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Correction of negative buoyancy in the phantom larva, Chaoborus americanus
J. Insect Physiol.,
1975, Vol. 21, pp. 1659 to 1670.
Pergamon
Press.
Printed
in Great Britain.
CORRECTION OF NEGATIVE BUOYANCY IN THE PHANTOM LARVA, CHAOBORUS AMERICANUS s. Department
TEFWXJCHI
of Zoology, University of Wisconsin, (Received
19 December
1974
*
Madison, Wisconsin 53706, U.S.A.
; revised 20 January
1975)
Abstract-Fourth instar larvae of Chaoborus americanus correct a condition of negative buoyancy by increasing the amount of gas held by two pairs of tracheal sacs. Mass spectrometric analysis of sac gas during the period following the addition of weights to larvae, reveal that the added gas is derived from the dissolved gases present in the surrounding medium and that it enters by physical means. The response is characterized by a transient decrease in the rigidity of the sac wall. Electron and light microscopy reveal that muscular tissue and motor terminals are associated with the sac wall and that a resilin-like protein may be present. These results suggest that the increase in the amount of gas held by the sacs is a secondary effect of an initial expansion of the sac wall itself and that the cuticular lining of the sacs is an active participant in the adjustment process.
INTRODUCTION CHAOBORUSlarvae exert control over density through gas-filled semi-rigid tracheal sacs (KROGH, 1911; FRANKENBERG,1915; DAMANT,1924; DIJHR, 195.5; TERAGUCHI, 1975). Hypotheses explaining the alteration of sac size by Chaoborus are of two kinds. One attributes changes to alteration of the sac contents, the other to alteration of the sac wall. Hypotheses implicating the sac contents propose either that liquid is pumped in and out or that a gas is secreted or excreted. Hypotheses implicating the sac wall suggest that either the properties of the sac wall are under direct control or that contraction of muscle, extrinsic or intrinsic to the sac wall, is involved.
KROGH (1911) has suggested that the sacs function as ballast tanks, Since processes involving fluid removal from a tracheal structure are well documented in insects (KEISTER and BUCK, 1949; WIGGLESWORTH,1953), the ability to move fluid across the tracheal wall could be a property of tracheal epithelia. De novo filling in Chaoborus can occur under pressure and without access to the surface (F RANKJJNBERG, 1915). However, Krogb’s hypothesis is rendered unlikely by the fact that fluid has not been seen in sacs after the first filling except under unusual conditions (KROGH, 1911; FRANKENBERG,1915; DAMANT,1924). Gas secretion has been implicated as the means by which neutral buoyancy may be recovered. FRANKENBERG(1915) observed that ligation of newly-hatched Chaoborus larvae prevented de nwo filling of more anterior tracheae provided that the * Present address: Department of Biology, Case Western Reserve University, and Cleveland Museum of Natural History, Cleveland, Ohio 44106, U.S.A.
interruption was forward of the eighth abdominal segment. This suggested a gas gland in the eighth abdominal segment (FRANKENBERG,1915 ; AKEHURST,1922). Gas produced by a metabolic process should have a single component, since examples so far elucidated follow this pattern (e.g. SCHOLANDER, 1956; LARIMER and ASHBY, 1962; CICAK et aE., 1963). Other components enter the gas space by diffusion, subsequent to initial secretion. KROGH (1911) could not detect any change in sac gas composition following adjustment to hydrostatic pressure. However, from 1 to 17 hr were given for adjustment, and since the sac gases rapidly equilibrate with gases dissolved in the medium (KROGH, 1911; DAMANT,1924), the expected enrichment of one of the components of sac gas by the addition of a single gas might have been obscured by subsequent diffusion. Hypotheses favouring control by direct action on the container rather than control by direct action on the contents have been common since FRANKENBERGin 1915 (BARDENFLETH and EGE, 1916 ; DAMANT, 1924; CHRISTENSEN, 1928; DUHR, 1955). The focus has been upon the properties of the intima lining the sacs. It is certainly true that most of the cuticle is not outside the metabolic pool of the insect and it may be that several of its intermoult properties are under direct physiological control. Volume and plasticity are two properties which can be considered. FRANKENBERG(1915) proposed that changes in sac dimensions resulted from changes in volume of a component of the wall. He thought these volume changes were dependent upon water movement since isolated sacs shrank when exposed to air, concentrated NaCl, or 95% ethyl alcohol, and swelled again when replaced in tap water.
1659
1660
S. TERAGUCHI
amounts of carbon dioxide, argon, oxygen, and A change in plasticity coupled with external force nitrogen. This reflection will be distorted by conis also a plausible mechanism for regulation of sac tamination with air during sampling and during size. FRANKENBERG(1915) found that sacs became analysis. Data are presented as relative peak heights more rigid soon after excision, although they realthough relative peak height is not equivalent to tained the ability to swell or shrink in response to percentage composition, since sensitivity to various various solutions. Control over plasticity of extragases differs. It was not necessary to convert data cellular material has been documented not only in insects but in echinoderms as well (BENNET-CLARK, to percentage composition, since conclusions are 1962; COTTRELL, 1962; NUREZ, 1963; MADDRELL, based upon a comparison between values obtained in an identical way. 1966; TAKAHASHI,1966). The changes in plasticity Samples were prepared and run in pairs, one do not in themselves effect changes in extension being from weighted animals and the other from but are accompanied by a stretching force provided weighted plus suspended animals, the two types by muscle contraction or elevated blood pressure. having been held in the same container. ComIn Chaoborus the force could be provided by muscle parisons were then made between groups of the contraction, gas pressure, or elastic recoil. two types. Water was routinely monitored by the This paper presents evidence that neither the Winkler analysis for dissolved oxygen. model of KROGH (1911) which depicts the tracheal sacs as ballast tanks nor the model evoking gas secretion are appropriate. The expansion of the Histology tracheal sac is attributed to changes in plasticity Larvae were fixed in glutaraldehyde (6% with of the sac wall coupled with an extending force. 0.08 M cacodylate buffer at pH 7.4 and 2% sucrose) This force may be generated by extrinsic muscles, and postfixed in 1% osmium tetroxide (in cacodygas pressure, or by an intimal component. late containing 4% sucrose). Specimens were inblock stained in saturated aqueous uranyl acetate MATERIALS AND METHODS for 2 hr at room temperature, dehydrated and, embedded in Araldite. Sections were double stained, Manipulation of sac properties first in a freshly prepared mixture of methanol and Experimental techniques for weighting, suspen70% ethanol (1 : 1) saturated with uranyl acetate sion, and sac volume measurement have already for 10 min at 60°C (LOCKE and KRISHNAN, 1971), been described in TEF~AGUCHI (1975). and then in lead citrate for 5 min at room temperaThe resistance of the sac wall to a net force ture (LOCKE and COLLINS, 1965). exerted by its gaseous contents was studied by The attempt to visualize possible endogenous exposing larvae to decrements in pressure applied peroxidase activity involved the oxidation of dito a glass tube filled with water and sealed by aminobenzidine (DAB) in the presence of H,O, as plastic membranes. Larvae were placed in tubes in the procedure for plant peroxidase used as a similar to those used for all measurements, and the tracer (LOCKE and COLLINS, 1968). Control tissues dimensions were drawn by camera lucida before, were incubated without H,Oz. during, and after exposure to a vacuum. The presUnfixed material was treated with a mixture of sure decrement was monitored with a mercury toluidine blue and fast green (each 5 mg per 100 ml manometer. of 0.05 M pH 7 phosphate buffer) in an attempt to detect the presence of resilin or resilin-like proteins Mass spectrometry (ANDERSENand WEIS-FOGH, 1964). Sacs from two larvae (eight sacs) were sealed in Statistics a glass capillary tube filled with helium. The total volume held by the sample capillary tubes was 8 Unless otherwise specified, data are presented as to 9 /-Al and of this about 0.24 ~1 was sac gas. Saca mean plus or minus the standard error of the gas samples were examined with an AEI M9 double mean, with the number of observations from which focusing mass spectrometer. Most scans were done the mean was calculated indicated in brackets. at a resolution of 1000 to 1500 ppm, although the peaks at mass numbers 44, 40, 32, and 28 were examined for heterogeneity at 4000 ppm. The RESULTS sample was introduced into the source with a Characteristics of the response direct probe modified aa described in TERACUCHI (1972), in which details of the scanning procedure Time course. Sac expansion in larvae weighted to are also given. give maximum rates of adjustment (T~XRAGUCHI, Data are expressed as sample peak height minus 1975) is essentially completed during the thirty background peak height relative to the sum of these minutes immediately following application of the increased peak heights at mass numbers of 44, 40, weight (Fig. 1). Sacs return to their original dimen32, and 28. The data reflect changes in the relative sions following removal of the weight (Fig. 2).
Correction
of negative buoyancy
in Chaoborus amevicanus
1661
Physical changes. Sac dimensions change entirely in the lengthwise direction; or at least no changes in other dimensions could be detected under a magnification of x 80. The band widths (see Sac anatomy section) nearer the sac tips increase relatively more than those nearer the sac midpoint (Fig. 3).
I “6
I
I
I
6
I
I
I
4 DISTANCE
I
2
r
I
0
I
If 2
FROM CENTRAL
II 4
1 6
I1
I 8
POINT
Fig. 3. Changes in band width in larvae bearing weight. Graph of number of bands per unit length vs. distance from central point. Number of bands per unit length was mapped in viva along central line between arrows as shown in diagram. Weight was added at 0 min. 3, Number of bands per unit length at t = 0; 0, number at t = 30; 0, theoretical number resulting from a relative decrease to 70 per cent of the initial. ---,Sacsizeatt=O; ----,t=30.
