (Table 2) which had been used tocollect fluid after application of known quantities of the experimental solutions to thebladder surface.

258 M. J. O'DONNELL

Table 3. Estimated maximum solute concentrations (min) in frontal bodies and bladdermfluid

Solute

SoldiumPotassiumChlorideAmino acids1

Peptides1

Reducing sugars'Polyhydroxyl alcohols4

Total Required

ntalbodies

611071 0 0

—

—

<O-I-I< i -6275

—

Hypopharynge

Absorbing

672 1

68<o-i<I-2

—

IS8II7OO*

al Bladders:

Washed

8a2S83————

190—

1-4 Concentrations listed are the limits of sensitivity of the corresponding analytical method; noneof these solutes were present in detectable quantities.

• A relative humidity equivalent to the absorption threshold (81 % RH) is maintained above an idealsolution of the concentration indicated.

There were no reducing sugars in the frontal bodies detectable by DANSYLhydrazine. The detectable limit of glucose (< 30 pmol/spot) was used to estimate themaximum undetectable amount of reducing sugar present in frontal bodies, 3-30pmol. The technique readily detected a sugar with the same Rf as glucose (0-18) inmuscle tissue. Reducing sugars were also detectable in haemolymph.

Any polyhydroxyl alcohols present in the frontal bodies were also at concentrationsbelow detectable limits. Limits of detection of glycerol and mannitol were 10 and1 nmol/spot, respectively.

It was concluded that amino acids, peptides, proteins and reducing sugars wereundetectable because they were either absent, or present in only trace amounts, andnot because of deficiencies in the analytical techniques.

Calculation of solute concentrations

Solute concentrations were calculated from the dry weights (Tables 1, 2) byestimating the appropriate solvent volumes. Measurements of a marker solutionindicated that for each microgram dry weight of bladder tissue, 6-2 nl of an aqueoussolution is retained by the cuticular hairs. As an example, the 801 fig sample ofbladder tissue (pooled from 20 absorbing animals) would retain a maximum of801 x 6-2 = 4966 nl of fluid. It was assumed that all solutes were dissolved in thisestimated volume, and that none were intrinsic to bladder tissue; estimated concentra-tions are therefore maximums.

Solvent volume of the frontal bodies was estimated from the formula:

solvent volume = wet weight — dry weight,

in which wet weights were calculated from a wet/dry weight ratio of 0-31, based onwhole animals (Edney, 1966). However, the exoskeleton, whose dry/wet weight ratio ishigher than 0-31, is a major fraction of the total weight of the animal. This value is

Vapour absorption by the desert cockroach 259

(a) Frontal bodies

ox 8

'£ 0

I 10

Alanine

I I 1

1

-

1

1 1

»3

1 I

3 t 4

Amino acids1 1 1 I

3 GlucoseI 1 1 1

30 50

(c) Haemolymph40

20

0

_D4

a 4

70

100

50

010

Alanine

a 5I 1

oI I I

40

20

0

-

11

o1

!

a

a

1

Amino acids

D1 1 1

a

1

D ° Glucose•

I I I I I

30 50 70

•a00

(6) HypophoryngeaJ bladders

Alanine

•ioj I

4 -

4 -

- •

-nl

O

-

*

1

1 o1 1 1

0 °

o oi o

1 1

- ; 3

Amino addsI i I

Glucose

1 1 1

10 30 50

(d) Muscle

4 -

•

••

1 1 1

Alanine

. 3 .

•

4 -

-

1 4

_

1 1

•

•

1

• 4

• 5

Amino acids

1 1 1 1

10 30 50 70

Time (h)Fig. 2. Incorporation of "C-labelled compounds into various tissues during absorption. In(6), the open circles indicate animals in which the bladders were forced to protrude by squeez-ing the abdomen, and then washed with distilled water applied with a syringe. Closed circlesin (6) indicate those animals fast-frozen during absorption. Muscles were excised from thehindleg femur. Points are the means of the indicated number of values; lengths of the barscorrespondent ± 1 S.D.

therefore an overestimate of the ratio for tissues such as the frontal bodies, whosesolvent volumes are correspondingly underestimated. Calculated solute concentrationsare therefore maximums. Maximum estimated concentrations of each solute (Table 3)are 2-4 orders of magnitude below those required for absorption.

