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Utilization of U14C amino acids or U14C protein by adult Glossina morsitans during in utero development of larva
r. Insect Pfq&l.,
1974, Vol. 20, pp. 2157 to 2170. Pergamon Press. Printed in Great Britain
UTILIZATION OF U-f*C AMINO ACIDS OR U-l*C PROTEIN BY ADULT GLOSSINA MORSITANS DURING 1N UTTER DE~LOP~ENT OF LARVA P. A. LANGLEY Tsetse Research Laboratory,
and R. W. PIMLEY
University of Bristol School of Veterinary Science, Langford. Bristol BS18 7DU, England (Received 3 April 1974)
Abstract-Administration of U-l*C protein hydrolysate in the diet of adult female GEoss57Iamorsr%ansat different times throughout the second reproductive cycle was followed by analysis of the distribution of radioactivity between the adult flies, their excreta, and the fully grown third instar larvae produced by these flies. A constant proportion of the total administered label was recoverable independently of the time lapse between adm~istration and assay. Peak incorporation of labelled material occurred in the larva between the seventh and eighth day of a 9 or 10 day interlarval period, indicating that the larva feeds avidly on recently synthesized maternal uterine gland secretion at this time. Haemocoelic injection of II-l% protein hydrolysate into similar adult females, between feeds, resulted in continued incorporation of labelled material by the larva to within 12 hr of parturition. Results are consistent with the hypothesis that uterine gland secretion and larval feeding continue throughout the intrauterine life of the larva. A constant and low proportion of detectable label remained in the adult fly while increased inco~omtion by the larva was paralleled by a reduction of detectable label in the adult excreta. This indicates direct competition between the uterine gland cells and those of the Malpighian tubules for free amino acids in the haemolymph. Administration of U-14C protein in the adult diet did not result in incorporation of label by the developing larva, and the bulk was excreted as protein by the adult fly. Apparently the midgut trypsin of G. morsitanr is incapable of splitting this labelled protein. Analysis of urine and haemolymph samples from flies in early pregnancy, recently fed on a diet containing U-‘*C protein hydrolysate or U-14C protein, shows that free labelled amino acids in the diet enter the adult haemol~ph almost immediately after feeding, and are excreted along with dietary water during initial diuresis. The labelled protein used in these experiments was not taken up by the haemolymph and consequently did not appear in the urine. Implications are that the adult female G. morsitum possesses little storage capacity for substances in the diet which are destined to provide nutrients for the developing larva. Assuming a 48 hr digestion time, the digestive products of a blood meal ingested on day 5 or 6 of a 9 day interlarval period will provide the bulk of nutrients for larval growth. It is therefore significant that blood meals ingested at this time are larger than those ingested earlier or later in the cycle. 2157
2158
P.
A.
LANGLEY
AND
R. w.
PIMLJIY
INTRODUCTION
THE MODEof reproduction in tsetse flies is adenotrophic viviparity, in which a fertilized adult female produces a single, fully fed third instar larva at regular intervals of approximately 9 days at a maintenance temperature of 25°C. The adult feeds exclusively on vertebrate blood and the developing larva feeds on a secretion produced by the uterine glands of the female: the free life of the larva consists only of a short burrowing period of about 1 hr after which pupariation occurs (BUXTON,1955). Thus the birth product of an adult female tsetse fly is larger than itself, since not only will the larva produce an adult of the same size as the parent after metamorphosis, but the larva will contain all the nutritional requirements for survival and metamorphosis throughout the relatively long (approx. 30 days at 25°C) intra-puparial life. On the basis of histological observation it was stated by HOFFMANN(1954) that uterine gland secretion in G. palpalis was of limited duration and occupied no more than 60 hr from the third day following ovulation. A similar interpretation was given by BURSELLand JACKSON(1957) for G. pallidipes. However, ROBERTS (1971) found uterine gland secretion at the mouth of the milk duct opening in G. austeni from the time of hatching of the first instar larva until parturition, and concluded that secretion was available throughout larval life. Recent work by TOBE et al. (1973) confirms the observations of ROBERTSfor G. au&k. In G. palpalis embryonic development at 26°C apparently occupies 50 to 60 hr (HOFFMANN, 1954) while ROBERTS(1973) has shown that the first instar larva of G. morsitans hatches only after 4 days. Thus larval growth is extremely rapid and is confined to the 4 or 5 days prior to larviposition in this species. TOBE and DAVEY(1972) have shown that the pregnant female G. austeni feeds to constant volume, which provides a diminishing food capacity as the larva grows. Using injections of labelled tyrosine and leucine, TOBEand DAVEY(1974a, b) have demonstrated a very rapid synthesis of protein within the uterine glands, and an equally rapid transfer of nutrients from the adult oenocytes and fat body to the secretory glands is implied. CMELIK et al. (1969) have concluded that sufficient nutritive secretion (except for aromatic amino acid requirements) for complete larval development could be provided by a single blood meal of 40 mg in G. monitans. The timing of events such as adult feeding, larval hatch, and transfer of nutrient to the larva is obviously critical in Gloss&a. It is therefore important to know the extent to which the many species of Glossina may differ in their solution to the problem of larval nutrition, or whether the sometimes conflicting information in the literature can be rationalized to provide a comprehensive theory of the dynamics of nutrition and reproduction. The present paper is a step towards such rationalization and concerns the timing of nutrient uptake by the larva of G. niorsitans during the second reproductive cycle. An attempt has been made to assess the relative importance of successive adult meals in larval development by determining the fate of labelled amino acids or protein after administration to the adult either in the diet or by injection.
UTILIZATION
OF AMINO ACIDS AND PROTEIN BY ADULT GLOSSINA
MATERIALS
2159
AND METHODS
Adult G. morsitans were maintained according to the techniques used routinely at Langford (NASH et al., 1971) and offered food daily on goats. Groups of fertilized adult females were isolated as soon as the black polypneustic lobes of their first third instar larvae were visible through the abdominal integument (day 15 or 16 of adult life). Flies which deposited their larvae within the next 24 hr were segregated and designated day 1 of the second reproductive cycle. These were transferred to the Agar/Parafilm membrane feeding system described by LANGLEY (1972) and modified by MEWS (unpublished) in that the blood was presented on a stippled or grooved glass plate overlain by the membranes and that the agar contained 2% glycerol to assist moisture retention in the membrane. Successive groups of 10 flies each were segregated at intervals of 24 hr for 9 days. During the second reproductive cycle the flies were fed on haemolysed bovine blood containing 10es M ATP. On the ninth day of collection groups of flies from 1 to 9 days old provided a full range of different larval development stages at intervals of 24 hr. All the flies in each daily age group were isolated and weighed before and after feeding. Each fly was fed on whole defibrinated goat’s blood containing 0.1 &i U-14C protein hydrolysate (Radiochemical Centre, Amersham) per 50 mg blood, and 1O-3 M ATP. Following the labelled meal all flies were isolated in 3 x 1 in. glass tubes closed with perforated Parafilm, and maintained in an incubator at 25°C. A small circle of filter paper was included to collect excreta. The flies were individually fed each day on haemolysed bovine blood and the glass tubes housing the flies were changed daily. Flies would only feed up to the completion of 70 to 75 per cent of the second interlarval period: those which produced a larva on day 9 refused to feed after the seventh day. As soon after the second larviposition as possible the distribution of U-14C label between adult excreta, the parent flies, and their larvae was determined using a scintillation counting method (Nuclear Enterprises NE250 scintillant and a Nuclear Chicago Scintillation Counter Model 6850). Aliquots of excreta (0.2 ml) from individual tubes were assayed after dissolving in 2 ml 0.4% L&CO, and a correction made for the quenching effect of haematin, uric acid, and salt, using an internal standard. Flies and larvae were individually hydrolysed in 6 N HCl in sealed tubes at 120°C for 1 to 3 days, evaporated to dryness at 50°C and the hydrolysate re-dissolved in 2 ml water before counting O-2 ml aliquots. Corrections were again made for the quenching effect of non-amino acid contaminants in samples. After individual assay of excreta samples, the remaining portions of each sample were pooled, taken to pH 2-O with 6 N HCl, and after centrifugation the supernatant was assayed for U- 1% activity, with appropriate corrections for quenching. The precipitate which consisted mainly of uric acid &,, 298 nm on a Pye Unicam Spectrophotometer) was washed several times with 0.1 N HCl, finally re-dissolved in 0.4% Li,CO,, and assayed for UJ4C activity.
