- Email: [email protected]
Water balance in Rhodnius prolixus during flight: Quantitative aspects of diuresis and its relation to changes in haemolymph and flight-muscle water
.I. lr~srcr Physiol. Printed
in Great
WATER
Vol. 28. No. 7. pp. 573-577.
1982
0022-1910~8’i070573~05$03.00!0 0 1982 Prrqamor~ Pwss Ltd
Britain.
BALANCE IN RHODNlUS PROLIXUS DURING FLIGHT: QUANTITATIVE ASPECTS OF DIURESIS AND ITS RELATION TO CHANGES IN HAEMOLYMPH AND FLIGHT-MUSCLE WATER J. L. GRINGORTEN* and W. G. FRIEND Department
of Zoology. (Rrceioed
University 14 September
of Toronto.
Toronto.
Ontario.
1981: revised 27 November
Canada
M5S 1Al
1981)
Abstract-The amount of water voided by male Rhodnius probus which were flown to exhaustion varied from 0 to over 10% of the initial live weight. It accounted for nearly all of the body water lost
during the Right period. Simultaneous measurements on the loss of haemolymph water and an estimate of the amount of faecal water in the excreta indicated that the source of the voided water was primarily the haemolymph. The total water content of the flight muscles changed very little in insects which flew to exhaustion. It is concluded that, despite the diuresis and loss of water. and the considerable reduction in haemolymph volume, dehydration of the flight muscles of male R. prolixus does not occur during these flight periods, and is not a factor contributing to ‘exhaustion’. The possibility that insufficient
haemolymph is a factor limiting the duration of flight is discussed. Key Word Index: Rhodnius prolixus, water balance. flight. diuresis,
INTRODUCI’ION TETHERED male Rhodnius prolixus St&l (Hemiptera: Reduviidae) can be stimulated to fly for periods of 2-3 hr before becoming exhausted (GRINGORTEN and FRIEND, 1979a). The stress of flight stimulates both diuresis and defaecation, and, during this time, the insects can undergo up to a 50% reduction in haemolymph volume (GRINGORTEN and FRIEND, 1979b). Neither phenomenon. we believe, has been described for any other insect in relation to flight stress. Both the excretion and decline in haemolymph volume are related to wing beating, and are not caused by the flight stimulus per se (GRINGORTEN and FRIEND, 1979b). The possibility that a quantitative relationship exists between the diuresis and reduction in haemolymph volume has been suggested previously (GRINGORTENand FRIEND, 1979b). Part of the decline in haemolymph volume may be attributed to a failure of the hindgut to resorb water secreted by the Malphighian tubules (cf. GOLDSWORTHY, 1976). The percentage water in the haemolymph and the specific gravity remain relatively constant during these long flight periods (GRINGORTEN and FRIEND, 1979b). However, a conspicuous feature of flight muscle from insects which have flown to exhaustion is the lack of surrounding haemolymph and the shrunken, ‘dehydrated’ appearance of the muscle tissue. Flight-induced tissue dehydration was considered a distinct possiblity in view of the diuresis and large reduction in haemolymph volume. In this study, we examine the effects of excretion on the water balance of R. prolixus flown to exhaustion, particularly the quantitative relationships between the amount of water voided, total body-water loss, the * Present address: International Atomic Energy Agency. P.O. Box 100. A-1400 Vienna. Austria. LP 28!7
-*
573
excretion.
haemolymph.
flight
decline in haemolymph volume, and the changes occurring in the water content of the flight muscles.
MATERIALS
AND METHODS
The rearing conditions and flight stimulus for experimental insects have been described previously by GRING~RTEN and FRIEND (1979b). All insects were unmated males which had completed their adult development, and were in a state of hunger when flown (a necessary stimulus for sustained flight). but not yet starving (GRINGORTENand FRIEND, 1979~). Insects were weighed prior to and immediately after being flown to determine live-weight loss. Direct measurement of total body water in flown insects was made by weighing before and after oven drying. Their pre-flight body water was determined indirectly. from a regression analysis of total body water on live weight, performed on 33 control (unflown) insects. The net loss in body water during the flight period was then calculated by subtraction. The water content of the flight muscles was determined by a liquid-paraffin technique similar to that described by B~JRSELL(1960). A correction factor was determined for dry-weight losses in the tissue which occur during the extraction of the paraffin by benzene. This factor was determined by performing the same paraffin-benzene procedure on muscle tissue which had been oven-dried and weighed. and then reweighed following the extraction. The correction factor is the ratio pre-extracted/post-extracted dry weight. Dry-weight values for the experimental tissue were multiplied by this factor to obtain corrected dry weights. Tissue samples were pooled from three insects for each water-content measurement and each correction-factor determination.