Incorporation of ^C-labelled compounds

Following their injection into the haemocoel, there was no significant incorporationof 14C-labelled glucose, alanine or amino acid mixture into the frontal bodies or

260 M. J. O'DONNELL

bladders (Fig. 2). Approximately the same level of each compound was present inwashed and unwashed bladders (Fig. 2b), indicating that most of the compound waspresent in bladder tissue rather than bladder fluid. Concentrations were below those inhaemolymph or muscle. Quantities of 14C-labelled amino acids in bladder fluidcollected on Millipore strips within 2 days of injection (35-43 cpm/strip; 46 stripsapplied 1-5 times to bladders; n = 6 animals) were not significantly elevated abovebackground levels of radioactivity (36 cpm/strip).

DISCUSSION

The absorption mechanism of Arenivaga differs fundamentally from the solutedependent systems which have been proposed for tenebrionids (Machin, 1979 a, b)and acarines (Rudolph & Knulle, 1974; Wharton& Furumizo, 1977).

A comparison of maximal solute concentrations in frontal bodies and bladder fluidshows that concentrations are insufficient to lower water activity to the absorptionthreshold, 81 % RH (Table 3), even when the most unfavourable allowances forexperimental error are used.

Inorganic solute concentrations are unexpectedly higher in washed bladders than inbladders from absorbing animals, presumably because haemolymph tended to adhereto the inner surface of bladders forcibly everted before freezing and lyophilizing.Haemolymph chloride concentration (i28meq/l; Edney, 1966) represents 6-7% ofthe dry weight of haemolymph, considerably more than is present in bladders (1-2%)and frontal bodies (o-8%). Because the calculated concentrations in bladder fluidassume all of each element is in the bladder fluid, whereas in fact most is in the tissue,the concentrations in Table 3 are overestimates.

Corroboration of the estimated potassium concentration from the % dry weightdetermined by INAA (107 nw) is provided by the similar value obtained by atomicabsorption spectrophotometry of wet tissue extracted from fast frozen animals.

The unexpectedly high amount of iodine in bladder samples (0-04-0-07% dryweight) is also more likely contained in the cuticle, rather than in the fluid layer.Iodinated amino acids are accumulated in cuticle (Limpel & Casida, 1957 a, b), andmay be involved in cuticle hardening (Tong & Chaikoff, 1961). If the iodine were inionic form, its maximum concentration would be 0-5 and 0-2 mM in absorbing andwashed bladders, respectively.

Nor can organic solutes lower water activity sufficiently for absorption. Only2-4% of the total dry weight of bladders and frontal bodies is inorganic; the majorproportion is organic. However, virtually all of this consists of chitin and structuralproteins, which have been demonstrated histochemically (O'Donnell, 1981). Inbladder fluid and frontal bodies, concentrations of free amino acids, glucose, or theirderivatives, as indicated by direct chemical analysis and by accumulation of thecorresponding 14C-labelled compounds, are negligible. Concentrations of the labelledmolecules per microgram dry weight of frontal bodies or bladders was generally one-fifth that in the haemolymph (Fig. 2), suggesting that there is no synthesis of proteins,glycoproteins or long-chain carbohydrates within the frontal bodies during absorption.

These results support the hypothesis, based on ultrastructural analysis and measure-ments of internal osmolalities (O'Donnell, 1980, 1981) that the frontal bodies do not

Vapour absorption by the desert cockroach 261

function as secretory structures or salt glands, but as hydrostatic pumps which producean ultrafiltrate of the haemolymph. The frontal body plate, across which filtrationoccurs, has a very low permeability to molecules as small as glucose (O'Donnell,1981). It is therefore unlikely that macromolecules could be transferred out of thefrontal bodies in high enough concentrations to form a solution of reduced wateractivity.

