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Changes in ribonuclease activity during development of the mosquito, Aedes aegypti
Comp. Biochem. PhysioL Vol. 84B, No. 3, pp. 355-361, 1986 Printed in Great Britain
0305-0491/86 $3.00+0.00 Pergamon Journals Ltd
CHANGES IN RIBONUCLEASE ACTIVITY DURING DEVELOPMENT OF THE MOSQUITO, AEDES AEG YPTI MARY ANN FRITZ, PHYLLIS G. HOTCHKIN and ANN MARIE FALLON Department of Microbiology, University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, P.O. Box 55, Piscataway, NJ 08854, USA
(Received 24 October 1985) Abstract--l. In the mosquito Aedes aegypti, quantitative and qualitative changes have been detected in
ribonuclease activity during development. 2. Ribonuclease activity is particularly high in extracts from larvae, relative to that in extracts from pupae or adults. 3. Larval extract is enriched for a ribonuclease that is heat-labile, has an alkaline pH optimum, and is extremely sensitive to the divalent cation, manganese. 4. Extract from adult females is enriched for a heat-stable component that has an acidic pH optimum and is more active at 56 than at 30°C. 5. Throughout the vitellogenic cycle, no major changes in ribonuclease activity were detected in fat body extracts.
INTRODUCTION Ribonucleases are essential to normal cell growth and metabolism. Their precise roles in the cell, and the mechanisms by which their activities are regulated, however, are poorly understood. We have recently shown that cultured Aedes albopictus (mosquito) cells contain multiple ribonucleases that degrade labelled r R N A from Escherichia coil (Fritz and Fallon, 1985). In mosquito cells, ribonuclease activities differed from each other with respect to heat stability, pH optima, sensitivity to cations, subcellular localization, and electrophoretic mobility. Studies using extracts from intact insects representing other dipteran species provide further evidence for a multiplicity of ribonucleases that function during development. Doenecke et al. (1972) suggested that the activity o f ribonuclease H (an enzyme that specifically degrades the R N A component of R N A / D N A hybrids) in the integument of Calliphora larvae may be influenced by 20-hydroxyecdysone. Although it is not clear whether ribonuclease activity is directly responsive to changes in the hormonal milieu, Garcia-Segura and Gavilanes (1982) have shown that in Ceratitis capitata, ribonuclease activity varied over a twelve-fold range during development. Total alkaline ribonuclease and phosphodiesterase activities were particularly high in homogenates from larvae; acid ribonuclease was prominent during the pharate adult stage. Aoki and Natori (1981, 1983) have described two distinct ribonucleases in Sarcophaga peregrina : a latent activity that could be activated by p-chloromercuribenzoate in crude extracts from larval fat body, and a membrane-associated, calciumdependent ribonuclease that appeared to be activated at pupation. We have assayed ribonuclease activity during development of the mosquito, Aedes aegypti, in extracts 355
from whole insects and from dissected tissues of adult females. The properties of ribonucleases from mosquito tissues were in general similar to those we have described from cultured cells. In this report, we describe evidence for several ribonucleases that differ from each other in biochemical properties and in activity during different stages of the mosquito life cycle. MATERIALS AND METHODS
Mosquitoes Aedes aegypti (Rockefeller strain) larvae were reared at 24°C and fed with pulverized rat chow as described by Hotchkin (1985). Adults were maintained at 27°C under a 14 hr light cycle. Guinea-pigs were used to provide the blood meal. Preparation of extracts Larvae (at the end of the last instar), pupae, adult mosquitoes, or dissected parts from adult female mosquitoes (see Table 2), were frozen on dry ice and stored at -70°C. Larvae and pupae were washed with distilled water and blotted dry before freezing. Females were dissected in Aedes saline (Hagedorn et al., 1977). Heads were removed at the neck; thoraces were separated from the abdomen at the junction of the thorax and the first abdominal segment; legs and wings were removed at thoracic junctures and discarded. The last two abdominal segments and cerci, and the attached hindgut (including Malpighian tubules), were removed and discarded. The abdomen was torn open along either pleural region; ovaries were removed; midgut from blood-fed females was torn, and allowed to contract away from the partially digested blood meal. Fat body was left attached to the abdominal wall as described by Hagedorn et al. (1973). Insects were disrupted using a motor-driven Teflon pestle and glass homogenizer (15-20 strokes) in cold extraction buffer (135-150 #1 per insect) containing 0.01 M Tris HCI, pH 8.0, 0.05 M KCI, 0.25 M sucrose (ribonuclease-free,
MARY ANN FRITZ et al.
356
Bethesda Research Laboratories, Inc.), 0.1mM phenylmethyl sulfonyl fluoride (PMSF) and 0.1% dimethylsulfoxide (DMSO). Homogenates were sonicated on ice (one or two 60 sec pulses) with a Kontes sonicator equipped with a microprobe. Particulate material was removed by centrifugation at approx. 12,000g for 10 min at 4°C. Supernatants (crude extracts) were stored at -70°C. Dissected tissues from adult female mosquitoes were sonicated directly, without prior homogenization, in 60 #1 of extraction buffer per body part, and processed as described above. Generally, samples represented pooled tissues from 5 insects.