TIME
(MINUTES)
Fig. 1. Changes in sac area in larvae bearing weight. Air-equilibrated larvae in air-equilibrated water. Weight was added at 0 min. - - - -, Larvae free in water; , suspended larvae. 130
2
r
WEIGHT] 90
0I
II
I 2 TIME
Fig. 2. Changes of gravity. Air applied at 0 hr changes of right changes in right
” ,,
, 22
I 23
I 24
(HOURS)
in sac area in larvae weighted at centre equilibrated larvae and water. Weight and removed at 1 hr. -, Averaged posterior and right anterior sacs; . - . -, posterior sac; - - - -, right anterior sac.
If the net force exerted by the gas enclosed by the sac is suddenly increased by a decrease in hydrostatic pressure, the resultant increase in sac size should be inversely related to the rigidity of the enclosing sac wall, provided that no gas is allowed to escape. When free larvae which have been required to carry weights for various lengths
of time are subjected to instantaneous decrements in pressure, the sacs expand more when the larvae have carried weights for 15 min than when larvae have carried weights for shorter or longer periods. In other words, the same net force exerted by the instantaneous decrement in pressure (about 40 cm Hg) is able to instantaneously expand the sac to a greater extent 15 min after weighting than it can at other times during the process of adjustment. The resistance to expansion (rigidity) seen before weighting (0 min) is regained by 1 hr after weighting (Fig. 4). Suspended larvae do not show this decrease in rigidity but in fact show an increase in rigidity at 15 and 30 min. Upon the release of the vacuum, the sacs immediately resume the dimensions present before the exposure to the pressure decrement. The sac well is therefore very elastic in its properties, as well as exhibiting the transient increase in plasticity during adjustment to added weight.
Independent adjustment of anterior and posterior sacs. If larvae are weighted at the first abdominal segment so that they remain horizontally placed in the water, the anterior and posterior sacs expand as a single set (Fig. 2). If, however, larvae are weighted asymmetrically (at the cervix or at the eighth abdominal segment) rather than symmetrically (at the first abdominal segment) the response by the two pairs of sacs is also asymmetrical. While the initial fast response reveals less of this asymmetry, the response by 48 hr is decidedly asymmetrical (Fig. 5). The initial response involves increases in both anterior and posterior sets of sacs, whether the weight is cervical or abdominal. Asymmetry involving increases in one set but decreases in the other set develops after the initial time period of 1 hr.
1662
S. TERAGUCHI
Test of Krogh hypothesis If the sac is a semi-rigid container from which a liquid is removed in order to allow a quantity of gas to expand to an appropriate volume, then the external dimensions of the sacs should not increase during compensation for added weight. The increase in gas volume necessary for compensation for weights of 0.05 to 0.055 mg by larvae of 5.0 to 5.5 mg can be calculated (TERAGUCHI, 1972). These calculations predict that the gas space must increase by between 18 and 22 per cent. Since the sac area of larvae weighted according to the above specificationf increases by 21 per cent (Fig. 2), the removal OS fluid ballast from the interior cannot be the means by which neutral buoyancy is regained.
0
7
5
I5
30
I 4s
I 60
TIME V~INUTES)
Fig. 4. Changes in sac rigidity during compensation for added weight. Weight added at 0 min. Degree of instantaneous expansion upon sudden exposure to reduced pressure (minus 40 cm Hg). - - - -, Larvae free in water; -, larvae suspended.
$__-_+“+ /’
zf
/t
TIME lHOURS1
Fig. 5. position. cervix or 0 hr and terior
Response to displacement from horizontal Changes in sac area in larvae weighted at eighth abdominal segment. Weight applied at removed at 48 hr. -, Changes in right ansac; - - - -, changes in right posterior sac.
Table
1.
Sac-gas
composition
Test of gas-secretion hypothesis If the control of sac volume is exerted by a mechanism which directly acts upon the gas added, then the gas added should be largely a single gas of some biological activity and the concentration of this gas should rise relative to the other components of the gas mixture in the sac during adjustment to a weight. If, on the other hand, the gas entering the sac is of the same composition as the mixture which is dissolved in the surrounding tissues and if it enters by physical forces rather than directly as a result of a metabolic process, then the changes in the composition of the sac contents should reflect the dissolved gases in the environment even when the gas in the environment is largely an inert gas not known to be handled by metabolic processes. The composition of sac gas does reflect the composition of that dissolved in the water (Table 1; also KROGH, 1911). When larvae are transferred from water saturated with air to water saturated with nitrogen, the relative peak height of nitrogen rises from 82 to 87% and that of oxygen falls from 13 to 7% (Table 1). When larvae are transferred from water saturated with air to water saturated with oxygen, the relative peak height of oxygen rises from 13 to 36% and that of nitrogen falls from 82 to 55% (Table 1). Carbon dioxide rises in both
of larvae equilibrated with water saturated Analysis by mass spectrometry
with various gases.
SAC GAS
NITROGEN
OXYGEN
(28)
(32)
ARMN (40)
DIOXIOE (44)
SAT!lsATING
GAS
DISSOLVED OXYGEN
IN WATER cdl>
REIATmE PEAX HEIGHTS (percentages)
82.0 c 0.4 ( 9)
13.0 + 1.0 ( 9)
1.0 + 0.2 ( 9)
3.0 f 0.5 ( 9)
87.0 i 2.0 ( 7)
7.0 i 1.0 ( 7)
0.9 i 0.1 ( 7)
5.0 f 0.1 ( 9)
nitrogen
36.0 t 4.0 (14) 1.0 t 0.1 (14)
7.0 f 1.0 (14)
oxygen
55.0 -t4.0 (14)
air
7.17 + 0.63 ( 2) 1.33 r 0.22 (24) 38.44 + 1.73 ( 5)
Correction of negative buoyancy in Chaoborus americanus transfers, but this event is probably the result of increased larval activity upon transfer. Sac gas from both expanding and static sacs was examined for changes in the relative amounts of nitrogen, carbon monoxide, oxygen, argon, and carbon dioxide. Entrance of any of the gases would increase its proportion in sac gas. Argon is used here as a tracer gas, since it should not be handled by any known metabolic process. Therefore, any increase in the proportion of argon in sac gas can be attributed to diffusion. Larvae weighing 5.0 to 5.5 mg contain about 0.12 ~1 of gas. The composition of sac gas from larvae held in air-water recorded as peak heights (expressed as percentage of sum of these peak heights) on a mass spectrograph is 82 : 13 : 1 : 3 for peaks at mass numbers of 28 : 32 : 40 : 44. The addition of about 0.03 ~1 of gas is required to compensate for a weight of 0.05 to 0.055 mg. If the gas in the sacs is initially ‘air’ (which gives relative peak height of 82 : 13 : 1 : 3 for Na-OS-A-CO, on the mass spectrometer), then the relative peak heights that would be approached during adjustment if the adjustment results from the addition of 0.03 ~1 of either air, argon, oxygen, carbon dioxide nitrogen, or carbon monoxide are shown in Table 2. In general, the sac should become enriched in the entering gas but diluted in all other components. Outward diffusion was not taken into account. Table 3 gives the changes in sac-gas composition of larvae which were first weighted and then either suspended or left free in water saturated with air. At 1.5 min the relative peak heights are 83 : 10 : 1 : 5 for free animals and 81 : 11 : 2 : 6 for suspended animals. The ratios obtained can be accounted for by assuming that 0.03 ~1 of a gas mixture similar to air entered the sacs during adjustment, and that
1663
Table 2. Predicted sac-gas composition resulting from the entrance of 0.03 ~1 of various gases into air-filledsacs MASS WMXR
--I14.0
1.0
66.1
10.5
20.9
66.1
30.6
0.8
66.1
10.5
0.8
22.5
86.3
I I 10.5
0.8
2.4
= MASS Nln43P.R
xIITROGEN (28)
LARVA
t free free suspended free suspended free suspended free suspended
OXYGEN (32)
ARGON (40)
CARBON DIOXIDE (40)
RCLATIVE PEAK HEIGHT As % OF SCM OF TAESE PEAK HEIGHTS
!
2.4 2.4
this mixture was somewhat enriched in CO2 and depleted of Oa due to the activity of the animal. This idea is supported by the similarity between the ratios for free and suspended animals, since although only free animals increase sac volume (Fig. l), both sets of animals have been subjected to similar manipulations. Figs. 6 to 9 give the changes in sac-gas composition of larvae held for adjustment in water saturated Larvae with argon (0, = 0.69 + 0.08 (10) mg/l.). were held in air-water until the application of the weight but were then transferred to argon-water for adjustment. Under these conditions, the responses as revealed by an increase in sac area reach a maximum at 15 min (Fig. 10). Since the relative proportion of mass 28 (nitrogen, Fig. 6) in the sac-gas decreases markedly during the initial 15 min interval, the gas entering cannot be largely nitrogen. The decrease from 82 to 52% in the proportion of nitrogen in static sacs (Fig. 6,
Table 3. Changes in sac-gas composition in larvae bearing weight. Air-equilibrated larvae in air equilibrated water. Analysis by mass spectrometry
TIME
3.0
81.9
82 + 0.4 ( 9)
13 r l( 9)
1 i 0.2( 9)
3 c 0.5( 9)
83 k 1
( 8)
10 e l( 8)
lf5
551
81 * I.
( 7)
11 f I( 7)
2 t 0.5( 7)
6+1
(7)
82 f 1
( 9)
11 i l( 9)
1 f 0.2( 9)
5Cl
(9) (7)
(8)
(8)
80 t 1
( 7)
13 2 1( 7)
2 t 0.2( 7)
5t1
83 t 2
( 8)
11 f 2( 8)
2 f 0.2( 8)
4 + 0.4( 8)
815
1
( 7)
12 f 1( 7)
2 f 0.2( 7)
5*1
(7)
80 * 1
(10)
13 +_l(l0)
2 k O.l(lO)
5 f 1
(10)
82 i 0.4 ( 6)
13 k 1( 6)
1 t 0.2( 6)
3 f:0.5( 6)
1664
S. TERACUCHI
16
6 0 80 -
II-7
20--I TIME
(MINUTES)
Fig. 6. Changes in amount of nitrogen in sac-gas relative to the total amount of nitrogen, oxygen, argon, and carbon dioxide. Larvae bearing weight. Weight added at 0 min. Air-equilibrated larvae in argonequilibrated water. - - - -, Larvae free in water; -, larvae suspended.
