Acetate transport by locust rectum in vitro

ACETATE

TRANSPORT

BY LOCUST RECTLJM IN VITRO

T. BAUMEISTER*,J. MEREDITH, W. JCLIEN and J. PHILLIPS Department of Zoology. Univeristy of British Columbia. Vancouver. BC VhT I W5. Canada

Abstract-Everted rectal sacs of Schisrocerca gregaria absorb ‘Y-acetate from the lumen side at high rate5 against large electrical and often small concentration differences. Most of the lJC-activity in the absorbed fluid remains as acetate, but small amounts serve as substrate for aerobic respiration within this tissue. When acetate is substituted for SOi- or Cl- in external salines. both short-circuit current (I,) and the open-circuit transepithelial potential (PD) increase by as much as 2- to 3-fold. The stimulatory effect of acetate on I,, and PD exhibits saturation kinetics. The ‘steady-state’ influx of ‘YY-acetate from lumen (L) to haemocoel (H) side greatly exceeds efflux (haemocoel to lumen) across short-circuited recta. Over the whole range of acetate concentrations tested. the resulting net flux of acetate is sufficient to explain all of the increase in I, caused b> this organic anion. Acetate was detected in moderate concentrations in body fluids of locusts. The possible significance of acetate transport in viva is discussed. Kc! w0rd I&,.\-: Locust rectum. acetate transport. insect excretion

INTRODUCTION

Schisrocerca

IN V~~~orecta of thedesert locust generate large shortcircuit currents (1,J and open-circuit transepithelial electrical differences (PD; haemocoel negative), indicating transport of Cl- and other anions from lumen to haemocoel,astirstdescribed byWILLIAMSCfu/. (1978). Cyclic-AMP and corpora cardiaca cause large increases in I, PD and net flux of ““Cl- across this epithelium (SPRING ~‘t ul.. 1978; HANRAHAN, 1978; SPRING and PHILLIPS, 1980a,b; PHILLIPS, 1980). One observation made during these studies was totally unexpected. When we replaced all of the external Cl with acetate. the ‘unstimulated’ /,,and PD both nearly doubled (WILLIAMS et d.. 1978) and CAMP or corpora cardiaca no longer caused stimulation (SPRING and PHILLIPS, 1980b). Three explanations for this action of acetate seemed possible: (1) acetate is actively transported from lumen to haemocoel by an electrogenic mechanism. (2) Oxidation of acetate enhances ATP synthesis in in vitro recta and this stimulates transport of other anions (e.g. HCO; or organic anions) from lumen to haemocoel, or cations (e.g. H+) from lumen to haemocoel. (3) Acetate is converted within the rectal tissue to another organic anion which is then transported to the haemocoel side. In this paper, we report experiments which clearly favour the first explanation as the predominant mechanism responsible for generating increased I,. The kinetics of acetate transport are reported and the possible significance of this process in viva is discussed.

MATERIALS

AND

METHODS

Material.?

The

experimental

* Reprint

requests

animals

should

were

adult

be sent to J. PHILLIPS.

female

gregaria Forskhl, 5 weeks past their final moult and starved for 1 day prior to use. The colony was maintained at 28°C and SO”;, relative humidity on a diet of fresh lettuce. bran, powdered milk, yeast and dried grass. Methods Everred recta/ sacs. Net absorption of ‘Y-acetate (uniformly labelled, 55 mCi;mmole: New England Nuclear, Inc.. Montreal)wasfollowedwith timeat 38 C acrosseverted rectal sacs. asdescribcd for inorgzanic ions by GOH and PHIL.LIPS(1978). The rectal sacs were bathed externally (lumen side) with complex acetate saline (Table 1) bubbled vigorously with 95”,, 0,-Y’,, CO,. The internal (haemocoel) compartment was initially free of solution and all of the absorbate was collected for analysis at 1-3 hr intervals using a ‘Hamilton’ micro-syringe. Water movement was measured gravimetrically as previously described by GOH and PHILLIPS (1978). The iaC-activity in external saline, absorbed fluid. column fractions or hyamine hydroxide samples (see below), was measured in a commercial xylene-based scintillation medium using an ‘Isocap’ liquid scintillation counter (Nuclear-Chicago. Ltd.. Chicago). Quench correctionswere made using thechanncls ratio method. Separation merhods. The chemical form of lJC-activity in absorbed fluid and the external media was determined by ion-exchange chromatography as described by VON KORFF (1969). Initially samples were chromatographed on a 4 x 30 cm column of Dowex 1-X8 eluted with a continuous Cl- gradient (O-0.01 N HCI). This separated carboxylic acids ot both the Krebs cycle and glycolytic pathways, as well as monoamino monocarboxylic acids from dicarboxylic amino acids. However aspartate. glutamate. /5hydroxybutyrate and bicarbonate all co-chromatograph with acetate by this method. Bicarbonate and CO, were driven off by the addition of HCI to the absorbed fluid and shaking for 2 hr.