J. L.
574
and W. G. FRIEND
GRINCORTEN
Table 1. Live-weight, total body-water and excretion losses in male R. prolixus flown to exhaustion
Insect 1 2 3 4 5 6 7
Flight duration (min)
Pre-flight live weight (mg)
Live-weight loss (mg)
Pre-flight body water* (mg)
115.1 94.8 101.8 114.8 116.1 111.2 72.9
16.3 15.6 13.7 13.2 11.8 6.0 0.9
77.2 63.6 68.2 77.0 77.8 74.5 48.9
207 153 200 184 135 151 106
Body-water loss (mg) 15.0 12.5 13.4 10.0 12.5 2.1 0.3
Excreta wet weight (mg) 14.2 t 11.2
f 9.9 4.0 0
Excreted water (mg) 13.5 12.91 11.1 10.7$ 9.9 3.3 0
* Determined from the regression curve in Fig. 1. i Insect excreted, but weight not obtained. $ Determined from the regression curve in Fig. 2. Excreta produced during the flight period were collected in vials containing liquid paraffin. The vial and paraffin together were pre-weighed, which permitted direct determination of the wet weight of the excreta at the end of the flight period. Water content was subsequently measured in a manner similar to that used for flight muscle. Dry weight of the excreta could then be calculated by subtracting water content from wet weight, which obviated the .need to perform a benzene extraction. Haemolymph volume was determined by isotope dilution using [carboxyl-‘4C]inulin. The technique has been described in detail elsewhere (GRINGORTEN and FRIEND. 1979b).
RESULTS General features of excretion The excretion pattern in insects which flew was quite variable. During flight experiments on approx. 150 insects in this and in related studies, excretion was never observed prior to 10 min of flight. Not all insects excrete. In a sample of 14 insects which were flown to exhaustion, and observed continuously throughout their flight periods (103-209 min), 2 failed to void. Among those that did void, the frequency ranged .from 1 to 6 eliminations, and ceased altogether within 20min prior to the end of the flight period. In most cases, the final elimination occurred more than one hour before the end of the flight period. Invariably, the first elimination was dark in colour, representing a clearing of the rectum of faecal matter. Subsequent eliminations became progressively Table 2. Haemolymph-water
Insect 1 2 6 Mean S.E.
lighter in colour until they were clear and colourless, an indication of diuresis. In a number of instances the diuresis was again followed by a defaecation, and in one insect with a high excretion frequency, urination and defaecation alternated several times. In two cases, insects which had excreted numerous times during the flight period were subsequently observed to open the anus without voiding anything. One of these kept the anus splayed for 4 min. Quantitative aspects of excretion and water balance
Excretion of water during flight was measured in seven insects (Table l), and in three of these, the loss of water from the haemolymph was also determined (Table 2). The relationship between live weight and total body water in control insects, which was used to determine the pre-flight, total body water of flown insects, is illustrated in Fig. 1. Attempts to collect the excreta from insects 2 and 4 (Table 1) were unsuccessful; r;timates of the amounts of water excreted by these insects were made from a linear regression of excreted water on live-weight loss,using data from the remaining insects (Table 1, Fig. 2). The amounts excreted during flight were quite variable (Table l), ranging from 0 (insect 7) to over 12% (insect 1) of the initial live weight. However, it is evident that where excretion did occur, it accounted for most (median 83%) of the live-weight loss incurred by the insects. It is also apparent that the excreta were quite dilute, averaging (median) 97% water. Excreted water accounted for approx. 90% (median) of the body water lost among all insects during the flight period, and virtually all (97%) of the body water lost among insects l-6 (excreted water > 0). The total amount of
loss in male R. prolixus flown to exhaustion
Pre-flight Post-flight haemolymph haemolymph volume* volume
Haemolymphvolume loss
Haemolymphwater loss
Flight duration
Pre-flight live weight
Excreted water
(min)
(mg)
(PI)
(PI)
(Pi)
(mg)
(mg)
207 153 151 -
115.1 94.8 111.2
31.7 24.7 30.4
19.6 15.2 23.4
12.1 9.5 7.0 -
11.3 8.9 6.6 8.9 1.4
13.5 12.9 3.3 9.9 3.3
* Determined from a linear regression of haemolymph volume on live weight (GRINGORTEN and FRIEND, 1979b).
Water balance in Rhodnius prolixus during Right
._ ” . ..
. .. . ’. :=* .
Fig. I. The relationship between live weight and total body in unflown male R. profixus. Each point is a single insect. The equation of the best-fit curve is: body water (mg) = 0.670 x live weight (mg) + 0.042: r = 0.989.
water
575
Table 3. Per cent water in the flight muscles of unflown male R. prolixus and of insects flown to exhaustion*
Mean S.E.
Unflown Cd)
Flownt (“&)
66.5 66.0 65.8 65.7 66.0 0.2
71.1 71.5 71.3 0.2
was * Each measurement performed on samples pooled from three insects. t Flight duration: 118-160 min. median 13I min.