2160
P. A. LANGLEYANDR. W.
PIMLEY
A similar experiment was conducted upon groups of flies which received a labelled meal containing 0.1 $Zi U-W protein (Radiochemical Centre, Amersham) per 50 mg blood instead of the labelled hydrolysate. Both experiments were replicated and the results were repeatable. However, before the second U-i*C protein experiment the labelled material was dissolved in Tris buffer at pH 8 and run three times through a 2.5 x 1 cm column of Sephadex G-25, in order to remove small molecules such as amino acids and peptides before adding it to the blood for fly feeding. The new concentration was therefore 0.05 $Zi UJ4C protein per 50 mg blood. A third experiment involved 9 similar groups of flies on their second reproductive cycle except that they were injected with 1~1 ( =O*OS &i) U-14C protein hydrolysate per fly. Injections were made 24 hr after the previous meal and injected flies were not offered food for at least 2 hr following the injection. Excreta, flies, and larvae were assayed for U-14C activity as before. The experiment was replicated. Finally, groups of flies were starved for 48 hr early in their second reproductive cycle and then fed on either haemolysed or whole defibrinated bovine blood containing appro~mately 5 $Zi UJ*C protein hydrolysate per ml. Urine and haemolymph were collected as 1 ~1 samples from flies at 5, 15, and 30 min after feeding. Both G. morsitans and G. au&k females were used. The experiment was repeated with G. morsitans only, using U-14C protein in the diet at a rate of 10 @i/ml of blood, only approximately 60 per cent of which was soluble in the blood serum. RESULTS
Fig. 1 shows the relationship between total counts recovered (excreta, fly, and larva) and the size of meal containing U-r*C protein hydrolysate. It is apparent that the relationship is close to linear. Therefore, metabolic losses as WO, were either negligible or at least they were a constant proportion of the flies’ intake of label regardless of the time lapse between administration of the label and assay. Thus any differences in total counts recovered were attributable only to differences in meal size and hence to label intake. A similar result was obtained with U-14C protein in the blood meals. Losses from the larva would be confined at most to WO, since the larva stores all nitrogenous waste and its anus is closed. Fig. 2 shows the distribution of label between the adult fly, the total amount of excreta collected, and the fully developed third instar larva produced by flies receiving UJ4C protein hydrolysate in their diet at the time indicated during their second reproductive cycle. Radioactivity is expressed as a percentage of the total counts recovered: the time of administration of the label is expressed as a percentage of the interlarval period which varied from 9 to 12 days. Each point represents the mean of between 3 and 12 observations. It is apparent (Fig. 2) that the amount of label retained by the fly remained constant at a low level regardless of the time during the cycle at which it was administered. The slight rise when label was administered after completion of
UTILIZATION
OF AMINO
ACIDS
AND
Meal
PROTEIN
size,
BY
ADULT
GLOSSINA
2161
mg.
FIG. 1. Relationship between total counts recovered (fly + excreta+ larva) and size of ingested blood meal containing U- 14C protein hydrolysate. Labelled meals administered at different times during 9 day interlarval period of G. morsitans females on their second reproductive cycle. 0, Day 1; 0, day 3 ; n , day 4; 0, day 5; x, day 7; A, day 8. Relationship is linear (J = 1.51x-6.91) and independent of time lapse between administration and assay.
I
0 Proportion
IO
I
I
I
I
I
I
20
30
40
50
60
70
of interlorvol
period
completed,
I_ 00 %
FIG. 2. Distribution of radioactivity between adult G. morsitans female, its excreta, and fully grown larva, of flies receiving U-14C protein hydrolysate in their diet at the time indicated during their second reproductive cycle, and assayed after la&position. 0-0, Adult fly; 0-0, fully grown larva; x - x , adult excreta.
2162
P. A.
LANGLEY
AND
R. w.
PIMLEY
70 per cent of the interlarval period was probably due to an inadequate period remaining for the excess to be excreted before la~position and assay occurred. The most significant feature of Fig, 2 is the rapid uptake of label by the larva when administered in a blood meal ingested by the female fly only 2 or 3 days before larviposition. The increased uptake by the larva was accompanied by a corresponding decrease in the amount of excreted label. Following acid precipitation of excreta samples, more than 90 per cent of the uric acid present was found in the precipitate. However, more than 90 per cent of the measured radioactivity was found in the supernatant. Thus we concluded that incorporation of label into the uric acid molecule was negligible and that most of the radioactivity in the excreta was present in amino acids. From Fig. 3 it is apparent that the administration of U-W protein in the diet resulted in most of the Iabel being excreted. There was little evidence of rapid
60-
Proportion of interlarvalperiod completed,
%
FIG. 3. Distribution of radioactivity between adult G. morsituns female, its excreta, and fully grown larva, of flies receiving U-l*C protein in their diet at the time indicated during their second reproductive cycle, and assayed after larviposition. O-O, Adult fly; O-O, fully grown larva; x - x , adult excreta.
incorporation of this protein by the larva regardless of its stage of development at the time of administration. After separation of the adult excreta on a Sephadex G-25 column most of the radioactivity was found to be still in the protein fraction. Hence it was concluded that the labelled protein (heat denatured and of plant origin) had remained largely undigested.
UTILIZATION
OF AMINO ACIDS AND PROTEIN BY ADULT GLOSSINA
2163
Fig. 4 shows the pattern of distribution of label following injection of UJ*C protein hydrolysate into adult females at various times during their second reproductive cycle. The distribution is similar to that obtained following administration of the hydrolysate in the diet, except that the massive uptake of label by the larva
Proportion
of interlorvol
period
completed,
%
FIG. 4. Distribution of radioactivity between adult G. snorsitans female, its excreta, and fully grown larva, of Aies injected with U-W protein hydrolysate at the time indicated during their second reproductive cycle, and assayed after larviposition. O-0, Adult fly; O-0, fully grown larva; x - x , adult excreta.
can be seen to continue to within 12 hr of larviposition. This means that the third instar larva normally feeds avidly on the nutritive secretion produced by the parent until very shortly before larviposition and that this secretion is synthesized very rapidly by the female fly. Furthermore, it suggested to us that UJ*C amino acids in gplution in blood meals were entering the haemolymph of the fly very soon after feeding (Fig. 2). This contention was reinforced by comparing the rate of excretion of label in U-W protein hydrolysate-fed and UJ4C protein-fed flies. More than 80 per cent of excreted label had been eliminated by the former within 24 hr of feeding while 48 hr was necessary for an equivalent amount to be excreted by the latter (Table 1). Table 2 shows the results of sampling urine and haemolymph from flies fed on blood meals containing U-W protein hydrolysate. It is apparent that there was little difference between diets of whole defibrinated blood or haemolysed blood. Also the indication is that G. austeni behaves similarly to G. morsituns.
2164
P. A. LANGLEYANDR. W. PIMLEV
TABLE ~-AMOUNT OF UJ4C ACTIVITYIN EXCRBTAOF G. morsitans FEMALESAT DIFFERENT TIME INTERVALSFOLLOWINGA BLOODMEAL CONTAININGU-i’c AMINOACID MIXTUREOR U-r’c PROTEIN, EXPRESSEDAS PERCENTAGEOF TOTALACTIVITYRECOVERED FROM EXCRETA Time interval after feeding (hr) Label in diet
O-24
2448
48-72
72-96
UJ4C ammo acid mixture U-14C protein
79.8
13.7
3.0
1.9
0.9
0.7
40.2
47.0
6.0
2.5
2.0
2.3
96-120
120-144
Result of running excreta through Sephadex G-25 column indicates that a significant proportion of activity in excreta of U-i4C protein-fed flies is due to undigested protein.
70 ti E
60
Age
P
following
first
lorviposition,
doy5.