T. BACMEISTEH et (11. Table 1. Composition of salines*

Constituent

Concentration (mM) Simple acetate Simple SO: saline saline

Simple Cl saline

Complex acetate salinet

185

NaCl

93

Na,SO, Na acetate NaHCO, KC1 K acetate KH PO, K $0 i&04, Mg (acetate)z CaCI.

23.8 11

23.8

1.2

1.2 5.4 2.4

2.4

185 23.8

24.5 10.5

11 1.2

8.5

2.4 13

2.1 2.1

CaSd

Ca(ac&ate), glucose L-glutamate sucrose

10 3.4

2.1 10 3.4

10 3.4 96.2

2.0 16.6 12.3 79.8

* pH of simple salines adjusted to 7.0 with small amounts of HNO, or NaOH. t Complex saline also contained 7.4 mM sodium succinate, 1.87mM tnsodium citrate, 12.8 mM malic acid, 5.56 mM maltose, 2.67 mM glycine, 4.61 mM proline, 2.64 mM glutamine, 30 mg/l. penicillin and 100 mg/l. streptomycin sulphate, pH adjusted to 7.0 with NaOH (after WILLIAMSet al., 1978). ‘Apparent acetate’ fractions from the Dowex I-X8 column were pooled, neutralized and re-chromatographed on a 0.5 x 5.5 cm column of Dowex 50-X8 cation exchanger eluted with 20 ml of distilled water (eluant contains acetate, b-hydroxybutyrate) followed by 20 ml of 0.10 N HCl (eluant contains glutamate. aspartate). The distilled water fraction was evaporated down and re-chromatographed on a 4 x 30 cm silicic acid column eluted with a chloroform to butanol-ehloroform (40:60; V/V) gradient to separate /I-hydroxybutyrate from acetate. Neutralized methyl red indicator solution (0.04%) was used to detect the position of cold standards. The success of all three separation procedures was confirmed by chromatographing known r4C-labelled organic acids in the presence of cold acetate and j-hydroxybutyrate. Acetate concentrations in some body fluids from locusts were measured chemically using an Acetate Assay Kit (enzymic-uv method) and instructions from Boehringer Mannheim, GmbH (Mannheim, West Germany). Flux studies across short-circuited recta. Recta

8 L ,= 6 E ? 4 .$

--

46

93

-

139

-

186 azlde

G

?2-m ;

H”

were mounted as flat sheets between two ‘Ussing chambers’ and short-circuited, as described by WILLIAMS et al. (1978). Experiments were normally started 2 hr after dissection, when preparations had reached the ‘steady-state’ phase (see WILLIAMS et al., 1978). To study the effect of acetate on Z, and PD, recta were first bathed bilaterally in a simple SO,-saline (Table 1). When steady I, and PD values were approximated, the bathing medium was replaced bilaterally (3 changes in 1 min) with salines in which acetate was substituted for some of the SO:-. Three or four different concentrations of acetate were tested in random order on individual recta, with acetate-free SO,-saline present between tests. A typical trace for such experiments is shown in Fig. 1. The stimulatory effect of some other organic acids on I and PD was tested in a similar manner, usizg salines (near pH 7) in which part of the SOiwas replaced by 93 mM fumarate, malate, oxaloacetate, citrate or propionate (osmotic concentration adjusted with sucrose). To measure unidirectional fluxes, short-circuited recta were bathed bilaterally in 5 ml of SO,-based