water excreted by different insects which flew to exhaustion could be more than 10% of the initial live weight (insects 1, 2 and 3). and perhaps as much as its dry weight, as a result of the benzene extraction. 14”j0(insect 2). whereas muscle from flown insects lost only 11.4%. A linear regression of haemolymph volume on live Changes in the per cent water of the flight muscles weight, computed previously by GRING~RTENand of insects which flew to exhaustion are given in FRIEND (1979b), was used to determine the pre-flight Table 3. It is evident that relative water content inhaemolymph volume of insects which were flown to creased during flight (r-test, P < 0.01). exhaustion. From direct measurement of the postflight volume, the specific gravity (1.03) and the per cent water (91.0~4) in the haemolymph (GRINGORTEN DISCUSSION and FRIEND, 1979b). the amount of water lost from Quantitative aspects of excretion and water balance the haemolymph could be determined (specific gravity and per cent water remain constant): In the present investigation, water voided by male haemolymph-water loss (mg) = [pre-flight-postR. prolixus which were flown to exhaustion accounted flight haemolymph volume (PI)] x S.G. x O/;water/ for nearly all of the body water lost. The similarity of 100. the average values for haemolymph-water loss and When these values were compared with the amount excreted water (Table 2) indicated that the principal of water lost by excretion (Table 2), it was apparent source of excreted water was the haemolymph, and that excreted water could account for the water lost that little of this haemolymph water was replenished from the haemolymph (t-test, P > 0.50). from other sources during the flight period. That the haemolymph was the source was consistent with the Flight-muscle water content observation that the excreta became progressively There was a considerable difference between the clear and colourless during flight, characteristic of a dry-weight correction factor for flight muscle from diuresis, and were found. in actual fact, to be primarunflown insects and that from insects flown to ily water. exhaustion. Muscle from unflown insects lost 34.6X of Several points should be noted in any quantitative comparison between excreted water and loss of haemolymph water. The first excretion may possibly 14 cause the expulsion of a quantity of water which exceeds the intitial amount of water removed from the 12 haemolymph by the Malpighian tubules. particularly if the rectum is relatively full and near a critical “voiding threshold” prior to the commencement of flight. In addition. some faecal water is expelled along with urine water. and, finally. the amount of water calculated to be lost from the haemolymph is a net quantity. There is some evidence, in fact, that the per cent water in the midgut declines during flight (GRINGORTEN and FRIEND, unpublished). Any transfer of water from the midgut (or other sources) to the haemolymph will reduce the net loss in haemolymph water. All of these factors could cause excreted water to exceed the net loss in haemolymph water. However, there is one factor which could reduce Fig. 2. The relationship between excreted water and livethis tendency, or even have the opposite effect. There weight loss in male R. prolixus flown to exhaustion. Each point is a single insect. The equation of the regression line is no certainty that the total amount of water filtered from the haemolymph by the Malpighian tubules is is: excreted water (mg) = 0.906 x live-weight loss (mg) actually voided. especially if there is relatively little 1.268; r = 0.995.
1
J. L. GRINC~RTEN and W.G. FRIEND
576
fluid in the rectum prior to flight. In this situation, haemolymph-water loss could exceed the quantity of water excreted. In addition, the relative contribution of faecal water to the total water excreted is generally expected to be small. The per cent water in the posterior midgut (tissue + luminal contents) of unflown insects, under present experimental. conditions, is less than 75% (GRINGORTEN and FRIEND, unpublished). Therefore, if the excreta are 97% water, not more than 10% of this water, on the average, could have been derived from faeces [i.e. maximum = (0.75’0.25) x (0.03/0.97) x 100% = 9.30@; over 907; must have come from the haemolymph. and is further evidence of a true diuresis. As a further consequence. from data in Table 2, it can be calculated that, on the average, less than 1% [i.e. (9.9 x 0.907 - 8.9)/9.9 x 100% = 0.8%] to a maximum of 10% C(9.9 - 8.9)/9.9 x 100% = lO.l%] of the haemolymph water lost through this diuresis was replaced by water from other sources. Flight-muscle
water content
The increase in water content in relation to the dry weight of the flight muscles of insects which flew to exhaustion indicates that these tissues remain adequately hydrated. Their dry appearance, therefore, is deceptive. In fact, their total water content changed very little, if at all. The average flight-muscle dry weights in unflown insects and in insects flown to exhaustion are 3.3 + 0.1 (S.E.) mg (n = 6) and 2.5 f 0.1 mg per (n = 6) insect, respectively (GRINGORTEN. 1979). Using these values and the values for mean per cent water in Table 3, total flightmuscle water can be computed from the following formula: water (mg)=dry
weight (mg) x “;water/(lOO-yf;water).
This produces mean values of 6.4 mg water per insect in the flight muscles of unflown insects and 6.2 mg water in the flight muscles of insects flown to exhaustion. The difference is not significant (r-test, P > 0.05). It is concluded, therefore, that despite the diuresis and loss of water, and the considerable reduction in haemolymph volume, dehydration of the flight muscles does not occur during flight, and is not a factor contributing to ‘exhaustion’. The increase in per cent water in the flight muscles of flown insects must be a result of dry-weight losses in the muscle tissue, rather than net uptake of water or net gain from metabolic water. The average dryweight IoSSis, in fact, considerable-almost 25%. This loss probably also accounts for both their shrunken appearance and for the large decrease in their benzene-extractable dry weight (34.67; of the weight in UnflOWn inSeCtSvs 11.4% in flown insects). It may be noted that the calculated values for total dry-weight loss (0.80 mg) and benzene-extractable dry-weight loss (0.85 mg) are very similar. The nature of this material is not known. Although it appears to be readily extracted in benzene and may, therefore, be lipid, it should be pointed out that the ratio of benzene to tissue during the extraction was very large, and even relatively ‘insoluble’ substances may also have been removed. A more selective extraction technique is required to verify that the substance is, in fact, lipid.