TIME IMINUTES)
Fig. 8. Changes in the amount of argon in sac-gas relative to the total amount of nitrogen, oxygen, argon, and carbon dioxide. Larvae bearing weight. Weight added at 0 min. Air-equilibrated larvae in argonequilibrated water. - - - -, Larvae free in water;--, larvae suspended.
TIME
WINUTES)
Fig. 7. Changes in the amount of oxygen in sac-gas relative to the total amount of nitrogen, oxygen, argon, and carbon dioxide. Larvae bearing weight. Weight added at 0 min. Air-equilibrated larvae in argonequilibrated water. - - - -, Larvae free in water; -, larvae suspended.
TIME (MINUTES) Fig. 9. Changes in the amount of carbon dioxide in sacgas relative to the total amount of nitrogen, oxygen, argon, and carbon dioxide. Larvae bearing weight. Weight added at 0 min. Air-equilibrated larvae in argonequilibrated water. - - - -, Larvae free in water ;larvae suspended.
Correction of negative buoyancy in Chaoborus americanus
/’
010
TIME (MINUTES)
Fig. 10. Changes in sac area in larvae bearing weight. Air-equilibrated larvae in argon-equilibrated water. Weight added at 0 min. - - - -, Larvae free in water; -, larvae suspended. suspended larvae) can probably be attributed to simple diffusion. If the initial sac-gas composition is expressed as 82% nitrogen and 18% other than nitrogen, and if the change in the sac-gas composition due to diffusion seen in static sacs also occurs in free animals, then without entrance of the additional volume of gas the composition at 1.5 min would be 52 o/0nitrogen and 48 ok other than nitrogen. If, in addition, a volume of something other than nitrogen equal to 13 o/o(Fig. 10) of the initial sac volume enters, the proportion of nitrogen will be further reduced to .52/113, or 46%. The proportion of nitrogen observed at 1.5 min is 44%, which suggests that no nitrogen enters the sac as a direct consequence of adjustment in argon water. Similar arguments lead to similar conclusions for oxygen (Fig. 7). Since the relative proportion of mass 40 (argon, Fig. 8) in the sac gas increases markedly during the initial 15 min interval, the gas entering may be largely argon. The increase from 1 to 3 1 y. in the proportion of argon in static sacs (Fig. 8, suspended larvae) can probably be attributed to simple diffusion. If the initial sac-gas composition is expressed as 1 ‘$6 argon and 99% other than argon, and if the change in the sac-gas composition due to simple diffusion seen in static sacs also occurs in free animals, then without entrance of the additional volume of gas the composition, at 15 min, would be 3 1 y. argon and 69yo other than argon. If, in addition, a volume of argon equal to 13% (Fig. 10) of the initial sac volume enters, the proportion of argon will be further increased to 44/113, or 39%. The proportion of argon observed is 4376, which suggests that in argon-water, argon is the gas that enters the sacs as a direct consequence of the adjustment process. The dip in the curve for suspended larvae and the plateau in the curve for free larvae between 15 and 45 min cannot be satisfactorily explained at this
1665
time, but it should be noted that a decrease in the sac volume of free larvae occurs during this same time interval (Fig. 10). Since the relative proportion of mass 44 (carbon dioxide, Fig. 9) increases during the initial 15 min interval, the entrance of carbon dioxide can be considered. The increase in the proportion of carbon dioxide in static sacs (Fig. 9, suspended larvae) can probably be attributed to simple diffusion, Since the increase in the proportion of carbon dioxide in expanding sacs (Fig. 9, free larvae) is identical at 15 min to that seen in static sacs, it is unlikely that carbon dioxide enters the sac as a direct consequence of the processes accomplishing adjustment. It is much more likely that the carbon dioxide enrichment is a consequence of increased larval activity, since both free and suspended animals were subjected to similar manipulations. Sac anatomy Light microscopy. Under phase contrast, the sac wall consists of thin, dark hoops (dark bands) separated by wider light bands (Fig. 11). The light bands (non-bulbed bands) become sky-blue when stained with toluidine blue-fast green (fresh tissue) or methylene blue (Araldite sections) and purple when stained with haematoxylin and eosin (BARDENFLETH and EGE, 1916). Toluidine blue does not react evenly over the entire sac; the midportion becomes intensely blue while the tips become only faintly blue. The dark bands (bulbed bands) become turquoise when stained with methylene blue (Araldite sections) and pink when stained with haematoxylin and eosin (BARDENFLETHand EGE, 1916). Since resilin stains with cations and basic dyes, the reactions to methylene blue, haematoxylin, eosin, and toluidine blue-fast green suggest that the dark bands (bulbed bands) do not contain resilin. The outside surface is formed by two layers of cells, an outer pigment cell layer and an inner tracheal cell layer. Narrow slips of muscle run from the sac wall to the body wall (Fig. 11). Electron microscopy. The intima is composed of alternating bulbed (dark) bands and non-bulbed (light) bands arranged perpendicularly to the axis of expansion. Folds in the cuticulin layer parallel both the bulbed and non-bulbed bands (Figs. 12, 13). Fibrils in both the non-bulbed and bulbed bands are preferentially oriented 90” with respect to the axis of expansion but those in the non-bulbed bands run vertically through the band while those in the bulbed bands run longitudinally through the band (Fig. 14). The gap between the heads of the bulbed bands may be wide or narrow. The weakness of the reaction of toluidine blue near the sac tips can be attributed to the decrease in the width of the non-bulbed bands relative to that of the stems of the bulbed bands. Coincident with the decrease in non-bulbed band width towards the
S. TERAGUCHI
1666
sac tip, is a decrease in bulbed band head width and a narrowing and thickening of the non-bulbed band pads (Figs. 12, 13). The sac was reacted with DAB with and without peroxide since peroxidases may be involved in the cross-linking of resilin (LOCKE, 1969). No part of the intima gave a positive reaction (Figs. 12, 13). The structure of the tracheal cell layer is not characteristic of a structure specialized for movement or of a structure specialized for transport. Few microtubules or filaments are present (Fig. 15) although in areas near muscle attachment (Fig. 16) or near tracheal cell-to-cell attachments, greater numbers of microtubules may be present. Neither the number nor the distribution of mitochondria suggest specialization by the tracheal cells for transport (Fig. 15). The cytoplasm is, however, rich in free ribosomes. The significance of this is not obvious. Perhaps these cells are involved in the manufacture of materials for intermoult sac growth. The attachment of the muscle slips to the sac wall differs from that at the body wall. The external attachments are accompanied by extensive systems of microtubules, interfolding of muscle-cell and and electron-dense epidermal-cell membranes, material along the region of contact. At the sac, a basement membrane of about 0.35 pm separates the muscle cell from the adjoining pigment cell (Fig. 17) or tracheal cell (Fig. 18) and accompanies deep infoldings of the muscle-cell membrane. Structures which are possibly neuromuscular junctions are sometimes seen at the muscle cell-tracheal cell interface (Fig. 18). Vesicles are present on the ‘axonal’ side of the areas of ‘synaptic’ contact. DISCUSSION Choborus larvae correct a condition of negative buoyancy by increasing the volume of gas held within the body in two pairs of tracheal sacs. The rapidity of the compensation for added weight and the independence between sacs during the compensation for added weight and the independence between sacs during the compensation for displacement from horizontal position suggest that sac volume may be under nervous control. Motor terminals are present at the muscle-tracheal cell interface. Since sac expansion occurs more rapidly than sac contraction and since asymmetrical responses to cervical or abdominal weights appear only after an initial symmetrical response, the compensatory process may occur in two phases: an initial fast phase and a subsequent ‘growth-like’ phase. Test of Krogh hypothesis The tracheal sacs do not function as ballast tanks from which a quantity of fluid is removed to increase
gas space. This hypothesis is refuted by two pieces of evidence. First, no fluid is seen inside the sacs and, second, sacs increase in size in response to added weight and in response to light fluids (DAMANT, 1924). Test of gas-secretion hypothesis A quantity of gas enters the sacs during the reduction of density, but the evidence does not support the hypothesis that this gas is produced by a metabolic process. Any gas (even an inert gas) present in the surrounding fluids enters probably because the force is provided by diffusion gradients. These gradients would be set up or magnified when the gas space is enlarged by action of the sac wall, since the enlargement would reduce the partial pressures of the confined gas mixture. The above scheme requires that the sac wall be readily permeated by available dissolved gases, and sac-gas composition does reflect dissolved gas composition. In addition, DAMANT (1924) observes buoyancy oscillation when larvae are transferred between waters containing various gases and concludes that sac walls are differentially permeable to the various gases. The hypothesis for gas secretion is also refuted by the results from mass spectrometric analysis of sac-gas composition during adjustment for weight. Sac-gas composition does not change when airequilibrated larvae adjust in air-equilibrated water. The expected enrichment of a single component of the mixture in larvae with expanding sacs is not seen. On the other hand, the entrance of air into an air-filled sac should not alter the component ratios. Sac-gas composition does change when airequilibrated larvae are transferred to argon-equilibrated water. The sac gas becomes enriched in argon and carbon dioxide and depleted of oxygen and nitrogen. The enrichment in argon and depletion of oxygen and nitrogen is magnified by concurrent sac expansion (induced by weighting). Argon is an inert gas, and it should not be handled by any known metabolic process. The enhanced enrichment (as compared with the control in which sac volume did not increase) can be accounted for by the hypothesis which attributes the entrance of gas to the physical force of diffusion. The increase in gas space resulting from sac-wall extension would decrease the partial pressure due to any amount of argon which enters. The diffusion of argon into an expanding sac should therefore take place more rapidly than into a static sac. In addition, rapidly eouilibrated gas in the capillary tracheae attached to the sac tips might be drawn into the main sac during the expansion. The gas-secretion hypothesis does not provide for these data. These data are consistent with an alternate hypothesis which attributes the entrance
1667
Fig. 11. Drawing of excised right anterior sac as seen with the phase-contrast microscope. 1 to V, Slips of tissue, probably muscular; VI, enlarged view of tissue seen at I; VII to VIII, capillar? tracheae; arrow, outlet of sac; arrowheads, dark bands; space between arrowheads, light band. Fig.