I

I

I

2

3

1

v I

I

4

Time after dIssectIon,

‘i 5

I

6

hr

Fig. I. Typical trace of short-circuit current across locust rectum to illustrate the method for studying the kinetics of acetate stimulation. Recta were bathed in a simple SO,-saline (Table l), which was exchanged bilaterally (3 rinses) for other salines in which some of the SO:- was replaced by acetate (presence indicated by solid horizontal bars; numbers indicate external acetate concentration, mM). During each ‘steady-state,’ the Iqcwas briefly shut off so that open-circuit PD could be measured. Azide (IO mM) was added at the end of all experiments to confirm the metabolic dependence of I,,

Acetate 75

transport

by locust

rectum

in vi/ro

~~ Alanlne

Glutamate

Lactate

(a)

Acetate 50

84i

-

8 %

(6)

Eluote

volume,

ml

Fig. 2. The profile of 14C-activity in eluate from ion-exchange chromatography columns used to separate carboxylic acids. In the initial separation (a), the distribution of ‘VJ-activity in pooled absorbed fluid from rectal sacs (solid histogram) is compared with that of l”C-labelled standard solutions (open histograms) run on the same column. Figures indicate the “4 of total 14C-activity (mean + S.D.. n) recovered in the acetate peak. In (b) and (c) the pooled ‘acetate’ fractions from (a) were re-chromatographed to separate those carboxylic acids which initially co-chromatographed with acetate (distribution of standards indicated by horizontal bars).

saline at selected acetate concentrations for 4 hr. Tracer amounts of ‘%-acetate were added to one side and the rate of appearance of radioactivity on the other side was followed by removing 4 ml of bathing saline at 0.5 hr intervals. Radioactivity in duplicate 2 ml samples of this fluid were estimated as described above. The volume of external fluid on the ‘cold’ side was restored after sampling with unlabelled saline, so that radioactivity remained less than 1:; of that on the ‘hot’ side. Average fluxes of ‘V-activity were calculated for the 4 hr period (8 duplicate samples) and compared to I, measured simultaneously, according to the method of WILLIAMS et al. (1978). Production of 14C0, was followed during some flux experiments by passing the gas exhaust from the two ‘Ussing chambers’ through a loo,/, hyamine hydroxide solution (in methanol). Residual dissolved CO, in the bathing saline was estimated at the end of experiments by acidification of the saline with 1 M HCl and trapping CO, as just described. 0, consumption by rectal tissue bathed in acetate saline was monitored, both before and after adding 10 mM sodium azide, using a Gilson ‘Oxygraph’ (Middleton. WI).

RESULTS

We were initially concerned whether 14C-activity in the absorbed fluid collected from rectal sacs remained largely in the form of acetate. Absorbed fluid was collected over a period of 3-4 hr from sacs bathed externally in complex acetate saline (63 mM; Table 1) and this fluid was subjected to column chromatography as described in the methods section. On average 87 + 8:: (n = 5) of the total 14C-activity in the absorbed fluid added to the column was recovered, compared to 87-98”; recovery for standard solutions of 14C-acetate. As shown in Fig. 2. 84&-8”; of the absorbed fluid ‘V-activity co-chromatographed with the acetate standard, while 80,d (range 5-14:;) was apparently converted to monoamino, monocarboxylic acids. The CO,/HCO J accounted for only 5s; or less of total 14C-activity in absorbed fluid before column chromatography, possibly because most 14C0, was lost to the atmosphere during experiments. When the pooled ‘acetate fractions’ from the initial separation were re-chromatographed as described in the methods. no

T. BACMEISTER ~‘1al.

198

Time,

hr

Fig. 3. (a) The rate ofacetatemovement from lumen to haemocoel side of everted rectal sacs, measured at hourly intervals. (b) The concentration of acetate in absorbed fluid from everted rectal sacs measured at hourly intervals during (a) (0). and in experiments done at a later time on a different population of locusts (0). The concentration in the ‘acetate’ fractions of rectal absorbed fluid (Fig. 2) are also shown (A). The broken horizontal line indicates the measured external acetate level in complex acetate saline. (Mean + S.E., n > 5).