(b)
IOOr
‘Z so-
.i? v) ? ; : t
604020 -
I z”
I I I 123456799
0
Age following
I first
I
I
I
lorviposition,
I
days.
50C--
(c)
1 , / ,
%
(
,
,
,
,
,
123456789
0 Age
following
first
lorviposition,
days.
FIG. 5(a). Sizes of labelled meals ingested by experimental G. morsitans on different days of their 9 to 12 day second interlarval period. Vertical lines represent fiducial limits of mean values. (b) Frequency of feeding by G. morsitans females during the 9 day period of their second reproductive cycle, and fed on rabbit. (c) Sizes of meals ingested by G. morsitans females during the 9 day period of their second reproductive cycle, and fed on rabbit.
Haemolysed bovine blood I- 1O-s M ATP
G. austeni
Species
(5) 164 k20.2
‘12p: rf:17.0 (11 300 -
(18) 268 244.2
(7) 262 + 70.0
15
Urine
-
(6) 1114 z!z535.2
(7) 1087 ?I 586.8
30
Food
TSETSE FLIESEARLY
OF U-r4C LABELLEDAMINO
FEMALE ACIDMIXTURB
IN TIIEIRSBC~ND
-
-
CONCENTRATION
OF u-‘*c
9162 + 382.0
(10) 9162 rt 382.0
(10) 8550 + 328.6
Blood mea1
PROTEIN
EARLY IN THEIR SECOND
(15) 3877 +_676.4
-
30
G. morsitafls
(6) 2707 t 1160.8
OF ADULT FEMALE
(4) 4111 k6.58.2
(5) 3828 f 952.8
-
15
Haemolymph
(7) 620.4 Z!I258.8
30
Figures in brackets = n. Values given are means & fiducial limits.
(13) 190.0 z!z52.0
15
Urine
(7) 271.2 z!I147.0
5
(6) 478.3 z!z130.4
15
Haemolymph
(10) 631.5 + 262.6
30
(5) 8971
Blood meal
Radioactivity in counts/min per ~1 at the timed intervals indicated (min)
(16) 82.2 * 18.8
5
5 (19) 3630 f451*0
A KNOWN
IN URINE AND HAEMOLYMPH
A BLOOD MEAL CONTAINING
MATERIAL
CYCLE, FOLLOWING
OF LABELLED
Fresh defibrinated bovine blood + 1O-3 M ATP
REPRODUCTIVE
3---CONCENTRATION
G. morsitans
TABLE
Haemolysed bovine blood + 1O-3 M ATP
G. morsitans
!l% rt:17.8
5
IN URINE AND HAEMOLYMPH 0P ADULT CONTAINING AKNOWN CONCENTRATION
Radioactivity in counts/min per ~1 at the timed intervals indicated (min)
MEAL
Figures in brackets = n, Values given are means + fiducial limits.
Fresh defibrinated bovine blood + 1O-3 M ATP
Food
A BLOOD
G. morsitans
Species
REPRODUCTIVECYCLE,FOLLOWING
TABLE ~-CONCENTRATIONOF LABELLED MATERIAL
z z
z
8
0
P
$
5
2
3 2
E
B
8
!!
z
%
$
5 i: Ki ?;
2166
P. A. LANGLEY ANDR. W. PIMLEY
The initial drops of urine contained only small quantities of label while the haemolymph of the flies contained 40 per cent of the label in the blood meal within 5 min of feeding. Thirty minutes after feeding the urine also contained significant quantities of label. This result shows that amino acids in solution in the blood meal were eliminated by passage across the anterior midgut wall and voided to the exterior via the Malpighian tubules during normal diuresis. Nevertheless, the 5 and 15 min urine samples contained few counts and one must postulate that the passage of amino acids through the Malpighian tubules is initially slower than that of water. Table 3 summarizes the results of experimental feeding of G. morsitans females on blood containing UJ4C protein. The amount of label in the urine and haemolymph of such flies increased during the 30 min following feeding, but the amount of label present in each did not exceed 7 per cent of the total label present in the blood meal. It is most likely that the presence of label in urine and haemolymph was due to amino acid or peptide contamination of the U-14C protein sample, which was not purified before use. The U-14C protein did not apparently enter the haemolymph of the fly as did UJ4C protein hydrolysate. The sizes of meals ingested by G. morsitans fed on the diet containing UJ4C protein hydrolysate are shown in Fig. 5(a). The largest meals were taken immediately after larviposition (day 1) and again between day 5 and 7. Large meals ingested at the later stages would be digested in time to provide the large quantities of nutritive secretion for growth of the third instar larva. Fig. 5(b) shows that between day 5 and 7 all flies would be expected to feed at least once, and on rabbit hosts a similar peak of meal size is discernable at this time (Fig. 5(c)).
DISCUSSION As yet, there are no data available on the concentration of free amino acids in tsetse haemolymph in relation to cycles of feeding and reproduction. However, it has recently been shown that the free amino acid concentration in the haemolymph of G. monitans falls from 20.21 to 14.86 g/l. during the first 2 days of adult life (CUNNINGHAMand SLATER,1974). If we take the lower value as representing the typical concentration in the adult and assume a haemolymph volume of about 5 ~1 which has been determined for the smaller G. austeni (TOBE and DAVEY, 1972) we obtain a value of 75 pg free amino acids in the adult haemolymph, which for pregnant G. morsitans females is probably an underestimate. Most carbon atoms in the U-14C protein hydrolysate used in the present experiments were labelled, because the mixture is derived from algae grown in media whose sole source of carbon is UJ4C bicarbonate. Since the specific activity in mCi/mAtom of carbon and the radioactive concentration in &Z/ml of solution is given, it has been possible to calculate the total concentration of amino acids present from the stated percentage composition by activity, of each of the 15 amino acids in the mixture, using the number of carbon atoms per amino acid molecule and the molecular weight of each amino acid.