12.
Cross-section of intima reacted with DAB but otherwise 0, opened fold; C, closed fold; Bb, bulbed band; P, pad;
Fig.
13.
Cross-section Non-bulbed
unstained. Without peroxide. Nb, non-bulbed band.
of intima reacted with DAB but otherwise unstained. With band; P, pad; arrow, fold over non-bulbed band; Bb, bulbed
peroxide. band.
Nb,
Fig. 14. Intima of sac near inner surface showing the fibrils of the bulbed-band in cross-section. Large dark fibrils are embedded in matrix which is in turn divided into honeycomb-like ‘cells’. Nb, Non-bulbed band; Bb, bulbed band.
1668
Fig. 15. Typical tracheal cell from layer covering Tracheal-cell mitochondrion; mt, microtubule; Fig.
16.
Tracheal
cell and inner
Fig.
17.
Junction
of muscle
Fig.
18.
Junction
of muscle chondrion;
sac. Cytoplasm tn. tracheal-cell
portion of intima near tubules; bh, bulbous
region head.
to sac bvith intervening pigment membrane; m, muscle. cell to tracheal cell showing t, tracheal cell ; NV, nerve:
is rich in free ribosomes. tm, nucleus; bh, bulbous head.
of muscle
cell.
attachment.
p, Pigment
neuromuscular junction. arrow, synaptic contact.
cell;
mt,
Bm,
,\Iicro-
basement
iX1, muscle
Xlito-
Correction of negative buoyancy in Ckaoborus americanus of gas to physical forces developed secondarily to an
initial action of the sac wall. The physical properties of the sac wall are not static and do in fact change during the adjustment to weight. In particular, there is a transient increase in the plasticity of the sac wall during adjustment. The sac wall is complex in structure. The intima is composed of several kinds of cuticle and the cellular coat is composed of two types of cells plus associated muscular and nervous tissue. The sac wall was examined for the structural basis of four sac properties: (1) the limitation of the direction of expansion ; (2) the regions of prospective expansion ; (3) the possible existence of two phases of expansion; (4) the development of expanding forces. The one-dimensional course of expansion would seem to have a multiple basis. The repeating units of ‘bulbed band-non-bulbed band’ are assembled perpendicularly to the axis of extension as are certain subunits of the bands. For example, the cuticulin layer (which is thought to limit intermoult expansion (BENNET-CLARK, 1963)) is preferentially folded perpendicularly to the axis of extension and the fibrils in the bulbed band are arranged in this same direction. Structural analysis does not localize the site of expansion in any one band. There are two lines of evidence which suggest that expansion during fast adjustment occurs mainly in the bulbed bands. First, deep folds or bulges in the cuticulin overly the stem portion. Second, the relative increase in the width of non-bulbed band-bulb stem pairs during sac expansion is greater near the sac tip than it is near the midsection. The contribution to the total width of the composite band by the two components is distinctly different in the middle than it is near the tip. The composition is about 50 per cent non-bulbed and 50 per cent bulb stem near the tip while near the middle the bulb stem composes more like 25 per cent of the total width. If expansion during fast adjustment is restricted to the bulbed bands and if all bulbed material swells to the same degree, the relative increase in width of each composite band would be greater near the tips. The evidence that associates expansion with the non-bulbed bands would suggest that at least the ‘growth-like’ step occurs here. Sacs grow between moults by increasing the width of the non-bulbed bands (FRANKENBERG, 1915). The sacs of very young fourth instar larvae resemble the tip regions of mature sacs in that the width of the non-bulbed bands is more nearly the same as that of the bulb step in most regions. In a mature larva, the differences between the wall near the tip and the wall nearer the middle suggest that material has been added to both the non-bulbed bands and the heads of the bulbed bands but not to the stems of the bulbed bands. Other aspects of structure which permit the postulation of expansion of non-bulbed bands are the appropriate wrinkling of the overlying
1669
cuticulin, the inverse correlation between the depth and width of the underlying pad, and the location of the inter-bulb-head gaps beneath the non-bulbed bands. In addition, while there is no evidence that resilin is a component of the bulbed bands, the possibility that this elastic protein is present in the non-bulbed bands has not yet been eliminated. The non-bulbed bands react with toluidine blue as would be expected of resilin. Physiological evidence suggests that the adjustment process may occur in two phases: a fast phase and a slow phase. This evidence is as follows: First, larvae which have carried weights for less than 24 hr can as rapidly return sacs to their original dimensions. However, if weights are left on for more than 48 hr or if floats of polyethylene are added to neutrally buoyant larvae, reduction in sac size is hardly detectable at 1 hr and takes 24 to 48 hr to complete. Second, asymmetry in sac response appears only subsequent to an initial symmetrical response. It is possible that the ambiguous result of the attempt to restrict the response to a simple component stems from an existence of a two-phase response since the sites of the two phases might differ. It can therefore be postulated that sac expansion can proceed through one or two phases depending on the persistence of the perturbation, the first phase being a fast, easily reversible step and the second being a ‘growth-like’ step in which material is added to the non-bulbed bands and/or the heads of the bulbed bands. In other systems involving cuticle expansion, although the cuticle itself participates dynamically in that it becomes less rigid, the actual force is provided by an extrinsic element. Neither the tracheal cell layer nor the pigment cell layer have the qualities of cells which would be expected to develop much force. Muscles (with innervation near to the sac) are present. However, the attachments to the sac are unlike those usually associated with muscles developing much force and the whole structure looks much like the stretch receptors described for the moth Anthereu (FINLAYSON and LOWENSTEIN, 1958). The muscle slips are probably the ‘tractus’ of POUCHET (1872) who suggested that they are involved in sac rotation or sac contraction. It is therefore possible that the force necessary for expansion is generated by an intimal component and that the muscular tissue associated with the sac wall serves some other function. An analysis of the material exchanges between the intima and its associated cells remains as a subject for further study. Acknowledgements-I thank Dr. L. M. PA~SANO for encouragement and assistance, Dr. H. SCHNOESfor his supervision of the mass spectrometric analyses, and Dr. M. LOCKEfor assistance with the electron microscopy. The work was supported by the Zoology Department of the University of Wisconsin and by the Biology Department of Case Western Reserve University.
1670
S. TERACUCHI REFERENCES
AKEHURST J. (1922) Larva of Chaoborus crystallinus (De Geer) (C. plumicornis F.). r. R. microsc. Sot. 15, 341-372. ANDERSEN G. 0. and WEIS-FOGH T. (1964) Resilin. A rubberlike protein in arthropod cuticle. &v. Insect Physiol. 2, l-65. BARDENFLETHD. S. and EGE R. (1916) On the anatomy and physiology of the air-sacs of the larva of Corethra plumicornis. Vidensk. Medr dansk naturh. Foren. 67, 25-42. BENNET-CLARK H. C. (1962) Active control of the mechanical properties of the insect endocuticle. J. Insect Physiol. 8, 627-633. BENNET-CLARK H. C. (1963) The relationship between epicuticular folding and subsequent size of an insect. 3. Insect Physiol. 9, 43-46. CHRISTENSENP. J. H. (1928) Bidrag til Kendskabet om Corethralarvens hydrostatiske Mekanisme. Vidensk. Medr dansk naturh. Foren. 86, 21-48. CICAK A., MCLAUGHLIN J. J. A., and WITTENBERGJ. B. (1963) Oxygen in the gas vacuole of rhizopod protozoan Arcella. Nature, Lond. 199, 983-985. COTTRELLC. B. (1962) The imaginal ecdysis of blowflies. Evidence for a change in the mechanical properties of the cuticle at expansion. 3. exp. Biol. 39, 449-458. DAMANTG. C. C. (1924) The adjustment of the buoyancy of the larva of Corethra plumicornis. 3. Physiol., Land. 59, 345-356. DUHR B. (1955) tiber Bewegung, Orientierung und Beutefang der Corethra larvae (Chaoborus crystallinus De Geer). Zool. Jb. (Physiol.) 65, 378-429. FINLAYSON L. H. and LOWENSTEIN 0. (1958) The structure and function of abdominal stretch receptors in insects. Proc. R. Sot. Lond. (B) 148, 433-449. FRANKENBERG B. (1915) Die Schwimmblasen von Corethra. Zool. Jb. (Physiol.) 35, 505-592. KEISTER M. L. and BUCK J. B. (1949) Tracheal filling in Sciara larvae. Biol. Bull., Woods Hole 97, 323-330. KROGH A. (1911) On the hydrostatic mechanism of the Corethra larva with an account of methods of microscopical gas analysis. Skand. Arch. Physiol. 25, 183-203.