Signifcunce rehc
c$ haemolymph-water
loss and
volume
t ion
The possibility that haemolymph-water loss is related to evaporative cooling was discussed in a previous communication and generally discounted (GRINGORTEN and FRIEND, 1979b). A possible advantage of such water losses in maintaining adequate substrate concentrations for muscle uptake was also mentioned previously (GRINGORTEN and FRIEND. 1979b). In addition, WIGGLESWORTH (1963) and EDWARDS (1964) point out that low haemolymph volume increases ‘aerodynamic efficiency’. However, to maintain or increase the efficiency of fuel transport to the flight muscles in this manner. a mechanism would have to be present which would compensate for the reduction in haemolymph volume in order to maintain circulation pressure. In certain adult Diptera which exhibit low levels of circulating haemolymph, this is achieved by air sacs which inflate during adult development (WIGGLESWORTH, 1963). However, neither such air sacs nor any other mechanism for elevating haemolymph pressure have been reported in R. prolixus. Therefore, it would appear that, from the point of view of fuel transport or aerodynamic efficiency, any benefit derived from a reduction in haemolymph volume must be rather limited. It seems likelier that the large reduction in haemolymph volume ultimately causes circutatory problems in the insect, and may be a factor in limiting the duration of flight. It is possible that a critically low level of circulating haemolymph is reached which prevents a sufficient amount of essential metabolites from reaching the flight muscles or adequate removal of waste products from them. In addition to these factors. R. prolixus may rely on circulating haemolymp? to conduct heat away from the flight muscles, and flight may terminate due to excess heat. Other studies have determined that flight-muscle proline is almost completely depleted in R. prolixus which are flown to exhaustion, and appears to be at least one source of energy in this insect (GRINGORTEN. 1979). Prior to flight, the molal concentrations of proline in haemolymph and flight musc!e are approximately the same. Although it decreases in both tissues during flight, proline begins to drop sharply in the flight muscles at a time when its decline in the haemolymph is already levelling off. At the end of the flight period. there is still a large proline reserve in the haemolymph and the molal concentration is three to six times higher than in the flight muscles. One explanation for these time-course differences and the apparent, ultimate inaccessibility of the haemolymph proline pool for continued uptake and metabolism by the flight muscles may be inadequate circulation, caused by the diuresis which reduces haemolymph volume. Although the time course for the decline in total haemolymph volume was not investigated, indirect evidence, based on changes in haemolymph glycine, indicates that the decline is most rapid during the first 20min of flight, and then tapers off during the remainder of the flight period. Total haemolymph glycine does not change appreciably during flightless than 51, (GRINGORTEN, 1979). so that changes in its molar concentration reflect changes in the haemolymph volume. Haemolymph glycine was found to
Water
balance
in Rhodnius
undergo nearly a 487, increase in concentration during flight. The most rapid increase-approx. 40% of the total--and, therefore, the most rapid decrease in total haemolymph volume, occurred during the first 20min. and the remaining 60% during the next 114 min (median exhaustion time). This pattern of change is similar to that observed for the decline in expressible haemolymph volume during flight (GRINGORTENand FRIEND, 1979b). Direct measurements of expressible volume showed that approx. 60”,, of the total decline occurred during the first 20 min. the rate then diminishing. with the remaining
40”,, occurring
during
the next-l
14 min.
.~~~no~~edyement.s-We are grateful to Ms. Rosemary Tanner for preparing the figures and editing the manuscript. We thank Professor E. Bursell, and Drs. P. A. Langley. J. J. B. Smith and S. S. Tobe for critical review of the manuscript. and Mr. F. W. Schueler for helpful comments. This work was supported by a grant to W. G. Friend from the National Research Council of Canada.
prolixus
during
flight
577
REFERENCES BURSELL E. (1960) Loss of water by excretion and defaecation in the tsetse fly. J. exp. Eiol. 37, 689-697. EDWARDS J. S. (1964) Diuretic function of the labial glands in adult giant silk moths. Hyulophoru cecropia. Nature. Land.
203, 668-669.
GOLDSWORTHY G. J. (1976) Hormones and flight in the locust. In Persprcricrs in Experimental Biology. Vol. 1. Zooloyy (Ed. by DAVIES P. S.). pp. 167 -177. Pergamon Press, Toronto. GRINGORTEN J. L. (1979) Aspects of the flight physiology of Rhodnius prolixus: wing-beat pattern. water-balance. and amino-acid changes during exhaustive flight. Ph.D. Thesis, University of Toronto. GRINGORTEN J. L. and FRIEND W. G. (1979a) Wing-beat pattern in Rhodnius prolixus Stil (Heteroptera: Reduviidae) during exhaustive Right. Can. J. Zool. 57. 391-395. GRINGOR~EN J. L. and FRIEND W. G. (1979bl Haemolymph-volume changes in Rhodnius prolixus during flight. J. exp. Biol. 83, 325-333. GRINGORTEN J. L. and FRIEND W. G. (1979~) Tissue development in Rhodnius prolixus (Hemiptera: Reduviidae): dry-weight changes in fed and unfed post-ecdysial males. Can. Enf. Ill. 735-740. WIGGLESWORTH V. B. (1963) A further function of the air sacs in some Insects. Nururr, Lond. 198. 106.
in Great
WATER
Vol. 28. No. 7. pp. 573-577.