UTILIZATION
OF AMINO ACIDS AND PROTEIN BY ADULT GLOSSINA
2167
The range of free amino acid concentrations in vertebrate plasma appears to be around 0.2 to O-3 g/l. (ALTMAN, 1964). Thus, the administration of O-1 &i U-14C protein hydrolysate per 50 mg of blood in the diet represented the addition of O-041 pg amino acid mixture per 6 to 9 pg amino acids in the serum. This is less than 0.7 per cent of the total free amino acids present in the plasma of such a volume of blood. Similarly, injection into the adult haemocoel of 1 ~1 U-14C protein hydrolysate (O-05 &i) represented the addition of 0.0205 pg amino acids to the haemolymph free amino acid pool of at least 75 pg. This is less than 0.03 per cent of the total free amino acids in the haemolymph. It is not likely that the addition of such small percentages of radioactively labelled amino acids to the totals present either in vertebrate blood or in the insect haemolymph would alter the physiology of the insect in relation to the fate of these nutrients during diuresis or intra-uterine larval growth. Our results show that the third instar larva of G. morsitans only ceases to feed some 12 hr before it is deposited by the parent female, which agrees with the observations of ROBERTS(1971) for G. austeni. Also the maximum rate of nutrient uptake occurs immediately before cessation of feeding (Fig. 4). Furthermore, the observation by TOBE et al. (1973) that the uterine gland of G. austeni continues to synthesize larval nutrient until a few hours before larviposition also applies to G. morsitans. The demonstration of a very rapid synthesis of protein by the uterine gland of G. austeni and its immediate transfer to the feeding larva (TOBE and DAVEY, 1974a, b) is also probably true for G. morsitans, although in the present experiments whole larvae were hydrolysed before assay and no attempt was made to distinguish whether the larvae initially contained labelled protein or amino acids. Clearly, the interpretations of histological data on cyclical secretory activity of the uterine gland in G. palpalis (HOFFMANN, 1954) and G. pallidipes (BURSELLand JACKSON,1957) in which it was suggested that the uterine gland secretes actively only for about 60 hr from the third day of a reproductive cycle, and that the developing larva only feeds during the first and second instar, are unlikely to be true. The results of experiments involving administration of U-14C protein hydrolysate either by injection or in the diet of the adult female demonstrate that she has little or no storage capacity for larval nutrients since it is only those amino acids which are available in the female haemolymph at the time of rapid larval growth which are transferred to the larva. Amino acid nutrients present in the haemolymph at other times are excreted. This progressive increase in the amount of labelled material reaching the larva at the expense of that which is excreted by the female must represent an increasing competition between the uterine glands and the Malpighian tubules for soluble nutrients in the haemolymph, as larval development proceeds (Figs. 2,4). The time of administration of U-14C protein hydrolysate which produces an increased label content of resultant larvae is when 40 to 50 per cent of the interlarval period has passed, which suggests that embryonic development in G. morsitans occupies the first half of each interlarval period, thus confirming the
2168
P. A. LANGLEY AND R. W. PIMLEY
observations of ROBERTS(1973). The low and constant level of incorporation of label by the adult fly is similar to that of larvae produced by flies receiving the label during the first 40 per cent of the interlarval period (Figs. 2, 4) and may represent the amount of label which is incorporated by inactive secretory glands by simple diffusion from the adult haemolymph. The result obtained following administration of UJ4C protein in the diet, to flies containing larvae at different stages of development, was unexpected. It was thought that a peak of incorporation into the resultant larva would be seen some 48 hr earlier on the time scale of the interlarval period than following adminisThis would have indicated the protein tration of UJ4C protein hydrolysate. digestion time and would represent more closely the uptake of nutrients supplied by the digested protein of a normal blood meal. It would then have been possible to predict accurately which adult meal was of greatest significance during larval growth and indicate that meals taken late in larval development could not be digested in time to provide nutrients for the development of that particular larva. However, it was found that the UJ4C protein used was poorly digested and that more than 70 per cent of the total recovered was excreted, there being no period of rapid incorporation by the developing larva (Fig. 3). Clearly the midgut trypsin of G. morsitans is unable to split heat-denatured plant protein and further experiments along these lines will necessitate the use of blood from an experimentally labelled host animal. Nevertheless, it is permissible from the data of LANGLEY(1967) to predict that digestion of a blood meal by G. morsitans females at 25°C takes around 48 hr (peak trypsin activity from 24 to 76 hr), and therefore that meals ingested on day 5 or 6 of a normal 9 day interlarval period will contribute most to the nutritional supply of a developing larva. An examination of Fig. 5(a) and (b) s h ows that, when fed through membranes at least, the adult female G. monitans ingests her largest meals between day 5 and day 7 of a 9 or 10 day larviposition cycle and that during this period all flies in an experimental group are expected to feed at least once. This is in keeping with the hypothesis that the largest meals are those which contribute most to larval nutrition and that flies have little capacity to store larval nutrients from earlier meals. The situation is similar in G. austeni when the largest adult meal is ingested approximately 4 days before parturition (TOBE and DAVEY,1972). The implication is that the largest meal is ingested at a time when the larva is still very small and that this meal provides the bulk of nutrients for larval growth. An examination of Tables 1 to 3 confirms the view that amino acids in solution in a blood meal enter the haemolymph of the adult fly very rapidly via the anterior midgut wall (the crop is impermeable (MOLOO and KUTUZA,1970)) to be disposed of according to the metabolic demands on the fly at the time of ingestion. The above findings raise interesting problems concerning the control of nutrient synthesis by the milk gland of the female G. morsitans, and in particular the apparent inability of flies to store amino acids which enter the haemolymph in advance of the normal products of digestion.
UTILIZATION OF AMINOACIDSANDPROTEINBY ADULTGLOSSINA
2169
Significant losses of label occurred in the urine of flies which did not contain rapidly growing larvae at the time of feeding on a labelled blood meal, and it is assumed that these represented significant losses of the total haemolymph pool of free amino acids. We must therefore postulate that either a mechanism exists to conserve these nutrients by reducing the free amino acid pool of the insect’s haemolymph to a low level immediately prior to feeding (which is unlikely in view of the amounts excreted following U -laC protein hydrolysate injection), or that such losses are an inevitable and acceptable consequence of the rapid diuresis which is characteristic of tsetse flies. Since it is known that diuresis in G. morsitans is affected by in vitro feeding (LANGLEY and PIMLEY, 1973) it may be that in part the present results are a reflection of abnormal physiology. Nevertheless, they do indicate that the fate of amino acids present in the haemolymph pool changes rapidly in relation to the demands upon the female fly made by the rapid growth of the larva. From the practical standpoint our results indicate that an approach to the solution of maintaining tsetse flies using in vitro methods should not involve the addition of proteins other than normal animal proteins to the diet, and that the addition of small soluble molecules such as amino acids may be of use only at critical times during larval development. Acknowledgements-We thank the Department of Animal Husbandry, University of Bristol School of Veterinary Science, for scintillation counting facilities, and in particular Dr. SIMON ROBBINSfor helpful discussion on technique. We also thank Dr. A. M. JORDAN, Mr. A. R. MEWS, and Dr. S. K. MOLOO for helpful comments on the manuscript. The financial support of the Overseas Development Administration of the U.K. Foreign and Commonwealth Office is gratefully acknowledged. REFERENCES ALTMANP. L. (1964) Blood and Other Body Fluids (Ed. by DITTMERD. S.). Fed. Amer. Sots. for Exp. Biol., Washington, D.C. BURSELL E. and JACKSONC. H. N. (1957) Notes on the Choriothete and miik gland of Glossina and Hippobosca (Diptera). Proc. R. ent. Sot. (A) 32, 30-34. BUXTONP. A. (1955) T7z Natural History of Tsetse Flies. London School of Hygiene and Tropical Medicine, Memoir 10. CMELIK S. W. H., BURSELLE., and SLACK E. (1969) Composition of the gut contents of third instar tsetse larvae (Glossina morsitans Westwood). Comp. .&o&m, Physiol. 29, 447453. CUNNXNGHAM I. and SLATERJ. S. (1974) Amino acid analyses of haemolymph of Glossina morsitans morsitans (Westwood). Acta trop. 31, 83-88. HOFFMANNR. (1954) Zur Fortpflanzungsbiologie und zur intra-uterinen Entwicklung von Gloss&z palpalis. Acta trop. 11, l-57. LANGLEY P. A. (1967) The control of digestion in the tsetse fly, Glossim morsitans: a comparison between field flies and flies reared in captivity. J. Insect Physiol. 13,477-486. LANGLEY P. A. (1972) The role of physical and chemical stimuli in the development of in vitro feeding techniques for tsetse flies Glossina spp. (Dipt., Glossinidae). Bull. ent. Res. 62, 215-228. LANGLEY P. A. and P~MLEY R. W. (1973) Influence of diet composition on feeding and water excretion by the tsetse Ay, Glossina morsitans. J. Insect Physiol. 19, 1097-l 109.
2170
P. A. LANGLEYAND R. W. PIMLEY
MOLOO S. K. and KUTUZA S. (1970) Feeding and crop emptying in Glossina brwipalpis Newstead. Acta trop. 27, 356-377. NASH T. A. M., JORDANA. M., and TREWERNM. A. (1971) Mass rearing of tsetse flies (Glossina spp.) : recent advances. In Sterility Principle for Insect Control or Eradication, pp. 99-110. I.A.E.A., Vienna. ROBERTSM. J. (1971) The functional anatomy of the head in the larvae of the tsetse fly, Glossina austeni Newstead (Diptera, Glossinidae). Entomologist 104, 190-203. ROBERTSM. J. (1973) Rate of embryonic development in the tsetse fly Glossina morsitans orientalis. Entomologia exp, appl. 16, 268-274. TOBE S. S. and DAVEY K. G. (1972) Volume relationships during the pregnancy cycle of the tsetse Ay Glossina austeni. Can. J. Zool. 50, 999-1010. TOBE S. S. and DAVEY K. G. (1974a) Nutrient transfer during the reproductive cycle in Glossina austeni Newst.-III. Protein synthesis in abdominal tissues. Tissue & Cell. In press. TOBE S. S. and DAVEY K. G. (1974b) Synthesis and turnover of haemolymph protein during the reproductive cycle of Glossina austeni. Can. J. Zool. In press. TOBE S. S., DAVEYK. G., and HUEBNERE. (1973) Nutrient transfer during the reproductive cycle in Glossina austeni Newst.-I. Histology and histochemistry of the milk gland, fat body and oenocytes. Tissue W Cell 5, 633-650.