LARIMER J. L. and ASHBY E. A. (1962) Float gases, gas secretion and tissue respiration in Portuguese man-ofwar, Physalia. 3. cell. camp. Physiol. 60, 41-97. LOCKE M. (1969) The localization of a peroxidase associated with hard cuticle formation in an insect, Calpodes ethlius Stoll, Lepidoptera, Hesperiidae. Tissue &f Cell 1, 555-574. LOCKE M. and COLLINS J. V. (1965) The structure and formation of protein granules in the fat body of an insect. 3. Cell Biol. 26, 857-884. LOCKE M. and COLLINS J. V. (1968) Protein uptake into multivesicular bodies and storage granules in the fat body of an insect. 3. Cell Biol. 36, 453-483. LOCKE M. and KRISHNAN N. (1971) Hot alcoholic phosphotungstic acid and uranyl acetate as routine stains for thick and thin sections. 3. Cell Biol. 50, 550-557. MADDRELL S. H. P. (1966) Nervous control of the mechanical properties of abdominal wall at feeding in Rhodnius. 3. exp. Biol. 44, 59-68. NUGEZ J. A. (1963) Central nervous control of the mechanical properties of the cuticle in Rhodnius prolixus. Nature, Lond. 199, 621-622. POUCHETG. (1972) DBvCloppement du systeme trachten de l’Anoph&le (Corethra plumicornis). Archs 2001. exp. ge’n. 1, 217-232. SCHOLANDER P. F. (1956) Observations of the gas gland in living fish. 3. cell. camp. Physiol. 48, 423-528. TAKAHASHIK. (1966) Muscle physiology. In Physiology of Echinodermata (Ed. by BOOLOOTIANR.), Vol. 22, pp. 513-527. Wiley, New York. TERAGUCHI S. (1972) Regulation of buoyancy by Chaoborus americanus (Joh.). Ph.D. Dissertation, University of Wisconsin, Madison. TERAGUCHIS. (1975) The detection of negative buoyancy by the phantom larva Chaoborus americanus (Joh.). 3. Insect Physiol. 21, 1265-1269. WIGGLE~WORTH V. B. (1953) Surface forces in the trr;~;;; system of insects. Quart. 3. micr. Sci. 94,
1975, Vol. 21, pp. 1659 to 1670.
Pergamon
Press.
Printed
in Great Britain.
CORRECTION OF NEGATIVE BUOYANCY IN THE PHANTOM LARVA, CHAOBORUS AMERICANUS s. Department
TEFWXJCHI
of Zoology, University of Wisconsin, (Received
19 December
1974
*
Madison, Wisconsin 53706, U.S.A.
; revised 20 January
1975)
Abstract-Fourth instar larvae of Chaoborus americanus correct a condition of negative buoyancy by increasing the amount of gas held by two pairs of tracheal sacs. Mass spectrometric analysis of sac gas during the period following the addition of weights to larvae, reveal that the added gas is derived from the dissolved gases present in the surrounding medium and that it enters by physical means. The response is characterized by a transient decrease in the rigidity of the sac wall. Electron and light microscopy reveal that muscular tissue and motor terminals are associated with the sac wall and that a resilin-like protein may be present. These results suggest that the increase in the amount of gas held by the sacs is a secondary effect of an initial expansion of the sac wall itself and that the cuticular lining of the sacs is an active participant in the adjustment process.
INTRODUCTION CHAOBORUSlarvae exert control over density through gas-filled semi-rigid tracheal sacs (KROGH, 1911; FRANKENBERG,1915; DAMANT,1924; DIJHR, 195.5; TERAGUCHI, 1975). Hypotheses explaining the alteration of sac size by Chaoborus are of two kinds. One attributes changes to alteration of the sac contents, the other to alteration of the sac wall. Hypotheses implicating the sac contents propose either that liquid is pumped in and out or that a gas is secreted or excreted. Hypotheses implicating the sac wall suggest that either the properties of the sac wall are under direct control or that contraction of muscle, extrinsic or intrinsic to the sac wall, is involved.
KROGH (1911) has suggested that the sacs function as ballast tanks, Since processes involving fluid removal from a tracheal structure are well documented in insects (KEISTER and BUCK, 1949; WIGGLESWORTH,1953), the ability to move fluid across the tracheal wall could be a property of tracheal epithelia. De novo filling in Chaoborus can occur under pressure and without access to the surface (F RANKJJNBERG, 1915). However, Krogb’s hypothesis is rendered unlikely by the fact that fluid has not been seen in sacs after the first filling except under unusual conditions (KROGH, 1911; FRANKENBERG,1915; DAMANT,1924). Gas secretion has been implicated as the means by which neutral buoyancy may be recovered. FRANKENBERG(1915) observed that ligation of newly-hatched Chaoborus larvae prevented de nwo filling of more anterior tracheae provided that the * Present address: Department of Biology, Case Western Reserve University, and Cleveland Museum of Natural History, Cleveland, Ohio 44106, U.S.A.
interruption was forward of the eighth abdominal segment. This suggested a gas gland in the eighth abdominal segment (FRANKENBERG,1915 ; AKEHURST,1922). Gas produced by a metabolic process should have a single component, since examples so far elucidated follow this pattern (e.g. SCHOLANDER, 1956; LARIMER and ASHBY, 1962; CICAK et aE., 1963). Other components enter the gas space by diffusion, subsequent to initial secretion. KROGH (1911) could not detect any change in sac gas composition following adjustment to hydrostatic pressure. However, from 1 to 17 hr were given for adjustment, and since the sac gases rapidly equilibrate with gases dissolved in the medium (KROGH, 1911; DAMANT,1924), the expected enrichment of one of the components of sac gas by the addition of a single gas might have been obscured by subsequent diffusion. Hypotheses favouring control by direct action on the container rather than control by direct action on the contents have been common since FRANKENBERGin 1915 (BARDENFLETH and EGE, 1916 ; DAMANT, 1924; CHRISTENSEN, 1928; DUHR, 1955). The focus has been upon the properties of the intima lining the sacs. It is certainly true that most of the cuticle is not outside the metabolic pool of the insect and it may be that several of its intermoult properties are under direct physiological control. Volume and plasticity are two properties which can be considered. FRANKENBERG(1915) proposed that changes in sac dimensions resulted from changes in volume of a component of the wall. He thought these volume changes were dependent upon water movement since isolated sacs shrank when exposed to air, concentrated NaCl, or 95% ethyl alcohol, and swelled again when replaced in tap water.
1659
1660
S. TERAGUCHI
amounts of carbon dioxide, argon, oxygen, and A change in plasticity coupled with external force nitrogen. This reflection will be distorted by conis also a plausible mechanism for regulation of sac tamination with air during sampling and during size. FRANKENBERG(1915) found that sacs became analysis. Data are presented as relative peak heights more rigid soon after excision, although they realthough relative peak height is not equivalent to tained the ability to swell or shrink in response to percentage composition, since sensitivity to various various solutions. Control over plasticity of extragases differs. It was not necessary to convert data cellular material has been documented not only in insects but in echinoderms as well (BENNET-CLARK, to percentage composition, since conclusions are 1962; COTTRELL, 1962; NUREZ, 1963; MADDRELL, based upon a comparison between values obtained in an identical way. 1966; TAKAHASHI,1966). The changes in plasticity Samples were prepared and run in pairs, one do not in themselves effect changes in extension being from weighted animals and the other from but are accompanied by a stretching force provided weighted plus suspended animals, the two types by muscle contraction or elevated blood pressure. having been held in the same container. ComIn Chaoborus the force could be provided by muscle parisons were then made between groups of the contraction, gas pressure, or elastic recoil. two types. Water was routinely monitored by the This paper presents evidence that neither the Winkler analysis for dissolved oxygen. model of KROGH (1911) which depicts the tracheal sacs as ballast tanks nor the model evoking gas secretion are appropriate. The expansion of the Histology tracheal sac is attributed to changes in plasticity Larvae were fixed in glutaraldehyde (6% with of the sac wall coupled with an extending force. 0.08 M cacodylate buffer at pH 7.4 and 2% sucrose) This force may be generated by extrinsic muscles, and postfixed in 1% osmium tetroxide (in cacodygas pressure, or by an intimal component. late containing 4% sucrose). Specimens were inblock stained in saturated aqueous uranyl acetate MATERIALS AND METHODS for 2 hr at room temperature, dehydrated and, embedded in Araldite. Sections were double stained, Manipulation of sac properties first in a freshly prepared mixture of methanol and Experimental techniques for weighting, suspen70% ethanol (1 : 1) saturated with uranyl acetate sion, and sac volume measurement have already for 10 min at 60°C (LOCKE and KRISHNAN, 1971), been described in TEF~AGUCHI (1975). and then in lead citrate for 5 min at room temperaThe resistance of the sac wall to a net force ture (LOCKE and COLLINS, 1965). exerted by its gaseous contents was studied by The attempt to visualize possible endogenous exposing larvae to decrements in pressure applied peroxidase activity involved the oxidation of dito a glass tube filled with water and sealed by aminobenzidine (DAB) in the presence of H,O, as plastic membranes. Larvae were placed in tubes in the procedure for plant peroxidase used as a similar to those used for all measurements, and the tracer (LOCKE and COLLINS, 1968). Control tissues dimensions were drawn by camera lucida before, were incubated without H,Oz. during, and after exposure to a vacuum. The presUnfixed material was treated with a mixture of sure decrement was monitored with a mercury toluidine blue and fast green (each 5 mg per 100 ml manometer. of 0.05 M pH 7 phosphate buffer) in an attempt to detect the presence of resilin or resilin-like proteins Mass spectrometry (ANDERSENand WEIS-FOGH, 1964). Sacs from two larvae (eight sacs) were sealed in Statistics a glass capillary tube filled with helium. The total volume held by the sample capillary tubes was 8 Unless otherwise specified, data are presented as to 9 /-Al and of this about 0.24 ~1 was sac gas. Saca mean plus or minus the standard error of the gas samples were examined with an AEI M9 double mean, with the number of observations from which focusing mass spectrometer. Most scans were done the mean was calculated indicated in brackets. at a resolution of 1000 to 1500 ppm, although the peaks at mass numbers 44, 40, 32, and 28 were examined for heterogeneity at 4000 ppm. The RESULTS sample was introduced into the source with a Characteristics of the response direct probe modified aa described in TERACUCHI (1972), in which details of the scanning procedure Time course. Sac expansion in larvae weighted to are also given. give maximum rates of adjustment (T~XRAGUCHI, Data are expressed as sample peak height minus 1975) is essentially completed during the thirty background peak height relative to the sum of these minutes immediately following application of the increased peak heights at mass numbers of 44, 40, weight (Fig. 1). Sacs return to their original dimen32, and 28. The data reflect changes in the relative sions following removal of the weight (Fig. 2).