1982
0022-1910~8’i070573~05$03.00!0 0 1982 Prrqamor~ Pwss Ltd
Britain.
BALANCE IN RHODNlUS PROLIXUS DURING FLIGHT: QUANTITATIVE ASPECTS OF DIURESIS AND ITS RELATION TO CHANGES IN HAEMOLYMPH AND FLIGHT-MUSCLE WATER J. L. GRINGORTEN* and W. G. FRIEND Department
of Zoology. (Rrceioed
University 14 September
of Toronto.
Toronto.
Ontario.
1981: revised 27 November
Canada
M5S 1Al
1981)
Abstract-The amount of water voided by male Rhodnius probus which were flown to exhaustion varied from 0 to over 10% of the initial live weight. It accounted for nearly all of the body water lost
during the Right period. Simultaneous measurements on the loss of haemolymph water and an estimate of the amount of faecal water in the excreta indicated that the source of the voided water was primarily the haemolymph. The total water content of the flight muscles changed very little in insects which flew to exhaustion. It is concluded that, despite the diuresis and loss of water. and the considerable reduction in haemolymph volume, dehydration of the flight muscles of male R. prolixus does not occur during these flight periods, and is not a factor contributing to ‘exhaustion’. The possibility that insufficient
haemolymph is a factor limiting the duration of flight is discussed. Key Word Index: Rhodnius prolixus, water balance. flight. diuresis,
INTRODUCI’ION TETHERED male Rhodnius prolixus St&l (Hemiptera: Reduviidae) can be stimulated to fly for periods of 2-3 hr before becoming exhausted (GRINGORTEN and FRIEND, 1979a). The stress of flight stimulates both diuresis and defaecation, and, during this time, the insects can undergo up to a 50% reduction in haemolymph volume (GRINGORTEN and FRIEND, 1979b). Neither phenomenon. we believe, has been described for any other insect in relation to flight stress. Both the excretion and decline in haemolymph volume are related to wing beating, and are not caused by the flight stimulus per se (GRINGORTEN and FRIEND, 1979b). The possibility that a quantitative relationship exists between the diuresis and reduction in haemolymph volume has been suggested previously (GRINGORTENand FRIEND, 1979b). Part of the decline in haemolymph volume may be attributed to a failure of the hindgut to resorb water secreted by the Malphighian tubules (cf. GOLDSWORTHY, 1976). The percentage water in the haemolymph and the specific gravity remain relatively constant during these long flight periods (GRINGORTEN and FRIEND, 1979b). However, a conspicuous feature of flight muscle from insects which have flown to exhaustion is the lack of surrounding haemolymph and the shrunken, ‘dehydrated’ appearance of the muscle tissue. Flight-induced tissue dehydration was considered a distinct possiblity in view of the diuresis and large reduction in haemolymph volume. In this study, we examine the effects of excretion on the water balance of R. prolixus flown to exhaustion, particularly the quantitative relationships between the amount of water voided, total body-water loss, the * Present address: International Atomic Energy Agency. P.O. Box 100. A-1400 Vienna. Austria. LP 28!7
-*
573
excretion.
haemolymph.
flight
decline in haemolymph volume, and the changes occurring in the water content of the flight muscles.
MATERIALS
AND METHODS
The rearing conditions and flight stimulus for experimental insects have been described previously by GRING~RTEN and FRIEND (1979b). All insects were unmated males which had completed their adult development, and were in a state of hunger when flown (a necessary stimulus for sustained flight). but not yet starving (GRINGORTENand FRIEND, 1979~). Insects were weighed prior to and immediately after being flown to determine live-weight loss. Direct measurement of total body water in flown insects was made by weighing before and after oven drying. Their pre-flight body water was determined indirectly. from a regression analysis of total body water on live weight, performed on 33 control (unflown) insects. The net loss in body water during the flight period was then calculated by subtraction. The water content of the flight muscles was determined by a liquid-paraffin technique similar to that described by B~JRSELL(1960). A correction factor was determined for dry-weight losses in the tissue which occur during the extraction of the paraffin by benzene. This factor was determined by performing the same paraffin-benzene procedure on muscle tissue which had been oven-dried and weighed. and then reweighed following the extraction. The correction factor is the ratio pre-extracted/post-extracted dry weight. Dry-weight values for the experimental tissue were multiplied by this factor to obtain corrected dry weights. Tissue samples were pooled from three insects for each water-content measurement and each correction-factor determination.