1974, Vol. 20, pp. 2157 to 2170. Pergamon Press. Printed in Great Britain
UTILIZATION OF U-f*C AMINO ACIDS OR U-l*C PROTEIN BY ADULT GLOSSINA MORSITANS DURING 1N UTTER DE~LOP~ENT OF LARVA P. A. LANGLEY Tsetse Research Laboratory,
and R. W. PIMLEY
University of Bristol School of Veterinary Science, Langford. Bristol BS18 7DU, England (Received 3 April 1974)
Abstract-Administration of U-l*C protein hydrolysate in the diet of adult female GEoss57Iamorsr%ansat different times throughout the second reproductive cycle was followed by analysis of the distribution of radioactivity between the adult flies, their excreta, and the fully grown third instar larvae produced by these flies. A constant proportion of the total administered label was recoverable independently of the time lapse between adm~istration and assay. Peak incorporation of labelled material occurred in the larva between the seventh and eighth day of a 9 or 10 day interlarval period, indicating that the larva feeds avidly on recently synthesized maternal uterine gland secretion at this time. Haemocoelic injection of II-l% protein hydrolysate into similar adult females, between feeds, resulted in continued incorporation of labelled material by the larva to within 12 hr of parturition. Results are consistent with the hypothesis that uterine gland secretion and larval feeding continue throughout the intrauterine life of the larva. A constant and low proportion of detectable label remained in the adult fly while increased inco~omtion by the larva was paralleled by a reduction of detectable label in the adult excreta. This indicates direct competition between the uterine gland cells and those of the Malpighian tubules for free amino acids in the haemolymph. Administration of U-14C protein in the adult diet did not result in incorporation of label by the developing larva, and the bulk was excreted as protein by the adult fly. Apparently the midgut trypsin of G. morsitanr is incapable of splitting this labelled protein. Analysis of urine and haemolymph samples from flies in early pregnancy, recently fed on a diet containing U-‘*C protein hydrolysate or U-14C protein, shows that free labelled amino acids in the diet enter the adult haemol~ph almost immediately after feeding, and are excreted along with dietary water during initial diuresis. The labelled protein used in these experiments was not taken up by the haemolymph and consequently did not appear in the urine. Implications are that the adult female G. morsitum possesses little storage capacity for substances in the diet which are destined to provide nutrients for the developing larva. Assuming a 48 hr digestion time, the digestive products of a blood meal ingested on day 5 or 6 of a 9 day interlarval period will provide the bulk of nutrients for larval growth. It is therefore significant that blood meals ingested at this time are larger than those ingested earlier or later in the cycle. 2157
2158
P.
A.
LANGLEY
AND
R. w.
PIMLJIY
INTRODUCTION
THE MODEof reproduction in tsetse flies is adenotrophic viviparity, in which a fertilized adult female produces a single, fully fed third instar larva at regular intervals of approximately 9 days at a maintenance temperature of 25°C. The adult feeds exclusively on vertebrate blood and the developing larva feeds on a secretion produced by the uterine glands of the female: the free life of the larva consists only of a short burrowing period of about 1 hr after which pupariation occurs (BUXTON,1955). Thus the birth product of an adult female tsetse fly is larger than itself, since not only will the larva produce an adult of the same size as the parent after metamorphosis, but the larva will contain all the nutritional requirements for survival and metamorphosis throughout the relatively long (approx. 30 days at 25°C) intra-puparial life. On the basis of histological observation it was stated by HOFFMANN(1954) that uterine gland secretion in G. palpalis was of limited duration and occupied no more than 60 hr from the third day following ovulation. A similar interpretation was given by BURSELLand JACKSON(1957) for G. pallidipes. However, ROBERTS (1971) found uterine gland secretion at the mouth of the milk duct opening in G. austeni from the time of hatching of the first instar larva until parturition, and concluded that secretion was available throughout larval life. Recent work by TOBE et al. (1973) confirms the observations of ROBERTSfor G. au&k. In G. palpalis embryonic development at 26°C apparently occupies 50 to 60 hr (HOFFMANN, 1954) while ROBERTS(1973) has shown that the first instar larva of G. morsitans hatches only after 4 days. Thus larval growth is extremely rapid and is confined to the 4 or 5 days prior to larviposition in this species. TOBE and DAVEY(1972) have shown that the pregnant female G. austeni feeds to constant volume, which provides a diminishing food capacity as the larva grows. Using injections of labelled tyrosine and leucine, TOBEand DAVEY(1974a, b) have demonstrated a very rapid synthesis of protein within the uterine glands, and an equally rapid transfer of nutrients from the adult oenocytes and fat body to the secretory glands is implied. CMELIK et al. (1969) have concluded that sufficient nutritive secretion (except for aromatic amino acid requirements) for complete larval development could be provided by a single blood meal of 40 mg in G. monitans. The timing of events such as adult feeding, larval hatch, and transfer of nutrient to the larva is obviously critical in Gloss&a. It is therefore important to know the extent to which the many species of Glossina may differ in their solution to the problem of larval nutrition, or whether the sometimes conflicting information in the literature can be rationalized to provide a comprehensive theory of the dynamics of nutrition and reproduction. The present paper is a step towards such rationalization and concerns the timing of nutrient uptake by the larva of G. niorsitans during the second reproductive cycle. An attempt has been made to assess the relative importance of successive adult meals in larval development by determining the fate of labelled amino acids or protein after administration to the adult either in the diet or by injection.
UTILIZATION
OF AMINO ACIDS AND PROTEIN BY ADULT GLOSSINA
MATERIALS
2159
AND METHODS
Adult G. morsitans were maintained according to the techniques used routinely at Langford (NASH et al., 1971) and offered food daily on goats. Groups of fertilized adult females were isolated as soon as the black polypneustic lobes of their first third instar larvae were visible through the abdominal integument (day 15 or 16 of adult life). Flies which deposited their larvae within the next 24 hr were segregated and designated day 1 of the second reproductive cycle. These were transferred to the Agar/Parafilm membrane feeding system described by LANGLEY (1972) and modified by MEWS (unpublished) in that the blood was presented on a stippled or grooved glass plate overlain by the membranes and that the agar contained 2% glycerol to assist moisture retention in the membrane. Successive groups of 10 flies each were segregated at intervals of 24 hr for 9 days. During the second reproductive cycle the flies were fed on haemolysed bovine blood containing 10es M ATP. On the ninth day of collection groups of flies from 1 to 9 days old provided a full range of different larval development stages at intervals of 24 hr. All the flies in each daily age group were isolated and weighed before and after feeding. Each fly was fed on whole defibrinated goat’s blood containing 0.1 &i U-14C protein hydrolysate (Radiochemical Centre, Amersham) per 50 mg blood, and 1O-3 M ATP. Following the labelled meal all flies were isolated in 3 x 1 in. glass tubes closed with perforated Parafilm, and maintained in an incubator at 25°C. A small circle of filter paper was included to collect excreta. The flies were individually fed each day on haemolysed bovine blood and the glass tubes housing the flies were changed daily. Flies would only feed up to the completion of 70 to 75 per cent of the second interlarval period: those which produced a larva on day 9 refused to feed after the seventh day. As soon after the second larviposition as possible the distribution of U-14C label between adult excreta, the parent flies, and their larvae was determined using a scintillation counting method (Nuclear Enterprises NE250 scintillant and a Nuclear Chicago Scintillation Counter Model 6850). Aliquots of excreta (0.2 ml) from individual tubes were assayed after dissolving in 2 ml 0.4% L&CO, and a correction made for the quenching effect of haematin, uric acid, and salt, using an internal standard. Flies and larvae were individually hydrolysed in 6 N HCl in sealed tubes at 120°C for 1 to 3 days, evaporated to dryness at 50°C and the hydrolysate re-dissolved in 2 ml water before counting O-2 ml aliquots. Corrections were again made for the quenching effect of non-amino acid contaminants in samples. After individual assay of excreta samples, the remaining portions of each sample were pooled, taken to pH 2-O with 6 N HCl, and after centrifugation the supernatant was assayed for U- 1% activity, with appropriate corrections for quenching. The precipitate which consisted mainly of uric acid &,, 298 nm on a Pye Unicam Spectrophotometer) was washed several times with 0.1 N HCl, finally re-dissolved in 0.4% Li,CO,, and assayed for UJ4C activity.