14C-activity was detected in the glutamate + asparate and 2-5% of activity was in the fractions, fl-hydroxybutyrate fractions, compared to 88-97x in the acetate fraction (Figs. 2b,c). In summary, most of the 14C-activity in absorbed fluid entering the haemocoel compartment of everted rectal sacs remains as acetate ( > 8 1%). Only small amounts of acetate were apparently used as substrate for aerobic respiration during experiments with rectal sacs. A quantitative estimate of i4C-acetate oxidation was subsequently made using short-circuited recta bathed in simple acetate saline (200 mM). When labelled acetate was added only to the lumen side, 0.19 + 0.02 pmoles 14C0,/hr/rectum (+ SD., n = 6) was evolved, mostly on the lumen side, and 0.002 pmoles 14COz/hr/rectum on the haemolymph side. These values include residual “CO, in the bathing saline. Since oxidation of each acetate molecule yields 2 CO, molecules, oxidation of acetate absorbed from the lumen was about 0.1 pmole/hr/rectum. This is only 1-3’4 of the unidirectional and net fluxes of Y-acetate from lumen to haemocoel under identical conditions, as discussed later. Therefore, i4C0, did not contribute significantly to estimated i4C-fluxes.

Fluid transport by these recta was similar in magnitude to that observed with NaCl-salines (GOH and PHILLIPS, 1978). Slightly different relative rates of fluid and acetate absorption could explain differences in acetate concentrations in experiments with different populations of locusts (Fig. 3b). Since external Na+ and K+ (on the lumen side) are both capable of sustaining prolonged fluid absorption across everted rectal sacs (PHILLIPS, 1980), we cannot conclude from the above experiments whether or not acetate transport can drive water absorption. Studies with short-circuited Typical

recta

traces of 1, exhibited

by individual

recta

Net absorption by everted rectal sacs The net rates of i4C-acetate movement from lumen to haemocoel side of rectal sacs increased gradually during experiments, and averaged 0.85 pmoles/hr/cm2 over the 2nd-4th hr (Fig. 3a). This rate is comparable to values for NaCl absorption by this in vitro preparation (GOH and PHILLIPS, 1978). The concentration of acetate in the absorbed fluid over this period (55-105 mM: Fig. 3b) did not vary greatly from that in complex acetate saline (66 mM) used to bathe the everted sacs in these experiments.

L 0

I I

Time after dissection,

I

2 hr

Fig. 4. The typical time course of short-circuit current (I ) across individual recta tathed in simple salines (Table ‘f) which differed in the principal anion they contain. Mean ‘steady-state‘ values for !,, at 2-4 hr are given in the text.

Acetate transport by locust rectum in vitro bathed in various simple salines are shown in Fig. 4. When acetate saline is present, the Z, does not decline over the first 1-2 hr as it does when Cl-saline or SO,-saline are used. In our studies, the average ‘steady-state’ (24 hr) values of Z, (pequiv./hr/cm2; mean + S.D., n > 8) were 4.7 k 1.2 for acetate-, 1.98 k 0.4 for Cl-, and 0.95 + 0.78 for SO,-saline: i.e. the presence of acetate increased Z, by at least 2.5-fold. This confirms preliminary reports by WILLIAMS(1975) and WILLIAMSet al. (1978) who used complex salines. A respiratory inhibitor, sodium azide (10 mM), caused Z, and 0, consumption of recta bathed in simple acetate saline to decrease rapidly by 80-90”; with a half-time of 3-5 min. Cyanide (10 mM) caused almost complete inhibition of 1,. but 50 mM iodoacetate was without effect after 0.5 hr. Acetazolamide (0.5 mM), which partially inhibits acetate transport in the rumen (STEVENS, 1973), caused only a 14% decrease in I, (i.e. -0.56 f 0.29 pequiv./hr/cm2; + SD., n = 12) across recta bathed in acetate-saline. This decrease in I, was similar to that reported by SPRING and PHILLIPS (1980a) when acetazolamide was added to simple NaCi-saline. We studied the influence of external acetate concentration (equal on lumen and haemocoel sides) on I, and PD (Fig. 5a,b). This was done by replacing some of the SOiin simple SO,-saline with equivalent amounts of acetate. Both Z, and PD were elevated significantly when 20 mM acetate was present externally. When 200 mM acetate was added, Z, increased by 2.8 pequiv/hr/cm2 and PD by 11 mV, indicating increased electrogenic transport of anions to the haemocoel side. The increase in Z, and PD tends toward saturation at high acetate concentrations. The