J. L.
574
and W. G. FRIEND
GRINCORTEN
Table 1. Live-weight, total body-water and excretion losses in male R. prolixus flown to exhaustion
Insect 1 2 3 4 5 6 7
Flight duration (min)
Pre-flight live weight (mg)
Live-weight loss (mg)
Pre-flight body water* (mg)
115.1 94.8 101.8 114.8 116.1 111.2 72.9
16.3 15.6 13.7 13.2 11.8 6.0 0.9
77.2 63.6 68.2 77.0 77.8 74.5 48.9
207 153 200 184 135 151 106
Body-water loss (mg) 15.0 12.5 13.4 10.0 12.5 2.1 0.3
Excreta wet weight (mg) 14.2 t 11.2
f 9.9 4.0 0
Excreted water (mg) 13.5 12.91 11.1 10.7$ 9.9 3.3 0
* Determined from the regression curve in Fig. 1. i Insect excreted, but weight not obtained. $ Determined from the regression curve in Fig. 2. Excreta produced during the flight period were collected in vials containing liquid paraffin. The vial and paraffin together were pre-weighed, which permitted direct determination of the wet weight of the excreta at the end of the flight period. Water content was subsequently measured in a manner similar to that used for flight muscle. Dry weight of the excreta could then be calculated by subtracting water content from wet weight, which obviated the .need to perform a benzene extraction. Haemolymph volume was determined by isotope dilution using [carboxyl-‘4C]inulin. The technique has been described in detail elsewhere (GRINGORTEN and FRIEND. 1979b).
RESULTS General features of excretion The excretion pattern in insects which flew was quite variable. During flight experiments on approx. 150 insects in this and in related studies, excretion was never observed prior to 10 min of flight. Not all insects excrete. In a sample of 14 insects which were flown to exhaustion, and observed continuously throughout their flight periods (103-209 min), 2 failed to void. Among those that did void, the frequency ranged .from 1 to 6 eliminations, and ceased altogether within 20min prior to the end of the flight period. In most cases, the final elimination occurred more than one hour before the end of the flight period. Invariably, the first elimination was dark in colour, representing a clearing of the rectum of faecal matter. Subsequent eliminations became progressively Table 2. Haemolymph-water
Insect 1 2 6 Mean S.E.
lighter in colour until they were clear and colourless, an indication of diuresis. In a number of instances the diuresis was again followed by a defaecation, and in one insect with a high excretion frequency, urination and defaecation alternated several times. In two cases, insects which had excreted numerous times during the flight period were subsequently observed to open the anus without voiding anything. One of these kept the anus splayed for 4 min. Quantitative aspects of excretion and water balance
Excretion of water during flight was measured in seven insects (Table l), and in three of these, the loss of water from the haemolymph was also determined (Table 2). The relationship between live weight and total body water in control insects, which was used to determine the pre-flight, total body water of flown insects, is illustrated in Fig. 1. Attempts to collect the excreta from insects 2 and 4 (Table 1) were unsuccessful; r;timates of the amounts of water excreted by these insects were made from a linear regression of excreted water on live-weight loss,using data from the remaining insects (Table 1, Fig. 2). The amounts excreted during flight were quite variable (Table l), ranging from 0 (insect 7) to over 12% (insect 1) of the initial live weight. However, it is evident that where excretion did occur, it accounted for most (median 83%) of the live-weight loss incurred by the insects. It is also apparent that the excreta were quite dilute, averaging (median) 97% water. Excreted water accounted for approx. 90% (median) of the body water lost among all insects during the flight period, and virtually all (97%) of the body water lost among insects l-6 (excreted water > 0). The total amount of
loss in male R. prolixus flown to exhaustion
Pre-flight Post-flight haemolymph haemolymph volume* volume
Haemolymphvolume loss
Haemolymphwater loss
Flight duration
Pre-flight live weight
Excreted water
(min)
(mg)
(PI)
(PI)
(Pi)
(mg)
(mg)
207 153 151 -
115.1 94.8 111.2
31.7 24.7 30.4
19.6 15.2 23.4
12.1 9.5 7.0 -
11.3 8.9 6.6 8.9 1.4
13.5 12.9 3.3 9.9 3.3
* Determined from a linear regression of haemolymph volume on live weight (GRINGORTEN and FRIEND, 1979b).
Water balance in Rhodnius prolixus during Right
._ ” . ..
. .. . ’. :=* .
Fig. I. The relationship between live weight and total body in unflown male R. profixus. Each point is a single insect. The equation of the best-fit curve is: body water (mg) = 0.670 x live weight (mg) + 0.042: r = 0.989.
water
575
Table 3. Per cent water in the flight muscles of unflown male R. prolixus and of insects flown to exhaustion*
Mean S.E.
Unflown Cd)
Flownt (“&)
66.5 66.0 65.8 65.7 66.0 0.2
71.1 71.5 71.3 0.2
was * Each measurement performed on samples pooled from three insects. t Flight duration: 118-160 min. median 13I min.