2160
P. A. LANGLEYANDR. W.
PIMLEY
A similar experiment was conducted upon groups of flies which received a labelled meal containing 0.1 $Zi U-W protein (Radiochemical Centre, Amersham) per 50 mg blood instead of the labelled hydrolysate. Both experiments were replicated and the results were repeatable. However, before the second U-i*C protein experiment the labelled material was dissolved in Tris buffer at pH 8 and run three times through a 2.5 x 1 cm column of Sephadex G-25, in order to remove small molecules such as amino acids and peptides before adding it to the blood for fly feeding. The new concentration was therefore 0.05 $Zi UJ4C protein per 50 mg blood. A third experiment involved 9 similar groups of flies on their second reproductive cycle except that they were injected with 1~1 ( =O*OS &i) U-14C protein hydrolysate per fly. Injections were made 24 hr after the previous meal and injected flies were not offered food for at least 2 hr following the injection. Excreta, flies, and larvae were assayed for U-14C activity as before. The experiment was replicated. Finally, groups of flies were starved for 48 hr early in their second reproductive cycle and then fed on either haemolysed or whole defibrinated bovine blood containing appro~mately 5 $Zi UJ*C protein hydrolysate per ml. Urine and haemolymph were collected as 1 ~1 samples from flies at 5, 15, and 30 min after feeding. Both G. morsitans and G. au&k females were used. The experiment was repeated with G. morsitans only, using U-14C protein in the diet at a rate of 10 @i/ml of blood, only approximately 60 per cent of which was soluble in the blood serum. RESULTS
Fig. 1 shows the relationship between total counts recovered (excreta, fly, and larva) and the size of meal containing U-r*C protein hydrolysate. It is apparent that the relationship is close to linear. Therefore, metabolic losses as WO, were either negligible or at least they were a constant proportion of the flies’ intake of label regardless of the time lapse between administration of the label and assay. Thus any differences in total counts recovered were attributable only to differences in meal size and hence to label intake. A similar result was obtained with U-14C protein in the blood meals. Losses from the larva would be confined at most to WO, since the larva stores all nitrogenous waste and its anus is closed. Fig. 2 shows the distribution of label between the adult fly, the total amount of excreta collected, and the fully developed third instar larva produced by flies receiving UJ4C protein hydrolysate in their diet at the time indicated during their second reproductive cycle. Radioactivity is expressed as a percentage of the total counts recovered: the time of administration of the label is expressed as a percentage of the interlarval period which varied from 9 to 12 days. Each point represents the mean of between 3 and 12 observations. It is apparent (Fig. 2) that the amount of label retained by the fly remained constant at a low level regardless of the time during the cycle at which it was administered. The slight rise when label was administered after completion of
UTILIZATION
OF AMINO
ACIDS
AND
Meal
PROTEIN
size,
BY
ADULT
GLOSSINA
2161
mg.
FIG. 1. Relationship between total counts recovered (fly + excreta+ larva) and size of ingested blood meal containing U- 14C protein hydrolysate. Labelled meals administered at different times during 9 day interlarval period of G. morsitans females on their second reproductive cycle. 0, Day 1; 0, day 3 ; n , day 4; 0, day 5; x, day 7; A, day 8. Relationship is linear (J = 1.51x-6.91) and independent of time lapse between administration and assay.
I
0 Proportion
IO
I
I
I
I
I
I
20
30
40
50
60
70
of interlorvol
period
completed,
I_ 00 %
FIG. 2. Distribution of radioactivity between adult G. morsitans female, its excreta, and fully grown larva, of flies receiving U-14C protein hydrolysate in their diet at the time indicated during their second reproductive cycle, and assayed after la&position. 0-0, Adult fly; 0-0, fully grown larva; x - x , adult excreta.
2162
P. A.
LANGLEY
AND
R. w.
PIMLEY
70 per cent of the interlarval period was probably due to an inadequate period remaining for the excess to be excreted before la~position and assay occurred. The most significant feature of Fig, 2 is the rapid uptake of label by the larva when administered in a blood meal ingested by the female fly only 2 or 3 days before larviposition. The increased uptake by the larva was accompanied by a corresponding decrease in the amount of excreted label. Following acid precipitation of excreta samples, more than 90 per cent of the uric acid present was found in the precipitate. However, more than 90 per cent of the measured radioactivity was found in the supernatant. Thus we concluded that incorporation of label into the uric acid molecule was negligible and that most of the radioactivity in the excreta was present in amino acids. From Fig. 3 it is apparent that the administration of U-W protein in the diet resulted in most of the Iabel being excreted. There was little evidence of rapid
60-
Proportion of interlarvalperiod completed,
%
FIG. 3. Distribution of radioactivity between adult G. morsituns female, its excreta, and fully grown larva, of flies receiving U-l*C protein in their diet at the time indicated during their second reproductive cycle, and assayed after larviposition. O-O, Adult fly; O-O, fully grown larva; x - x , adult excreta.
incorporation of this protein by the larva regardless of its stage of development at the time of administration. After separation of the adult excreta on a Sephadex G-25 column most of the radioactivity was found to be still in the protein fraction. Hence it was concluded that the labelled protein (heat denatured and of plant origin) had remained largely undigested.
UTILIZATION
OF AMINO ACIDS AND PROTEIN BY ADULT GLOSSINA
2163
Fig. 4 shows the pattern of distribution of label following injection of UJ*C protein hydrolysate into adult females at various times during their second reproductive cycle. The distribution is similar to that obtained following administration of the hydrolysate in the diet, except that the massive uptake of label by the larva
Proportion
of interlorvol
period
completed,
%
FIG. 4. Distribution of radioactivity between adult G. snorsitans female, its excreta, and fully grown larva, of Aies injected with U-W protein hydrolysate at the time indicated during their second reproductive cycle, and assayed after larviposition. O-0, Adult fly; O-0, fully grown larva; x - x , adult excreta.
can be seen to continue to within 12 hr of larviposition. This means that the third instar larva normally feeds avidly on the nutritive secretion produced by the parent until very shortly before larviposition and that this secretion is synthesized very rapidly by the female fly. Furthermore, it suggested to us that UJ*C amino acids in gplution in blood meals were entering the haemolymph of the fly very soon after feeding (Fig. 2). This contention was reinforced by comparing the rate of excretion of label in U-W protein hydrolysate-fed and UJ4C protein-fed flies. More than 80 per cent of excreted label had been eliminated by the former within 24 hr of feeding while 48 hr was necessary for an equivalent amount to be excreted by the latter (Table 1). Table 2 shows the results of sampling urine and haemolymph from flies fed on blood meals containing U-W protein hydrolysate. It is apparent that there was little difference between diets of whole defibrinated blood or haemolysed blood. Also the indication is that G. austeni behaves similarly to G. morsituns.
2164
P. A. LANGLEYANDR. W. PIMLEV
TABLE ~-AMOUNT OF UJ4C ACTIVITYIN EXCRBTAOF G. morsitans FEMALESAT DIFFERENT TIME INTERVALSFOLLOWINGA BLOODMEAL CONTAININGU-i’c AMINOACID MIXTUREOR U-r’c PROTEIN, EXPRESSEDAS PERCENTAGEOF TOTALACTIVITYRECOVERED FROM EXCRETA Time interval after feeding (hr) Label in diet
O-24
2448
48-72
72-96
UJ4C ammo acid mixture U-14C protein
79.8
13.7
3.0
1.9
0.9
0.7
40.2
47.0
6.0
2.5
2.0
2.3
96-120
120-144
Result of running excreta through Sephadex G-25 column indicates that a significant proportion of activity in excreta of U-i4C protein-fed flies is due to undigested protein.
70 ti E
60
Age
P
following
first
lorviposition,
doy5.