IW

transepithelial resistance increased by less than 50”, over the whole range of acetate concentrations tested. When identical experiments were conducted using 93 mequiv./l. of oxaloacetate, fumarate, malate. citrate or propionate, increases in Z, and PD were not observed over the next 0.5 hr. The contribution of acetate transport to AZ,, was estimated by measuring unidirectional fluxes of 14C-acetate under short-circuit conditions. After the first 1.5 hr, flux rates were reasonably constant with time for 24 hr (Fig. 6a). Average ‘steady-state’ fluxes are plotted as a function of external acetate concentration in Fig. 6b. The lumen to haemacoel and haemacoel to lumen fluxes ( + S.D.. II = 0) were 120 & 20 and 26 i 3 nequiv;hr:cm’ respective11

8c

(a)

T

6-

4-

2-

I

2

4

3 Time,

5

hr

(b)

(a)

11 c-

A---i

./

f

_. /

I

I

I

(h)

$7 ,

I 100

50

External

i I

I

50

100

External

acetate

I I50

cant,

I 200

mM

Fig. 5. The increase in short-circuit current (a) and transepithelial open-circuit PD (b) as a function of the external acetate concentration. These increases were estimated in experiments similar to the one illustrated in Fig. 1. (Mean + LE.. n = 10).

acetate

Cone,

I50

I ?OO

mM

Fig. 6. Studies of 14C-acetate flux across locust recta bathed in simple acetate-SO,saline mixtures under short-circuit conditions. (a) The time course of unidirectional influx (lumen to haemocoel, 0) and efflux (haemocoel to lumen, 0) for recta in 201 mM acetate-saline, to illustrate attainment of a ‘steady-state’ (mean + SE., n = S-9). (b) Unidirectional fluxes as a function of the external acetate concentration. (c) Net flux rates of 14C-acetate (0) calculated from (b) are compared to the mean increase in short-circuit current (0) measured simultaneously across the same rectal preparations (Mean + S.E., n = 4-10).

T. BACMEISTEK et ul.

200

at the lowest acetate concentration tested (5 mM). With 10 mM acetate the comparable values were 280 + 94 and 71 k 22 nequiv./hr/cm2. Thus, significant net flux of acetate (94 f 20 nequiv./hr/cm2) to the haemocoel side occurred when levels of this anion (5 mM) were close to physiological levels (see Table 2). Surprisingly, the net flux did not reach a limiting value even when external acetate levels were unnaturally high (e.g. 200 mM) The influx: efflux ratio was about 4: 1 at all external acetate concentrations tested between 5 and 46 mM. The average net flux of ‘“C-acetate was more than sufficient to account for the AI, measured simultaneously in these experiments (Fig. 6~); however, there was considerable variability at the higher external acetates concentrations. At an external concentration of 63-69 mM. the measured rate of net acetate movement across short-circuited recta (Fig. 6) is twice that estimated for everted rectal sacs in the open circuit state (Fig. 3). This ratio for the two preparations is similar to that previously reported for inorganic ions (discussed by WILLIAMSrt ul., 1978). Acetate

in body jluids

Some preliminary chemical measurements were made to determine if acetate is normally present in body fluids of locusts under different physiological conditions, e.g. during flight when lipid metabolism is stimulated. The results are presented in Table 2. Low levels of acetate were detected in haemolymph under all conditions (as was citrate, 2 mM; malate < 0.1 mM). As expected for a small molecule, acetate from the haemolymph appears in the excretory fluid. We do not wish to stress differences in haemolymph concentrations of acetate between physiological states, because mean values in Table 2 were obtained at different times and with different populations. When tracer amounts of 14C-acetate were introduced into the mouth, radioactivity could be detected in the haemolymph, unaltered in chemical form.