water excreted by different insects which flew to exhaustion could be more than 10% of the initial live weight (insects 1, 2 and 3). and perhaps as much as its dry weight, as a result of the benzene extraction. 14”j0(insect 2). whereas muscle from flown insects lost only 11.4%. A linear regression of haemolymph volume on live Changes in the per cent water of the flight muscles weight, computed previously by GRING~RTENand of insects which flew to exhaustion are given in FRIEND (1979b), was used to determine the pre-flight Table 3. It is evident that relative water content inhaemolymph volume of insects which were flown to creased during flight (r-test, P < 0.01). exhaustion. From direct measurement of the postflight volume, the specific gravity (1.03) and the per cent water (91.0~4) in the haemolymph (GRINGORTEN DISCUSSION and FRIEND, 1979b). the amount of water lost from Quantitative aspects of excretion and water balance the haemolymph could be determined (specific gravity and per cent water remain constant): In the present investigation, water voided by male haemolymph-water loss (mg) = [pre-flight-postR. prolixus which were flown to exhaustion accounted flight haemolymph volume (PI)] x S.G. x O/;water/ for nearly all of the body water lost. The similarity of 100. the average values for haemolymph-water loss and When these values were compared with the amount excreted water (Table 2) indicated that the principal of water lost by excretion (Table 2), it was apparent source of excreted water was the haemolymph, and that excreted water could account for the water lost that little of this haemolymph water was replenished from the haemolymph (t-test, P > 0.50). from other sources during the flight period. That the haemolymph was the source was consistent with the Flight-muscle water content observation that the excreta became progressively There was a considerable difference between the clear and colourless during flight, characteristic of a dry-weight correction factor for flight muscle from diuresis, and were found. in actual fact, to be primarunflown insects and that from insects flown to ily water. exhaustion. Muscle from unflown insects lost 34.6X of Several points should be noted in any quantitative comparison between excreted water and loss of haemolymph water. The first excretion may possibly 14 cause the expulsion of a quantity of water which exceeds the intitial amount of water removed from the 12 haemolymph by the Malpighian tubules. particularly if the rectum is relatively full and near a critical “voiding threshold” prior to the commencement of flight. In addition. some faecal water is expelled along with urine water. and, finally. the amount of water calculated to be lost from the haemolymph is a net quantity. There is some evidence, in fact, that the per cent water in the midgut declines during flight (GRINGORTEN and FRIEND, unpublished). Any transfer of water from the midgut (or other sources) to the haemolymph will reduce the net loss in haemolymph water. All of these factors could cause excreted water to exceed the net loss in haemolymph water. However, there is one factor which could reduce Fig. 2. The relationship between excreted water and livethis tendency, or even have the opposite effect. There weight loss in male R. prolixus flown to exhaustion. Each point is a single insect. The equation of the regression line is no certainty that the total amount of water filtered from the haemolymph by the Malpighian tubules is is: excreted water (mg) = 0.906 x live-weight loss (mg) actually voided. especially if there is relatively little 1.268; r = 0.995.
1
J. L. GRINC~RTEN and W.G. FRIEND
576
fluid in the rectum prior to flight. In this situation, haemolymph-water loss could exceed the quantity of water excreted. In addition, the relative contribution of faecal water to the total water excreted is generally expected to be small. The per cent water in the posterior midgut (tissue + luminal contents) of unflown insects, under present experimental. conditions, is less than 75% (GRINGORTEN and FRIEND, unpublished). Therefore, if the excreta are 97% water, not more than 10% of this water, on the average, could have been derived from faeces [i.e. maximum = (0.75’0.25) x (0.03/0.97) x 100% = 9.30@; over 907; must have come from the haemolymph. and is further evidence of a true diuresis. As a further consequence. from data in Table 2, it can be calculated that, on the average, less than 1% [i.e. (9.9 x 0.907 - 8.9)/9.9 x 100% = 0.8%] to a maximum of 10% C(9.9 - 8.9)/9.9 x 100% = lO.l%] of the haemolymph water lost through this diuresis was replaced by water from other sources. Flight-muscle
water content
The increase in water content in relation to the dry weight of the flight muscles of insects which flew to exhaustion indicates that these tissues remain adequately hydrated. Their dry appearance, therefore, is deceptive. In fact, their total water content changed very little, if at all. The average flight-muscle dry weights in unflown insects and in insects flown to exhaustion are 3.3 + 0.1 (S.E.) mg (n = 6) and 2.5 f 0.1 mg per (n = 6) insect, respectively (GRINGORTEN. 1979). Using these values and the values for mean per cent water in Table 3, total flightmuscle water can be computed from the following formula: water (mg)=dry
weight (mg) x “;water/(lOO-yf;water).
This produces mean values of 6.4 mg water per insect in the flight muscles of unflown insects and 6.2 mg water in the flight muscles of insects flown to exhaustion. The difference is not significant (r-test, P > 0.05). It is concluded, therefore, that despite the diuresis and loss of water, and the considerable reduction in haemolymph volume, dehydration of the flight muscles does not occur during flight, and is not a factor contributing to ‘exhaustion’. The increase in per cent water in the flight muscles of flown insects must be a result of dry-weight losses in the muscle tissue, rather than net uptake of water or net gain from metabolic water. The average dryweight IoSSis, in fact, considerable-almost 25%. This loss probably also accounts for both their shrunken appearance and for the large decrease in their benzene-extractable dry weight (34.67; of the weight in UnflOWn inSeCtSvs 11.4% in flown insects). It may be noted that the calculated values for total dry-weight loss (0.80 mg) and benzene-extractable dry-weight loss (0.85 mg) are very similar. The nature of this material is not known. Although it appears to be readily extracted in benzene and may, therefore, be lipid, it should be pointed out that the ratio of benzene to tissue during the extraction was very large, and even relatively ‘insoluble’ substances may also have been removed. A more selective extraction technique is required to verify that the substance is, in fact, lipid.