Correction
of negative buoyancy
in Chaoborus amevicanus
1661
Physical changes. Sac dimensions change entirely in the lengthwise direction; or at least no changes in other dimensions could be detected under a magnification of x 80. The band widths (see Sac anatomy section) nearer the sac tips increase relatively more than those nearer the sac midpoint (Fig. 3).
I “6
I
I
I
6
I
I
I
4 DISTANCE
I
2
r
I
0
I
If 2
FROM CENTRAL
II 4
1 6
I1
I 8
POINT
Fig. 3. Changes in band width in larvae bearing weight. Graph of number of bands per unit length vs. distance from central point. Number of bands per unit length was mapped in viva along central line between arrows as shown in diagram. Weight was added at 0 min. 3, Number of bands per unit length at t = 0; 0, number at t = 30; 0, theoretical number resulting from a relative decrease to 70 per cent of the initial. ---,Sacsizeatt=O; ----,t=30.
TIME
(MINUTES)
Fig. 1. Changes in sac area in larvae bearing weight. Air-equilibrated larvae in air-equilibrated water. Weight was added at 0 min. - - - -, Larvae free in water; , suspended larvae. 130
2
r
WEIGHT] 90
0I
II
I 2 TIME
Fig. 2. Changes of gravity. Air applied at 0 hr changes of right changes in right
” ,,
, 22
I 23
I 24
(HOURS)
in sac area in larvae weighted at centre equilibrated larvae and water. Weight and removed at 1 hr. -, Averaged posterior and right anterior sacs; . - . -, posterior sac; - - - -, right anterior sac.
If the net force exerted by the gas enclosed by the sac is suddenly increased by a decrease in hydrostatic pressure, the resultant increase in sac size should be inversely related to the rigidity of the enclosing sac wall, provided that no gas is allowed to escape. When free larvae which have been required to carry weights for various lengths
of time are subjected to instantaneous decrements in pressure, the sacs expand more when the larvae have carried weights for 15 min than when larvae have carried weights for shorter or longer periods. In other words, the same net force exerted by the instantaneous decrement in pressure (about 40 cm Hg) is able to instantaneously expand the sac to a greater extent 15 min after weighting than it can at other times during the process of adjustment. The resistance to expansion (rigidity) seen before weighting (0 min) is regained by 1 hr after weighting (Fig. 4). Suspended larvae do not show this decrease in rigidity but in fact show an increase in rigidity at 15 and 30 min. Upon the release of the vacuum, the sacs immediately resume the dimensions present before the exposure to the pressure decrement. The sac well is therefore very elastic in its properties, as well as exhibiting the transient increase in plasticity during adjustment to added weight.
Independent adjustment of anterior and posterior sacs. If larvae are weighted at the first abdominal segment so that they remain horizontally placed in the water, the anterior and posterior sacs expand as a single set (Fig. 2). If, however, larvae are weighted asymmetrically (at the cervix or at the eighth abdominal segment) rather than symmetrically (at the first abdominal segment) the response by the two pairs of sacs is also asymmetrical. While the initial fast response reveals less of this asymmetry, the response by 48 hr is decidedly asymmetrical (Fig. 5). The initial response involves increases in both anterior and posterior sets of sacs, whether the weight is cervical or abdominal. Asymmetry involving increases in one set but decreases in the other set develops after the initial time period of 1 hr.
1662
S. TERAGUCHI
Test of Krogh hypothesis If the sac is a semi-rigid container from which a liquid is removed in order to allow a quantity of gas to expand to an appropriate volume, then the external dimensions of the sacs should not increase during compensation for added weight. The increase in gas volume necessary for compensation for weights of 0.05 to 0.055 mg by larvae of 5.0 to 5.5 mg can be calculated (TERAGUCHI, 1972). These calculations predict that the gas space must increase by between 18 and 22 per cent. Since the sac area of larvae weighted according to the above specificationf increases by 21 per cent (Fig. 2), the removal OS fluid ballast from the interior cannot be the means by which neutral buoyancy is regained.
0
7
5
I5
30
I 4s
I 60
TIME V~INUTES)
Fig. 4. Changes in sac rigidity during compensation for added weight. Weight added at 0 min. Degree of instantaneous expansion upon sudden exposure to reduced pressure (minus 40 cm Hg). - - - -, Larvae free in water; -, larvae suspended.
$__-_+“+ /’
zf
/t
TIME lHOURS1
Fig. 5. position. cervix or 0 hr and terior
Response to displacement from horizontal Changes in sac area in larvae weighted at eighth abdominal segment. Weight applied at removed at 48 hr. -, Changes in right ansac; - - - -, changes in right posterior sac.
Table
1.
Sac-gas
composition
Test of gas-secretion hypothesis If the control of sac volume is exerted by a mechanism which directly acts upon the gas added, then the gas added should be largely a single gas of some biological activity and the concentration of this gas should rise relative to the other components of the gas mixture in the sac during adjustment to a weight. If, on the other hand, the gas entering the sac is of the same composition as the mixture which is dissolved in the surrounding tissues and if it enters by physical forces rather than directly as a result of a metabolic process, then the changes in the composition of the sac contents should reflect the dissolved gases in the environment even when the gas in the environment is largely an inert gas not known to be handled by metabolic processes. The composition of sac gas does reflect the composition of that dissolved in the water (Table 1; also KROGH, 1911). When larvae are transferred from water saturated with air to water saturated with nitrogen, the relative peak height of nitrogen rises from 82 to 87% and that of oxygen falls from 13 to 7% (Table 1). When larvae are transferred from water saturated with air to water saturated with oxygen, the relative peak height of oxygen rises from 13 to 36% and that of nitrogen falls from 82 to 55% (Table 1). Carbon dioxide rises in both
of larvae equilibrated with water saturated Analysis by mass spectrometry
with various gases.
SAC GAS
NITROGEN
OXYGEN
(28)
(32)
ARMN (40)
DIOXIOE (44)
SAT!lsATING
GAS
DISSOLVED OXYGEN
IN WATER cdl>
REIATmE PEAX HEIGHTS (percentages)
82.0 c 0.4 ( 9)
13.0 + 1.0 ( 9)
1.0 + 0.2 ( 9)
3.0 f 0.5 ( 9)
87.0 i 2.0 ( 7)
7.0 i 1.0 ( 7)
0.9 i 0.1 ( 7)
5.0 f 0.1 ( 9)
nitrogen
36.0 t 4.0 (14) 1.0 t 0.1 (14)
7.0 f 1.0 (14)
oxygen
55.0 -t4.0 (14)
air
7.17 + 0.63 ( 2) 1.33 r 0.22 (24) 38.44 + 1.73 ( 5)
Correction of negative buoyancy in Chaoborus americanus transfers, but this event is probably the result of increased larval activity upon transfer. Sac gas from both expanding and static sacs was examined for changes in the relative amounts of nitrogen, carbon monoxide, oxygen, argon, and carbon dioxide. Entrance of any of the gases would increase its proportion in sac gas. Argon is used here as a tracer gas, since it should not be handled by any known metabolic process. Therefore, any increase in the proportion of argon in sac gas can be attributed to diffusion. Larvae weighing 5.0 to 5.5 mg contain about 0.12 ~1 of gas. The composition of sac gas from larvae held in air-water recorded as peak heights (expressed as percentage of sum of these peak heights) on a mass spectrograph is 82 : 13 : 1 : 3 for peaks at mass numbers of 28 : 32 : 40 : 44. The addition of about 0.03 ~1 of gas is required to compensate for a weight of 0.05 to 0.055 mg. If the gas in the sacs is initially ‘air’ (which gives relative peak height of 82 : 13 : 1 : 3 for Na-OS-A-CO, on the mass spectrometer), then the relative peak heights that would be approached during adjustment if the adjustment results from the addition of 0.03 ~1 of either air, argon, oxygen, carbon dioxide nitrogen, or carbon monoxide are shown in Table 2. In general, the sac should become enriched in the entering gas but diluted in all other components. Outward diffusion was not taken into account. Table 3 gives the changes in sac-gas composition of larvae which were first weighted and then either suspended or left free in water saturated with air. At 1.5 min the relative peak heights are 83 : 10 : 1 : 5 for free animals and 81 : 11 : 2 : 6 for suspended animals. The ratios obtained can be accounted for by assuming that 0.03 ~1 of a gas mixture similar to air entered the sacs during adjustment, and that
1663
Table 2. Predicted sac-gas composition resulting from the entrance of 0.03 ~1 of various gases into air-filledsacs MASS WMXR
--I14.0
1.0
66.1
10.5
20.9
66.1
30.6
0.8
66.1
10.5
0.8
22.5
86.3
I I 10.5
0.8
2.4
= MASS Nln43P.R
xIITROGEN (28)
LARVA
t free free suspended free suspended free suspended free suspended
OXYGEN (32)
ARGON (40)
CARBON DIOXIDE (40)
RCLATIVE PEAK HEIGHT As % OF SCM OF TAESE PEAK HEIGHTS
!
2.4 2.4
this mixture was somewhat enriched in CO2 and depleted of Oa due to the activity of the animal. This idea is supported by the similarity between the ratios for free and suspended animals, since although only free animals increase sac volume (Fig. l), both sets of animals have been subjected to similar manipulations. Figs. 6 to 9 give the changes in sac-gas composition of larvae held for adjustment in water saturated Larvae with argon (0, = 0.69 + 0.08 (10) mg/l.). were held in air-water until the application of the weight but were then transferred to argon-water for adjustment. Under these conditions, the responses as revealed by an increase in sac area reach a maximum at 15 min (Fig. 10). Since the relative proportion of mass 28 (nitrogen, Fig. 6) in the sac-gas decreases markedly during the initial 15 min interval, the gas entering cannot be largely nitrogen. The decrease from 82 to 52% in the proportion of nitrogen in static sacs (Fig. 6,
Table 3. Changes in sac-gas composition in larvae bearing weight. Air-equilibrated larvae in air equilibrated water. Analysis by mass spectrometry
TIME
3.0
81.9
82 + 0.4 ( 9)
13 r l( 9)
1 i 0.2( 9)
3 c 0.5( 9)
83 k 1
( 8)
10 e l( 8)
lf5
551
81 * I.