Measurements of osmolalities within the frontal bodies have been reported else-where (O'Donnell, 1981); they support the hypothesis that these structures do notproduce a solution which is sufficiently concentrated to directly absorb water vapour.Measured values (645 mOsm/kg; O'Donnell, 1981) exceed the value approximated bysumming the solute concentrations (275-300 mOsm/kg), presumably because of theeffects of proteins or other macromolecular components on ice crystal formation andtherefore, freezing point depression.

It may be argued that as yet unconsidered solutes, particularly those of low molecu-lar weight, are involved during absorption byArenivaga. However, osmolalities withinfrontal bodies, the source of bladder fluid, indicate that solute concentrations are notelevated. Moreover, many solutes can be excluded on the basis of their physicalproperties and physiological effects. Many of the alcohols are either too toxic or toovolatile to be present in a solution of low vapour pressure (Machin, 1979a). Althoughamino acid concentrations of 0-29-2-43 g % occur in insect haemolymph (Wyatt,1961), most of the amino acids are too insoluble to be present in the concentrationsrequired for water vapour absorption. Concentrations at saturation range from 4250 mMfor glycine and 2250 mM for alanine, to as low as 70 mM for aspartic acid (Weast, 1968).A saturated solution of proline, the most soluble amino acid (saturated at 12400 mM)could absorb water vapour from humidities as low as 78% RH (Machin, 1979 a).

A number of aspects of the behaviour of the absorption system are inconsistent withthe lowering of water activity by high solute concentrations, and therefore support theresults above. The drying of the bladder surface which follows interrupting thesupply of frontal body fluid to the bladders is incompatible with a solute dependentabsorption mechanism. A suitably concentrated solution on the bladder surface wouldnot dry out, but would equilibrate with ambient humidity.

A further observation concerns the appearance of dried bladders. No crystallinedeposits are evident; such deposits have been found on the absorbing surfaces ofacarines (Rudolph & Knulle, 1974; Wharton & Furumizo, 1977) and are cited asevidence for the use of concentrated solutions during absorption.

Clearly, the results presented here strongly imply that fluid, produced by thefrontal bodies and conveyed to the bladder surface, plays a subordinate role in themechanism of water vapour absorption, perhaps aiding condensate removal so as tofacilitate continuous absorption. The primary function of reducing water activity may,therefore, reside in the cuticular hairs which cover the bladder surface. The wateraffinity of the hairs is currently under study.

I am grateful to A. J. Forester, J. Machin, and S. H. P. Maddrell for their criticalreadings of the manuscript. INAA measurements were performed by R. Hancock.This research was supported by a grant from the Natural Sciences and EngineeringResearch Council (Canada) to J. Machin.

262 M. J. O'DONNELL

REFERENCES

AVIGAD, G. (1977). DANSYL hydrazine as a fluorometric reagent for thin layer chromatographicanalysis of reducing sugars. Chromatography io, 343-347.

DIAMOND, J. M. & BOSSERT, W. H. (1967). Standing gradient osmotic flow. A mechanism for couplingwater and solute transport in epithelia. J. gen. Phytiol. 50, 2061-2083.

ECKHARDT, A. E., HAYER, C. E. & GOLDSTEIN, I. J. (1976). A sensitive fluorometric method for thedetection of glycoproteins in polyacrylamide gels. Analyt. Biochem. 73, 192-197.

EDNEY, E. B. (1966). Absorption of water vapour from uraaturated air by Arenivaga sp. (Polyphgidae,Dictyoptera). Comp. Biochem. Phytiol. 19, 387-408.

HANCOCK, R. G. V. (1976). Low flux multielement instrumental neutron activation analysis in archaeo-metry. Analyt. Chem. 48, 1443-1445.