Signifcunce rehc
c$ haemolymph-water
loss and
volume
t ion
The possibility that haemolymph-water loss is related to evaporative cooling was discussed in a previous communication and generally discounted (GRINGORTEN and FRIEND, 1979b). A possible advantage of such water losses in maintaining adequate substrate concentrations for muscle uptake was also mentioned previously (GRINGORTEN and FRIEND. 1979b). In addition, WIGGLESWORTH (1963) and EDWARDS (1964) point out that low haemolymph volume increases ‘aerodynamic efficiency’. However, to maintain or increase the efficiency of fuel transport to the flight muscles in this manner. a mechanism would have to be present which would compensate for the reduction in haemolymph volume in order to maintain circulation pressure. In certain adult Diptera which exhibit low levels of circulating haemolymph, this is achieved by air sacs which inflate during adult development (WIGGLESWORTH, 1963). However, neither such air sacs nor any other mechanism for elevating haemolymph pressure have been reported in R. prolixus. Therefore, it would appear that, from the point of view of fuel transport or aerodynamic efficiency, any benefit derived from a reduction in haemolymph volume must be rather limited. It seems likelier that the large reduction in haemolymph volume ultimately causes circutatory problems in the insect, and may be a factor in limiting the duration of flight. It is possible that a critically low level of circulating haemolymph is reached which prevents a sufficient amount of essential metabolites from reaching the flight muscles or adequate removal of waste products from them. In addition to these factors. R. prolixus may rely on circulating haemolymp? to conduct heat away from the flight muscles, and flight may terminate due to excess heat. Other studies have determined that flight-muscle proline is almost completely depleted in R. prolixus which are flown to exhaustion, and appears to be at least one source of energy in this insect (GRINGORTEN. 1979). Prior to flight, the molal concentrations of proline in haemolymph and flight musc!e are approximately the same. Although it decreases in both tissues during flight, proline begins to drop sharply in the flight muscles at a time when its decline in the haemolymph is already levelling off. At the end of the flight period. there is still a large proline reserve in the haemolymph and the molal concentration is three to six times higher than in the flight muscles. One explanation for these time-course differences and the apparent, ultimate inaccessibility of the haemolymph proline pool for continued uptake and metabolism by the flight muscles may be inadequate circulation, caused by the diuresis which reduces haemolymph volume. Although the time course for the decline in total haemolymph volume was not investigated, indirect evidence, based on changes in haemolymph glycine, indicates that the decline is most rapid during the first 20min of flight, and then tapers off during the remainder of the flight period. Total haemolymph glycine does not change appreciably during flightless than 51, (GRINGORTEN, 1979). so that changes in its molar concentration reflect changes in the haemolymph volume. Haemolymph glycine was found to
Water
balance
in Rhodnius
undergo nearly a 487, increase in concentration during flight. The most rapid increase-approx. 40% of the total--and, therefore, the most rapid decrease in total haemolymph volume, occurred during the first 20min. and the remaining 60% during the next 114 min (median exhaustion time). This pattern of change is similar to that observed for the decline in expressible haemolymph volume during flight (GRINGORTENand FRIEND, 1979b). Direct measurements of expressible volume showed that approx. 60”,, of the total decline occurred during the first 20 min. the rate then diminishing. with the remaining
40”,, occurring
during
the next-l
14 min.
.~~~no~~edyement.s-We are grateful to Ms. Rosemary Tanner for preparing the figures and editing the manuscript. We thank Professor E. Bursell, and Drs. P. A. Langley. J. J. B. Smith and S. S. Tobe for critical review of the manuscript. and Mr. F. W. Schueler for helpful comments. This work was supported by a grant to W. G. Friend from the National Research Council of Canada.
prolixus
during
flight
577
REFERENCES BURSELL E. (1960) Loss of water by excretion and defaecation in the tsetse fly. J. exp. Eiol. 37, 689-697. EDWARDS J. S. (1964) Diuretic function of the labial glands in adult giant silk moths. Hyulophoru cecropia. Nature. Land.
203, 668-669.
GOLDSWORTHY G. J. (1976) Hormones and flight in the locust. In Persprcricrs in Experimental Biology. Vol. 1. Zooloyy (Ed. by DAVIES P. S.). pp. 167 -177. Pergamon Press, Toronto. GRINGORTEN J. L. (1979) Aspects of the flight physiology of Rhodnius prolixus: wing-beat pattern. water-balance. and amino-acid changes during exhaustive flight. Ph.D. Thesis, University of Toronto. GRINGORTEN J. L. and FRIEND W. G. (1979a) Wing-beat pattern in Rhodnius prolixus Stil (Heteroptera: Reduviidae) during exhaustive Right. Can. J. Zool. 57. 391-395. GRINGOR~EN J. L. and FRIEND W. G. (1979bl Haemolymph-volume changes in Rhodnius prolixus during flight. J. exp. Biol. 83, 325-333. GRINGORTEN J. L. and FRIEND W. G. (1979~) Tissue development in Rhodnius prolixus (Hemiptera: Reduviidae): dry-weight changes in fed and unfed post-ecdysial males. Can. Enf. Ill. 735-740. WIGGLESWORTH V. B. (1963) A further function of the air sacs in some Insects. Nururr, Lond. 198. 106.