( 7)
11 f I( 7)
2 t 0.5( 7)
6+1
(7)
82 f 1
( 9)
11 i l( 9)
1 f 0.2( 9)
5Cl
(9) (7)
(8)
(8)
80 t 1
( 7)
13 2 1( 7)
2 t 0.2( 7)
5t1
83 t 2
( 8)
11 f 2( 8)
2 f 0.2( 8)
4 + 0.4( 8)
815
1
( 7)
12 f 1( 7)
2 f 0.2( 7)
5*1
(7)
80 * 1
(10)
13 +_l(l0)
2 k O.l(lO)
5 f 1
(10)
82 i 0.4 ( 6)
13 k 1( 6)
1 t 0.2( 6)
3 f:0.5( 6)
1664
S. TERACUCHI
16
6 0 80 -
II-7
20--I TIME
(MINUTES)
Fig. 6. Changes in amount of nitrogen in sac-gas relative to the total amount of nitrogen, oxygen, argon, and carbon dioxide. Larvae bearing weight. Weight added at 0 min. Air-equilibrated larvae in argonequilibrated water. - - - -, Larvae free in water; -, larvae suspended.
TIME IMINUTES)
Fig. 8. Changes in the amount of argon in sac-gas relative to the total amount of nitrogen, oxygen, argon, and carbon dioxide. Larvae bearing weight. Weight added at 0 min. Air-equilibrated larvae in argonequilibrated water. - - - -, Larvae free in water;--, larvae suspended.
TIME
WINUTES)
Fig. 7. Changes in the amount of oxygen in sac-gas relative to the total amount of nitrogen, oxygen, argon, and carbon dioxide. Larvae bearing weight. Weight added at 0 min. Air-equilibrated larvae in argonequilibrated water. - - - -, Larvae free in water; -, larvae suspended.
TIME (MINUTES) Fig. 9. Changes in the amount of carbon dioxide in sacgas relative to the total amount of nitrogen, oxygen, argon, and carbon dioxide. Larvae bearing weight. Weight added at 0 min. Air-equilibrated larvae in argonequilibrated water. - - - -, Larvae free in water ;larvae suspended.
Correction of negative buoyancy in Chaoborus americanus
/’
010
TIME (MINUTES)
Fig. 10. Changes in sac area in larvae bearing weight. Air-equilibrated larvae in argon-equilibrated water. Weight added at 0 min. - - - -, Larvae free in water; -, larvae suspended. suspended larvae) can probably be attributed to simple diffusion. If the initial sac-gas composition is expressed as 82% nitrogen and 18% other than nitrogen, and if the change in the sac-gas composition due to diffusion seen in static sacs also occurs in free animals, then without entrance of the additional volume of gas the composition at 1.5 min would be 52 o/0nitrogen and 48 ok other than nitrogen. If, in addition, a volume of something other than nitrogen equal to 13 o/o(Fig. 10) of the initial sac volume enters, the proportion of nitrogen will be further reduced to .52/113, or 46%. The proportion of nitrogen observed at 1.5 min is 44%, which suggests that no nitrogen enters the sac as a direct consequence of adjustment in argon water. Similar arguments lead to similar conclusions for oxygen (Fig. 7). Since the relative proportion of mass 40 (argon, Fig. 8) in the sac gas increases markedly during the initial 15 min interval, the gas entering may be largely argon. The increase from 1 to 3 1 y. in the proportion of argon in static sacs (Fig. 8, suspended larvae) can probably be attributed to simple diffusion. If the initial sac-gas composition is expressed as 1 ‘$6 argon and 99% other than argon, and if the change in the sac-gas composition due to simple diffusion seen in static sacs also occurs in free animals, then without entrance of the additional volume of gas the composition, at 15 min, would be 3 1 y. argon and 69yo other than argon. If, in addition, a volume of argon equal to 13% (Fig. 10) of the initial sac volume enters, the proportion of argon will be further increased to 44/113, or 39%. The proportion of argon observed is 4376, which suggests that in argon-water, argon is the gas that enters the sacs as a direct consequence of the adjustment process. The dip in the curve for suspended larvae and the plateau in the curve for free larvae between 15 and 45 min cannot be satisfactorily explained at this
1665
time, but it should be noted that a decrease in the sac volume of free larvae occurs during this same time interval (Fig. 10). Since the relative proportion of mass 44 (carbon dioxide, Fig. 9) increases during the initial 15 min interval, the entrance of carbon dioxide can be considered. The increase in the proportion of carbon dioxide in static sacs (Fig. 9, suspended larvae) can probably be attributed to simple diffusion, Since the increase in the proportion of carbon dioxide in expanding sacs (Fig. 9, free larvae) is identical at 15 min to that seen in static sacs, it is unlikely that carbon dioxide enters the sac as a direct consequence of the processes accomplishing adjustment. It is much more likely that the carbon dioxide enrichment is a consequence of increased larval activity, since both free and suspended animals were subjected to similar manipulations. Sac anatomy Light microscopy. Under phase contrast, the sac wall consists of thin, dark hoops (dark bands) separated by wider light bands (Fig. 11). The light bands (non-bulbed bands) become sky-blue when stained with toluidine blue-fast green (fresh tissue) or methylene blue (Araldite sections) and purple when stained with haematoxylin and eosin (BARDENFLETH and EGE, 1916). Toluidine blue does not react evenly over the entire sac; the midportion becomes intensely blue while the tips become only faintly blue. The dark bands (bulbed bands) become turquoise when stained with methylene blue (Araldite sections) and pink when stained with haematoxylin and eosin (BARDENFLETHand EGE, 1916). Since resilin stains with cations and basic dyes, the reactions to methylene blue, haematoxylin, eosin, and toluidine blue-fast green suggest that the dark bands (bulbed bands) do not contain resilin. The outside surface is formed by two layers of cells, an outer pigment cell layer and an inner tracheal cell layer. Narrow slips of muscle run from the sac wall to the body wall (Fig. 11). Electron microscopy. The intima is composed of alternating bulbed (dark) bands and non-bulbed (light) bands arranged perpendicularly to the axis of expansion. Folds in the cuticulin layer parallel both the bulbed and non-bulbed bands (Figs. 12, 13). Fibrils in both the non-bulbed and bulbed bands are preferentially oriented 90” with respect to the axis of expansion but those in the non-bulbed bands run vertically through the band while those in the bulbed bands run longitudinally through the band (Fig. 14). The gap between the heads of the bulbed bands may be wide or narrow. The weakness of the reaction of toluidine blue near the sac tips can be attributed to the decrease in the width of the non-bulbed bands relative to that of the stems of the bulbed bands. Coincident with the decrease in non-bulbed band width towards the
S. TERAGUCHI
1666
sac tip, is a decrease in bulbed band head width and a narrowing and thickening of the non-bulbed band pads (Figs. 12, 13). The sac was reacted with DAB with and without peroxide since peroxidases may be involved in the cross-linking of resilin (LOCKE, 1969). No part of the intima gave a positive reaction (Figs. 12, 13). The structure of the tracheal cell layer is not characteristic of a structure specialized for movement or of a structure specialized for transport. Few microtubules or filaments are present (Fig. 15) although in areas near muscle attachment (Fig. 16) or near tracheal cell-to-cell attachments, greater numbers of microtubules may be present. Neither the number nor the distribution of mitochondria suggest specialization by the tracheal cells for transport (Fig. 15). The cytoplasm is, however, rich in free ribosomes. The significance of this is not obvious. Perhaps these cells are involved in the manufacture of materials for intermoult sac growth. The attachment of the muscle slips to the sac wall differs from that at the body wall. The external attachments are accompanied by extensive systems of microtubules, interfolding of muscle-cell and and electron-dense epidermal-cell membranes, material along the region of contact. At the sac, a basement membrane of about 0.35 pm separates the muscle cell from the adjoining pigment cell (Fig. 17) or tracheal cell (Fig. 18) and accompanies deep infoldings of the muscle-cell membrane. Structures which are possibly neuromuscular junctions are sometimes seen at the muscle cell-tracheal cell interface (Fig. 18). Vesicles are present on the ‘axonal’ side of the areas of ‘synaptic’ contact. DISCUSSION Choborus larvae correct a condition of negative buoyancy by increasing the volume of gas held within the body in two pairs of tracheal sacs. The rapidity of the compensation for added weight and the independence between sacs during the compensation for added weight and the independence between sacs during the compensation for displacement from horizontal position suggest that sac volume may be under nervous control. Motor terminals are present at the muscle-tracheal cell interface. Since sac expansion occurs more rapidly than sac contraction and since asymmetrical responses to cervical or abdominal weights appear only after an initial symmetrical response, the compensatory process may occur in two phases: an initial fast phase and a subsequent ‘growth-like’ phase. Test of Krogh hypothesis The tracheal sacs do not function as ballast tanks from which a quantity of fluid is removed to increase
gas space. This hypothesis is refuted by two pieces of evidence. First, no fluid is seen inside the sacs and, second, sacs increase in size in response to added weight and in response to light fluids (DAMANT, 1924). Test of gas-secretion hypothesis A quantity of gas enters the sacs during the reduction of density, but the evidence does not support the hypothesis that this gas is produced by a metabolic process. Any gas (even an inert gas) present in the surrounding fluids enters probably because the force is provided by diffusion gradients. These gradients would be set up or magnified when the gas space is enlarged by action of the sac wall, since the enlargement would reduce the partial pressures of the confined gas mixture. The above scheme requires that the sac wall be readily permeated by available dissolved gases, and sac-gas composition does reflect dissolved gas composition. In addition, DAMANT (1924) observes buoyancy oscillation when larvae are transferred between waters containing various gases and concludes that sac walls are differentially permeable to the various gases. The hypothesis for gas secretion is also refuted by the results from mass spectrometric analysis of sac-gas composition during adjustment for weight. Sac-gas composition does not change when airequilibrated larvae adjust in air-equilibrated water. The expected enrichment of a single component of the mixture in larvae with expanding sacs is not seen. On the other hand, the entrance of air into an air-filled sac should not alter the component ratios. Sac-gas composition does change when airequilibrated larvae are transferred to argon-equilibrated water. The sac gas becomes enriched in argon and carbon dioxide and depleted of oxygen and nitrogen. The enrichment in argon and depletion of oxygen and nitrogen is magnified by concurrent sac expansion (induced by weighting). Argon is an inert gas, and it should not be handled by any known metabolic process. The enhanced enrichment (as compared with the control in which sac volume did not increase) can be accounted for by the hypothesis which attributes the entrance of gas to the physical force of diffusion. The increase in gas space resulting from sac-wall extension would decrease the partial pressure due to any amount of argon which enters. The diffusion of argon into an expanding sac should therefore take place more rapidly than into a static sac. In addition, rapidly eouilibrated gas in the capillary tracheae attached to the sac tips might be drawn into the main sac during the expansion. The gas-secretion hypothesis does not provide for these data. These data are consistent with an alternate hypothesis which attributes the entrance
1667
Fig. 11. Drawing of excised right anterior sac as seen with the phase-contrast microscope. 1 to V, Slips of tissue, probably muscular; VI, enlarged view of tissue seen at I; VII to VIII, capillar? tracheae; arrow, outlet of sac; arrowheads, dark bands; space between arrowheads, light band. Fig.