DISCUSSION Experiments described in this paper clearly demonstrate that net absorption of acetate occurs from lumen to haemocoel side of rectal sacs against both a large PD and often small concentration differences. The active nature of this process is confirmed by the large flux ratios of 14C-acetate Table

2. Chemical

determinations

under short-circuit conditions. The resultant net flux of *aC-acetate is sufficient to explain the large increase in I,, caused by the presence of external acetate. A simultaneous increase in open-circuit PD confirms that acetate transport is electrogenic. While acetate transport can completely explain some of the acetate absorbed by rectal AI,, tissue also serves as substrate in aerobic respiration. This is evident from the appearance of small amounts of “Y-activity in CO,, amino acids, B-hydroxybutyrate and other unidentified organic acids (probably acetoacetate and krebs cycle intermediates as judged from their elution volumes). Clearly this tissue, like others in insects, ccntains an acetyl thiokinase capable of converting acetate to acetyl-CoA (reviewed in CANDY and KILBY, 1975). For example, it is known that 14C-acetate is readily incorporated into lipids in locusts. Rectal transport in vitro seems more than sufficient to explain recovery of the small amounts of acetate secreted by the Malpighian tubules in situ (Table 2). The estimated net reabsorption of acetate in vitro was 94 nequiv/hrjcm’ when the luminal level of this anion was 5 mM. This rate is 3 times the estimated value for Malpighian tubule secretion of acetate (e.g. 8-24 pl/hr containing 3.5 mM (Table 2) = 28-84 nequiv/hr acetate secreted). Low levels of acetate in faeces compared to Malpighian tubule fluid (Table 3) support this view. (It should be noted that lluid reabsorption in the rectum will tend to raise acetate levels in the lumen.) The question remains as to why the acetate transport system in the in vitro rectum does not saturate at lower concentrations. Possibly this transport mechanism in vivo carried other solutes or, on occasion, larger amount of acetate. These points are discussed below. Conceivably, acetate might make use of a general transport mechanism which normally moves other organic molecules, possibly one which is recycled to drive fluid absorption when Cl- is in short supply (see WALL, 1977). Glyciqe differs from acetate only by addition of an amino group. Both these organic acids are actively absorbed by locust rectum at similar rates when luminal levels are about 5-10 mM, but glycine transport saturates at low concentrations (BALSHIN and PHILLIPS, 1971; BALSHIN, 1973; PHILLIPS. 1980). G. JARON, in our laboratory, observed no decrease in 14C-glycine influx when large amounts of acetate (100 mM ) were added bilaterally, indicating that these two organic acids do not share a common carrier (preliminary observation). Acetate crosses brush border membranes of rat

of acetate concentrations physiological states

in locust

body

fluids

under

different

Acetate concentration Body fluid Haemolymph Haemolymph Haemolymph Haemolymph Malpighian tubule Faeces

The values shown

Pre-treatment

(mM)

secretion

(pooled)

are means f SD.

5.7 k 1.4+ 8.7 + 2.9 f 3.5 1.2 + U.&i Number

1.2 0.4 1.1 1.3

(8) (4) (7) (8)

0.5 I:; drv wt

unfed 24 hr recently fed after flight (4 hr) dehydrated over acid and starved unfed 24 hr unfed 24 hr

of observations

shown in parentheses.

(4 days)