(b)
IOOr
‘Z so-
.i? v) ? ; : t
604020 -
I z”
I I I 123456799
0
Age following
I first
I
I
I
lorviposition,
I
days.
50C--
(c)
1 , / ,
%
(
,
,
,
,
,
123456789
0 Age
following
first
lorviposition,
days.
FIG. 5(a). Sizes of labelled meals ingested by experimental G. morsitans on different days of their 9 to 12 day second interlarval period. Vertical lines represent fiducial limits of mean values. (b) Frequency of feeding by G. morsitans females during the 9 day period of their second reproductive cycle, and fed on rabbit. (c) Sizes of meals ingested by G. morsitans females during the 9 day period of their second reproductive cycle, and fed on rabbit.
Haemolysed bovine blood I- 1O-s M ATP
G. austeni
Species
(5) 164 k20.2
‘12p: rf:17.0 (11 300 -
(18) 268 244.2
(7) 262 + 70.0
15
Urine
-
(6) 1114 z!z535.2
(7) 1087 ?I 586.8
30
Food
TSETSE FLIESEARLY
OF U-r4C LABELLEDAMINO
FEMALE ACIDMIXTURB
IN TIIEIRSBC~ND
-
-
CONCENTRATION
OF u-‘*c
9162 + 382.0
(10) 9162 rt 382.0
(10) 8550 + 328.6
Blood mea1
PROTEIN
EARLY IN THEIR SECOND
(15) 3877 +_676.4
-
30
G. morsitafls
(6) 2707 t 1160.8
OF ADULT FEMALE
(4) 4111 k6.58.2
(5) 3828 f 952.8
-
15
Haemolymph
(7) 620.4 Z!I258.8
30
Figures in brackets = n. Values given are means & fiducial limits.
(13) 190.0 z!z52.0
15
Urine
(7) 271.2 z!I147.0
5
(6) 478.3 z!z130.4
15
Haemolymph
(10) 631.5 + 262.6
30
(5) 8971
Blood meal
Radioactivity in counts/min per ~1 at the timed intervals indicated (min)
(16) 82.2 * 18.8
5
5 (19) 3630 f451*0
A KNOWN
IN URINE AND HAEMOLYMPH
A BLOOD MEAL CONTAINING
MATERIAL
CYCLE, FOLLOWING
OF LABELLED
Fresh defibrinated bovine blood + 1O-3 M ATP
REPRODUCTIVE
3---CONCENTRATION
G. morsitans
TABLE
Haemolysed bovine blood + 1O-3 M ATP
G. morsitans
!l% rt:17.8
5
IN URINE AND HAEMOLYMPH 0P ADULT CONTAINING AKNOWN CONCENTRATION
Radioactivity in counts/min per ~1 at the timed intervals indicated (min)
MEAL
Figures in brackets = n, Values given are means + fiducial limits.
Fresh defibrinated bovine blood + 1O-3 M ATP
Food
A BLOOD
G. morsitans
Species
REPRODUCTIVECYCLE,FOLLOWING
TABLE ~-CONCENTRATIONOF LABELLED MATERIAL
z z
z
8
0
P
$
5
2
3 2
E
B
8
!!
z
%
$
5 i: Ki ?;
2166
P. A. LANGLEY ANDR. W. PIMLEY
The initial drops of urine contained only small quantities of label while the haemolymph of the flies contained 40 per cent of the label in the blood meal within 5 min of feeding. Thirty minutes after feeding the urine also contained significant quantities of label. This result shows that amino acids in solution in the blood meal were eliminated by passage across the anterior midgut wall and voided to the exterior via the Malpighian tubules during normal diuresis. Nevertheless, the 5 and 15 min urine samples contained few counts and one must postulate that the passage of amino acids through the Malpighian tubules is initially slower than that of water. Table 3 summarizes the results of experimental feeding of G. morsitans females on blood containing UJ4C protein. The amount of label in the urine and haemolymph of such flies increased during the 30 min following feeding, but the amount of label present in each did not exceed 7 per cent of the total label present in the blood meal. It is most likely that the presence of label in urine and haemolymph was due to amino acid or peptide contamination of the U-14C protein sample, which was not purified before use. The U-14C protein did not apparently enter the haemolymph of the fly as did UJ4C protein hydrolysate. The sizes of meals ingested by G. morsitans fed on the diet containing UJ4C protein hydrolysate are shown in Fig. 5(a). The largest meals were taken immediately after larviposition (day 1) and again between day 5 and 7. Large meals ingested at the later stages would be digested in time to provide the large quantities of nutritive secretion for growth of the third instar larva. Fig. 5(b) shows that between day 5 and 7 all flies would be expected to feed at least once, and on rabbit hosts a similar peak of meal size is discernable at this time (Fig. 5(c)).
DISCUSSION As yet, there are no data available on the concentration of free amino acids in tsetse haemolymph in relation to cycles of feeding and reproduction. However, it has recently been shown that the free amino acid concentration in the haemolymph of G. monitans falls from 20.21 to 14.86 g/l. during the first 2 days of adult life (CUNNINGHAMand SLATER,1974). If we take the lower value as representing the typical concentration in the adult and assume a haemolymph volume of about 5 ~1 which has been determined for the smaller G. austeni (TOBE and DAVEY, 1972) we obtain a value of 75 pg free amino acids in the adult haemolymph, which for pregnant G. morsitans females is probably an underestimate. Most carbon atoms in the U-14C protein hydrolysate used in the present experiments were labelled, because the mixture is derived from algae grown in media whose sole source of carbon is UJ4C bicarbonate. Since the specific activity in mCi/mAtom of carbon and the radioactive concentration in &Z/ml of solution is given, it has been possible to calculate the total concentration of amino acids present from the stated percentage composition by activity, of each of the 15 amino acids in the mixture, using the number of carbon atoms per amino acid molecule and the molecular weight of each amino acid.