HOUSE, C. R. (1974). Water Transport in Cells and Tissues. London: Edward Arnold.KAY, R. E., STEVENS-GUILLE, P. D., HILBORN, J. W. & JESVIS, R. E. (1973). SLOWPOKE: A new low-cost

laboratory reactor. Int.J. appl. Radial. Isotopes £4, 509—518.LIMPEL, L. E. & CASIDA, J. E. (1957a). Iodine metabolism in insects: I. In vivo metabolism of radio-

iodide. J'. exp.Zool. 135, 19-28.LIMPEL, L. E. & CASIDA, J. E. (1957b). Iodine metabolism in insects. II. In vivo distribution of iodo-

amino acids and related study with Periplaneta.J. exp. Zocl. 136, 595-613.LOCKE, M. (1964). The structure and function of the integument in insects. In The Physiology of Insects,

2nd ed., vol. vi (ed. M. Rockstein), pp. 123-213.MACHIN, ] . (1979a). Atmospheric water absorption in arthropods. Adv. Insect Physiol. 14, 1-48.MACHIN, J. (19796). Compartmental osmotic pressures in the rectal complex of Tenebrio larvae:

evidence for a single tubular pumping site.J'. exp. Biol. 8a, 123-138.MADDRELL, S. H. P. (197:). The mechanisms of insect excretory systems. Adv. Insect. Physiol. 8,

200-33' •NOBLE-NESBITT, J. (1977). Active transport of water vapour. In Transport of Ions and Water in Animals

(ed. B. L. Gupta, R. B. Moreton, J. L. Oschman and B. J. Wall), pp. 571-598.NOBLE-NESBITT, J. (1978). Absorption of water vapour by Thermobia domestica and other insects. In

Comparative Physiology—Water, Ions, and Fluid Mechanics (ed. K. Schmidt-Nielsen, L. Bolis andS. H. P. Maddrell), pp. 53-66. Cambridge University Press.

O'DONNELL, M. J. (1977a). Site of water vapour absorption in the desert cockroach, Arenivaga investi-gata. Proc. Natl. Acad. Sci. U.S.A. 74, 1757-1760.

O'DONNELL, M. J. (19776). Hypopharyngeal bladders and frontal glands: Novel structures involved inwater vapour absorption in the desert cockroach Arenivaga investigata. Am. Zool. 17, 902.

O'DONNELL, M. J. (1978). The site of water vapour absorption in Arenivaga investigata. In ComparativePhysiology—Water, Ions and Fluid Mechanics (ed. K. Schmidt-Nielsen, L. Bolis and S. H. P.Maddrell), pp. 115-121. Cambridge University Press.

O'DONNELL, M. J. (1980). Water vapour absorption by the desert burrowing cockroach Arenivagainvestigata (Dictyoptera: Polyphagidae). Ph.D. Thesis, University of Toronto.

O'DONNELL, M. J. (1981). Frontal bodies: Novel structures involved in water vapour absorption in thedesert burrowing cockroach, Arenivaga investigata. Tissue and Cell. (In the Press.)

RAMSAY, J. A. (1964). The rectal complex of the mealworm Tenebrio molitor, L. (Coleoptera, Tene-brionidae). Phil. Trans. R. Soc. Lond. B 248, 279-314.

ROTH, M. (1971). Fluorescent reaction for amino acids. Analyt. Chem. 43, 880-882.RUDOLPH, D. & KNULLE, W. (1974). Site and mechanism of water vapour uptake from the atmosphere

in ixodid ticks. Nature, London 349, 84-85.STEPHENS, G. C , VOLK, M. J., WRIGHT, S. H. & BACKLUND P. S. (1978). Transepidermal accumulation

of naturally occurring amino acids in the sand dollar, Dendraster excentricus. Biol. Bull. mar. biol.Lab., Woods Hole 154, 335~347-

Tong, W. & Chaikoff, I. L. (1961). 1J1Iodine utilization by the aquarium snail and the cockroach.Biochem. biophyt. Acta 48, 347-351.

Weast, R. C. (1968). Handbook of Chemistry and Physics, 49th ed. Cleveland. Chemical Rubber Company.Wharton, G. W. & Furumizo, R. T. (1977). Supracoxal gland secretions as a source of fresh water for

Acaridei. Acarolgia 19, 112—116.WYATT, G. R. (1961). The biochemistry of insect haemolymph. Ann. Rev. Ent. 6, 75-102.