Acetate

transport

by locust rectum

intestine three times more quickly than Cl- (LIEDTKE and HOPFER, 1977). A similar situation in locust rectum might permit more rapid access of acetate to a general anion pump in the baso-lateral membrane that normally moves Cl-. However, Cl- transport is stimulated by CAMP. whereas acetate transport is not (SPRING and PHILLIPS, 1980b). HANRAHAN (personal communication) in our laboratory has recently shown that high external acetate does not inhibit net 36Clflux across short-circuited locust recta; therefore, separate mechanisms must exist for these two anions. Citrate, malate, fumarate, oxaloacetate and propionate did not stimulate rectal I,, which indicates that acetate is not transported by a completely non-specific mechanism for weak organic acids, as proposed for vertebrate intestine (JA(IW)N and DLUXK. 1979). We have not yet tested the effect of ketone bodies (acetoacetate and P-hydroxybutyrate) on rectal I,,, These substances are produced and also oxidized by several locust tissues, and they have been detected in haemolymph (HILL (‘I al.. 1972). Conceivably these ketones are lost from haemolymph in the malpighian tubule secretion and are recovered from the rectal lumen by the ‘acetate’ transport mechanism. A parallel situation is found in proximal tubules of rat kidney. which actively absorb lSC-acetate in vitro (also by an acetazolamide-insensitive process); it is proposed that some of this reabsorbed acetate and other organic acids (e.g. lactate) serve as respiratory \uh\lr;llc> I’or the kranspol-ting epithelium (SC HAEF~K and ANDKEOIL 1976). We have observed moderate amounts of acetate in body fluids from locusts (Table 2). TULP and VAN KA%~(1970) report that isolated mitochondria from housefly flight muscle produce large quantities of acetate by decarboxylation of pyruvate, even when Krehs cycle is uninhibited. Conceivably acetate levels in haemolymph may also rise during flight or starvation when lipid is metabolized. Moreover, insect physiologists may have overlooked an important end-product of microbial digestion in locusts. Volatile fatty acids (acetate, propionate, butyrate) are the major end-products of digestion amongst vertebrate herbivores and these species possess transport systems for acetate in the rumen or colon (reviewed by PROSSER, 1973; also STEVENS, 1973). Perhaps these end-products are also produced in large amounts in locust gut, particularly during starvation when the last meal is retained for long periods. BRAC~;E and MARKOVETZ (1980) report that bacterial end-products (i.e. tracer amounts; including acetate) can cross the colon wall of Pcrip/tsnr/a americana from the lumen and most of rhe ‘Y-activity remains unaltered in chemical form. The mechanism was not studied nor were net rates of absorption against electrochemical gradients reported. HUNGATI; ( 1946) showed that cellulose was fermented to CO,, Hz and acetate in the gut of termites and he proposed that acetate absorption occurred in the hindgut. The possibility that similar events occur in locusts and other species has not been thoroughly investigated. In summary, the demonstration of acetate transport in locust rectum raises several interesting and important questions which require investigation.

II) I

bI ~‘i/ro

Ackno~~ied~ement.F-The work was supported by operating grants to J. PHILLIPSfrom the National Sciences and Engineering Research Council of Canada.

REFERENCES BALSHIP; M. (1Y7?) AhsorpIlon by

01’

the rectum of the desert locust

amino

acids ;,I \,,ro gr~yyzr~(~).

(Sc/li.c/otrrc,cr

Ph.D. thesis. University of British Columbia. Vancouver. Canada. BALSHIN M. and PHILLIPS J. E. llY7ll Active absorption of amino acids in the rectum of the desert locust (Schis/ocerca greguria). Yarurc~. Land. 233, 53-55. RRACKEJ. W. and MARE;• V~TZ A. J. (1’80) Transport of bacterial end products from the colon of Prr$anera umrricana. J. Itwct Ptlj,\ioi. 26, 85-X’). CANDY D. J. and KILRY B. A. ( 1975) In.rcc,r Biwtwmirrr~~ wul Funcrion. Chapman and Hall. London. GOH S. and PHILLIPS J. E. (197X) Dependence of prolonged water absorption by in vitro locust rectum on ion transport. J. e\-p. Biol. 72, 25--11_ HANRAHAV J. W. (1978) Hormonal regularion ofchlorIde in locusIs. T/w PII) ~io/o,~/\r. .,li~r. Pizw,~/. 5~. 21. 51). HILL L.. I~ATT M E. G.. lloR\t J. A. and BhlI I \ E. (IL)721 Factors affectIn conccntr;~tIon~ of acc’loacclalc and II-~hydroxq butbrate in the hacmolymph and &sue\ ol‘ the desert locusts. J. In.src~r Ph~sioi. 18, 1765-11X5. HUNCATF R. E. (lY46) The symbiotw utilization of cellulose. J. Eli.shn Mitchell

~~~iwzt. Sot. 62, 9-74.

JACKSON M. J. and D~DFK J. A. (107Y) Epithetial transport of weak electrolytes. Fe,
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