UTILIZATION
OF AMINO ACIDS AND PROTEIN BY ADULT GLOSSINA
2167
The range of free amino acid concentrations in vertebrate plasma appears to be around 0.2 to O-3 g/l. (ALTMAN, 1964). Thus, the administration of O-1 &i U-14C protein hydrolysate per 50 mg of blood in the diet represented the addition of O-041 pg amino acid mixture per 6 to 9 pg amino acids in the serum. This is less than 0.7 per cent of the total free amino acids present in the plasma of such a volume of blood. Similarly, injection into the adult haemocoel of 1 ~1 U-14C protein hydrolysate (O-05 &i) represented the addition of 0.0205 pg amino acids to the haemolymph free amino acid pool of at least 75 pg. This is less than 0.03 per cent of the total free amino acids in the haemolymph. It is not likely that the addition of such small percentages of radioactively labelled amino acids to the totals present either in vertebrate blood or in the insect haemolymph would alter the physiology of the insect in relation to the fate of these nutrients during diuresis or intra-uterine larval growth. Our results show that the third instar larva of G. morsitans only ceases to feed some 12 hr before it is deposited by the parent female, which agrees with the observations of ROBERTS(1971) for G. austeni. Also the maximum rate of nutrient uptake occurs immediately before cessation of feeding (Fig. 4). Furthermore, the observation by TOBE et al. (1973) that the uterine gland of G. austeni continues to synthesize larval nutrient until a few hours before larviposition also applies to G. morsitans. The demonstration of a very rapid synthesis of protein by the uterine gland of G. austeni and its immediate transfer to the feeding larva (TOBE and DAVEY, 1974a, b) is also probably true for G. morsitans, although in the present experiments whole larvae were hydrolysed before assay and no attempt was made to distinguish whether the larvae initially contained labelled protein or amino acids. Clearly, the interpretations of histological data on cyclical secretory activity of the uterine gland in G. palpalis (HOFFMANN, 1954) and G. pallidipes (BURSELLand JACKSON,1957) in which it was suggested that the uterine gland secretes actively only for about 60 hr from the third day of a reproductive cycle, and that the developing larva only feeds during the first and second instar, are unlikely to be true. The results of experiments involving administration of U-14C protein hydrolysate either by injection or in the diet of the adult female demonstrate that she has little or no storage capacity for larval nutrients since it is only those amino acids which are available in the female haemolymph at the time of rapid larval growth which are transferred to the larva. Amino acid nutrients present in the haemolymph at other times are excreted. This progressive increase in the amount of labelled material reaching the larva at the expense of that which is excreted by the female must represent an increasing competition between the uterine glands and the Malpighian tubules for soluble nutrients in the haemolymph, as larval development proceeds (Figs. 2,4). The time of administration of U-14C protein hydrolysate which produces an increased label content of resultant larvae is when 40 to 50 per cent of the interlarval period has passed, which suggests that embryonic development in G. morsitans occupies the first half of each interlarval period, thus confirming the
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observations of ROBERTS(1973). The low and constant level of incorporation of label by the adult fly is similar to that of larvae produced by flies receiving the label during the first 40 per cent of the interlarval period (Figs. 2, 4) and may represent the amount of label which is incorporated by inactive secretory glands by simple diffusion from the adult haemolymph. The result obtained following administration of UJ4C protein in the diet, to flies containing larvae at different stages of development, was unexpected. It was thought that a peak of incorporation into the resultant larva would be seen some 48 hr earlier on the time scale of the interlarval period than following adminisThis would have indicated the protein tration of UJ4C protein hydrolysate. digestion time and would represent more closely the uptake of nutrients supplied by the digested protein of a normal blood meal. It would then have been possible to predict accurately which adult meal was of greatest significance during larval growth and indicate that meals taken late in larval development could not be digested in time to provide nutrients for the development of that particular larva. However, it was found that the UJ4C protein used was poorly digested and that more than 70 per cent of the total recovered was excreted, there being no period of rapid incorporation by the developing larva (Fig. 3). Clearly the midgut trypsin of G. morsitans is unable to split heat-denatured plant protein and further experiments along these lines will necessitate the use of blood from an experimentally labelled host animal. Nevertheless, it is permissible from the data of LANGLEY(1967) to predict that digestion of a blood meal by G. morsitans females at 25°C takes around 48 hr (peak trypsin activity from 24 to 76 hr), and therefore that meals ingested on day 5 or 6 of a normal 9 day interlarval period will contribute most to the nutritional supply of a developing larva. An examination of Fig. 5(a) and (b) s h ows that, when fed through membranes at least, the adult female G. monitans ingests her largest meals between day 5 and day 7 of a 9 or 10 day larviposition cycle and that during this period all flies in an experimental group are expected to feed at least once. This is in keeping with the hypothesis that the largest meals are those which contribute most to larval nutrition and that flies have little capacity to store larval nutrients from earlier meals. The situation is similar in G. austeni when the largest adult meal is ingested approximately 4 days before parturition (TOBE and DAVEY,1972). The implication is that the largest meal is ingested at a time when the larva is still very small and that this meal provides the bulk of nutrients for larval growth. An examination of Tables 1 to 3 confirms the view that amino acids in solution in a blood meal enter the haemolymph of the adult fly very rapidly via the anterior midgut wall (the crop is impermeable (MOLOO and KUTUZA,1970)) to be disposed of according to the metabolic demands on the fly at the time of ingestion. The above findings raise interesting problems concerning the control of nutrient synthesis by the milk gland of the female G. morsitans, and in particular the apparent inability of flies to store amino acids which enter the haemolymph in advance of the normal products of digestion.
UTILIZATION OF AMINOACIDSANDPROTEINBY ADULTGLOSSINA
2169
Significant losses of label occurred in the urine of flies which did not contain rapidly growing larvae at the time of feeding on a labelled blood meal, and it is assumed that these represented significant losses of the total haemolymph pool of free amino acids. We must therefore postulate that either a mechanism exists to conserve these nutrients by reducing the free amino acid pool of the insect’s haemolymph to a low level immediately prior to feeding (which is unlikely in view of the amounts excreted following U -laC protein hydrolysate injection), or that such losses are an inevitable and acceptable consequence of the rapid diuresis which is characteristic of tsetse flies. Since it is known that diuresis in G. morsitans is affected by in vitro feeding (LANGLEY and PIMLEY, 1973) it may be that in part the present results are a reflection of abnormal physiology. Nevertheless, they do indicate that the fate of amino acids present in the haemolymph pool changes rapidly in relation to the demands upon the female fly made by the rapid growth of the larva. From the practical standpoint our results indicate that an approach to the solution of maintaining tsetse flies using in vitro methods should not involve the addition of proteins other than normal animal proteins to the diet, and that the addition of small soluble molecules such as amino acids may be of use only at critical times during larval development. Acknowledgements-We thank the Department of Animal Husbandry, University of Bristol School of Veterinary Science, for scintillation counting facilities, and in particular Dr. SIMON ROBBINSfor helpful discussion on technique. We also thank Dr. A. M. JORDAN, Mr. A. R. MEWS, and Dr. S. K. MOLOO for helpful comments on the manuscript. The financial support of the Overseas Development Administration of the U.K. Foreign and Commonwealth Office is gratefully acknowledged. REFERENCES ALTMANP. L. (1964) Blood and Other Body Fluids (Ed. by DITTMERD. S.). Fed. Amer. Sots. for Exp. Biol., Washington, D.C. BURSELL E. and JACKSONC. H. N. (1957) Notes on the Choriothete and miik gland of Glossina and Hippobosca (Diptera). Proc. R. ent. Sot. (A) 32, 30-34. BUXTONP. A. (1955) T7z Natural History of Tsetse Flies. London School of Hygiene and Tropical Medicine, Memoir 10. CMELIK S. W. H., BURSELLE., and SLACK E. (1969) Composition of the gut contents of third instar tsetse larvae (Glossina morsitans Westwood). Comp. .&o&m, Physiol. 29, 447453. CUNNXNGHAM I. and SLATERJ. S. (1974) Amino acid analyses of haemolymph of Glossina morsitans morsitans (Westwood). Acta trop. 31, 83-88. HOFFMANNR. (1954) Zur Fortpflanzungsbiologie und zur intra-uterinen Entwicklung von Gloss&z palpalis. Acta trop. 11, l-57. LANGLEY P. A. (1967) The control of digestion in the tsetse fly, Glossim morsitans: a comparison between field flies and flies reared in captivity. J. Insect Physiol. 13,477-486. LANGLEY P. A. (1972) The role of physical and chemical stimuli in the development of in vitro feeding techniques for tsetse flies Glossina spp. (Dipt., Glossinidae). Bull. ent. Res. 62, 215-228. LANGLEY P. A. and P~MLEY R. W. (1973) Influence of diet composition on feeding and water excretion by the tsetse Ay, Glossina morsitans. J. Insect Physiol. 19, 1097-l 109.
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MOLOO S. K. and KUTUZA S. (1970) Feeding and crop emptying in Glossina brwipalpis Newstead. Acta trop. 27, 356-377. NASH T. A. M., JORDANA. M., and TREWERNM. A. (1971) Mass rearing of tsetse flies (Glossina spp.) : recent advances. In Sterility Principle for Insect Control or Eradication, pp. 99-110. I.A.E.A., Vienna. ROBERTSM. J. (1971) The functional anatomy of the head in the larvae of the tsetse fly, Glossina austeni Newstead (Diptera, Glossinidae). Entomologist 104, 190-203. ROBERTSM. J. (1973) Rate of embryonic development in the tsetse fly Glossina morsitans orientalis. Entomologia exp, appl. 16, 268-274. TOBE S. S. and DAVEY K. G. (1972) Volume relationships during the pregnancy cycle of the tsetse Ay Glossina austeni. Can. J. Zool. 50, 999-1010. TOBE S. S. and DAVEY K. G. (1974a) Nutrient transfer during the reproductive cycle in Glossina austeni Newst.-III. Protein synthesis in abdominal tissues. Tissue & Cell. In press. TOBE S. S. and DAVEY K. G. (1974b) Synthesis and turnover of haemolymph protein during the reproductive cycle of Glossina austeni. Can. J. Zool. In press. TOBE S. S., DAVEYK. G., and HUEBNERE. (1973) Nutrient transfer during the reproductive cycle in Glossina austeni Newst.-I. Histology and histochemistry of the milk gland, fat body and oenocytes. Tissue W Cell 5, 633-650.