12.
Cross-section of intima reacted with DAB but otherwise 0, opened fold; C, closed fold; Bb, bulbed band; P, pad;
Fig.
13.
Cross-section Non-bulbed
unstained. Without peroxide. Nb, non-bulbed band.
of intima reacted with DAB but otherwise unstained. With band; P, pad; arrow, fold over non-bulbed band; Bb, bulbed
peroxide. band.
Nb,
Fig. 14. Intima of sac near inner surface showing the fibrils of the bulbed-band in cross-section. Large dark fibrils are embedded in matrix which is in turn divided into honeycomb-like ‘cells’. Nb, Non-bulbed band; Bb, bulbed band.
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Fig. 15. Typical tracheal cell from layer covering Tracheal-cell mitochondrion; mt, microtubule; Fig.
16.
Tracheal
cell and inner
Fig.
17.
Junction
of muscle
Fig.
18.
Junction
of muscle chondrion;
sac. Cytoplasm tn. tracheal-cell
portion of intima near tubules; bh, bulbous
region head.
to sac bvith intervening pigment membrane; m, muscle. cell to tracheal cell showing t, tracheal cell ; NV, nerve:
is rich in free ribosomes. tm, nucleus; bh, bulbous head.
of muscle
cell.
attachment.
p, Pigment
neuromuscular junction. arrow, synaptic contact.
cell;
mt,
Bm,
,\Iicro-
basement
iX1, muscle
Xlito-
Correction of negative buoyancy in Ckaoborus americanus of gas to physical forces developed secondarily to an
initial action of the sac wall. The physical properties of the sac wall are not static and do in fact change during the adjustment to weight. In particular, there is a transient increase in the plasticity of the sac wall during adjustment. The sac wall is complex in structure. The intima is composed of several kinds of cuticle and the cellular coat is composed of two types of cells plus associated muscular and nervous tissue. The sac wall was examined for the structural basis of four sac properties: (1) the limitation of the direction of expansion ; (2) the regions of prospective expansion ; (3) the possible existence of two phases of expansion; (4) the development of expanding forces. The one-dimensional course of expansion would seem to have a multiple basis. The repeating units of ‘bulbed band-non-bulbed band’ are assembled perpendicularly to the axis of extension as are certain subunits of the bands. For example, the cuticulin layer (which is thought to limit intermoult expansion (BENNET-CLARK, 1963)) is preferentially folded perpendicularly to the axis of extension and the fibrils in the bulbed band are arranged in this same direction. Structural analysis does not localize the site of expansion in any one band. There are two lines of evidence which suggest that expansion during fast adjustment occurs mainly in the bulbed bands. First, deep folds or bulges in the cuticulin overly the stem portion. Second, the relative increase in the width of non-bulbed band-bulb stem pairs during sac expansion is greater near the sac tip than it is near the midsection. The contribution to the total width of the composite band by the two components is distinctly different in the middle than it is near the tip. The composition is about 50 per cent non-bulbed and 50 per cent bulb stem near the tip while near the middle the bulb stem composes more like 25 per cent of the total width. If expansion during fast adjustment is restricted to the bulbed bands and if all bulbed material swells to the same degree, the relative increase in width of each composite band would be greater near the tips. The evidence that associates expansion with the non-bulbed bands would suggest that at least the ‘growth-like’ step occurs here. Sacs grow between moults by increasing the width of the non-bulbed bands (FRANKENBERG, 1915). The sacs of very young fourth instar larvae resemble the tip regions of mature sacs in that the width of the non-bulbed bands is more nearly the same as that of the bulb step in most regions. In a mature larva, the differences between the wall near the tip and the wall nearer the middle suggest that material has been added to both the non-bulbed bands and the heads of the bulbed bands but not to the stems of the bulbed bands. Other aspects of structure which permit the postulation of expansion of non-bulbed bands are the appropriate wrinkling of the overlying
1669
cuticulin, the inverse correlation between the depth and width of the underlying pad, and the location of the inter-bulb-head gaps beneath the non-bulbed bands. In addition, while there is no evidence that resilin is a component of the bulbed bands, the possibility that this elastic protein is present in the non-bulbed bands has not yet been eliminated. The non-bulbed bands react with toluidine blue as would be expected of resilin. Physiological evidence suggests that the adjustment process may occur in two phases: a fast phase and a slow phase. This evidence is as follows: First, larvae which have carried weights for less than 24 hr can as rapidly return sacs to their original dimensions. However, if weights are left on for more than 48 hr or if floats of polyethylene are added to neutrally buoyant larvae, reduction in sac size is hardly detectable at 1 hr and takes 24 to 48 hr to complete. Second, asymmetry in sac response appears only subsequent to an initial symmetrical response. It is possible that the ambiguous result of the attempt to restrict the response to a simple component stems from an existence of a two-phase response since the sites of the two phases might differ. It can therefore be postulated that sac expansion can proceed through one or two phases depending on the persistence of the perturbation, the first phase being a fast, easily reversible step and the second being a ‘growth-like’ step in which material is added to the non-bulbed bands and/or the heads of the bulbed bands. In other systems involving cuticle expansion, although the cuticle itself participates dynamically in that it becomes less rigid, the actual force is provided by an extrinsic element. Neither the tracheal cell layer nor the pigment cell layer have the qualities of cells which would be expected to develop much force. Muscles (with innervation near to the sac) are present. However, the attachments to the sac are unlike those usually associated with muscles developing much force and the whole structure looks much like the stretch receptors described for the moth Anthereu (FINLAYSON and LOWENSTEIN, 1958). The muscle slips are probably the ‘tractus’ of POUCHET (1872) who suggested that they are involved in sac rotation or sac contraction. It is therefore possible that the force necessary for expansion is generated by an intimal component and that the muscular tissue associated with the sac wall serves some other function. An analysis of the material exchanges between the intima and its associated cells remains as a subject for further study. Acknowledgements-I thank Dr. L. M. PA~SANO for encouragement and assistance, Dr. H. SCHNOESfor his supervision of the mass spectrometric analyses, and Dr. M. LOCKEfor assistance with the electron microscopy. The work was supported by the Zoology Department of the University of Wisconsin and by the Biology Department of Case Western Reserve University.
1670
S. TERACUCHI REFERENCES
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LARIMER J. L. and ASHBY E. A. (1962) Float gases, gas secretion and tissue respiration in Portuguese man-ofwar, Physalia. 3. cell. camp. Physiol. 60, 41-97. LOCKE M. (1969) The localization of a peroxidase associated with hard cuticle formation in an insect, Calpodes ethlius Stoll, Lepidoptera, Hesperiidae. Tissue &f Cell 1, 555-574. LOCKE M. and COLLINS J. V. (1965) The structure and formation of protein granules in the fat body of an insect. 3. Cell Biol. 26, 857-884. LOCKE M. and COLLINS J. V. (1968) Protein uptake into multivesicular bodies and storage granules in the fat body of an insect. 3. Cell Biol. 36, 453-483. LOCKE M. and KRISHNAN N. (1971) Hot alcoholic phosphotungstic acid and uranyl acetate as routine stains for thick and thin sections. 3. Cell Biol. 50, 550-557. MADDRELL S. H. P. (1966) Nervous control of the mechanical properties of abdominal wall at feeding in Rhodnius. 3. exp. Biol. 44, 59-68. NUGEZ J. A. (1963) Central nervous control of the mechanical properties of the cuticle in Rhodnius prolixus. Nature, Lond. 199, 621-622. POUCHETG. (1972) DBvCloppement du systeme trachten de l’Anoph&le (Corethra plumicornis). Archs 2001. exp. ge’n. 1, 217-232. SCHOLANDER P. F. (1956) Observations of the gas gland in living fish. 3. cell. camp. Physiol. 48, 423-528. TAKAHASHIK. (1966) Muscle physiology. In Physiology of Echinodermata (Ed. by BOOLOOTIANR.), Vol. 22, pp. 513-527. Wiley, New York. TERAGUCHI S. (1972) Regulation of buoyancy by Chaoborus americanus (Joh.). Ph.D. Dissertation, University of Wisconsin, Madison. TERAGUCHIS. (1975) The detection of negative buoyancy by the phantom larva Chaoborus americanus (Joh.). 3. Insect Physiol. 21, 1265-1269. WIGGLE~WORTH V. B. (1953) Surface forces in the trr;~;;; system of insects. Quart. 3. micr. Sci. 94,