,~
Electrophoretic analysis Polyacrylamide gels containing [nP]rRNA were used to detect ribonuclease activity after electrophoresis. Gels were prepared by a modification of the method of Huet et al. (1978) as described in the legend to Table 5. The crude whole-cell extract from cultured Ae. albopictus cells was prepared as previously described (Fritz and Fallon, 1985).
RESULTS
Changes in ribonudease activity during development T o establish the developmental profile of ribonuclease activity in Ae. aegypti, we m e a s u r e d hydrolysis o f [3H]rRNA substrate at 30°C by crude extracts p r e p a r e d from larvae, pupae, a n d adult male a n d female mosquitoes (Fig. 1). O n a per animal basis, ribonuclease activity in extracts from larvae after a one h o u r i n c u b a t i o n was more t h a n 4-fold higher t h a n t h a t in pupae, a b o u t 7-fold higher t h a n t h a t in adult females, a n d a b o u t 10-fold higher t h a n t h a t in adult males. The levels of ribonuclease activity at different
i
I
30
60
90
6
4 e ,-r
E
2
0
Time
RNA, l~g ~ r animal
Larva
19.0
]41
Pupa
10.2
115
6.9
1Ol
2.8
Adult
female
Adult male
2.8
Protein~ L~E per anima]
74
RNase 30*C c~/anlmal
(minutes)
developmental stages as they are represented in Fig. 1 are n o t corrected for the " e n d o g e n o u s " R N A c o n t r i b u t e d by the crude extracts a n d the c o n s e q u e n t variation in the specific activity of the labelled R N A substrate in the different assay mixtures. The R N A a n d protein c o n t e n t o f m o s q u i t o extracts are s h o w n in Table 1. The R N A c o n t e n t per larval m o s q u i t o was almost 7-fold higher t h a n t h a t per adult male, 3-fold higher t h a n t h a t per adult female, and 2-fold higher t h a n t h a t per pupa. In contrast, the total protein c o n t e n t in extracts from the various stages varied by less t h a n 2-fold. O n the basis of the relative R N A c o n t e n t in extracts from larvae, pupae, and adults, it can be estimated t h a t the absolute level of ribo-
~atlo, relative to adult male%
RNase, 56aC, cpm/an~mal
1,7 ~ 106
64.2
6.3 x 105
4.5 x 105
9.0
2.2 x 105
4.0
3.9
5.5
6.9
1.0
2,0 x i05
× 10 5
1.8 x 105
120
Fig. 1. Hydrolysis of [3H]rRNA by extracts from larvae (O), pupae (A), adult female (~') and adult male (11) Ae. aegypti. Crude extracts (see Materials and Methods) were diluted 30-fold in buffer (50 mM Tris-HCl, pH 8.0, 83 mM sucrose, 17 mM KC1, 0.03 mM PMSF and 0.03% DMSO) and incubated at 30°C for the indicated time. Blank values, obtained from samples assayed in the absence of extract (approx. 500cpm) have been subtracted. Similar results were obtained in 3 independent experiments.
Table 1. Changes in RNA and protein levels and in ribonuclease activity at different stages of the mosquito life cycle Sta~e
I
U
Ribonuclease assay Ribonuclease activity was assayed by the hydrolysis of [3H]rRNA from E. coli as described previously (Fritz and Fallon, 1985). Briefly, crude extract (22.5 #1) was incubated with 2.5 #1 (10,000 cpm; 4 × 104 cpm/#g) of the [3H]rRNA substrate at 30 or at 56°C, as indicated in the legends to figures and tables. The reaction was stopped by addition of an equal volume (25/~1) of cold 10% trichloroacetic acid (TCA). Samples were maintained on ice for at least 10 min, centrifuged at 10,000g for 10 min, and radioactivity in the supernatant was determined by scintillation counting. Total protein concentration was determined by the Coomassie blue method of Brogdon (1984), using bovine serum albumin (BSA) as the standard. Total RNA in the crude extracts was determined spectrophotometrically after precipitation with perchloric acid and hydrolysis in potassium hydroxide essentially as described previously (Hagedorn et al., 1973).
I
x 10 5
Ratio, relative to adult male% 21.4
I.N
RNA and protein in crude soluble extracts were determined as described in the Materials and Methods. Ribonuclease (RNase) activities are the average of duplicate experiments similar to the one as shown in Fig. 1; values at 60 min were used to calculate activity per animal, tRatios represent activity relative to that in adult males, corrected for the endogenous RNA contributed by the extract. Ratios therefore are the product of [RNase activity (cpm) at stage S/RNase activity in males (I .8 x 10~cpm)] × [RNA(/~g) at stage S/RNA in males (2.8 #g)].
Ribonucleases in Aedes aegypti
Larvae 8
Adult
30 °
56 °
357
Females
30 •
56 °
7
"
6
i $}
' 4
____t 0
30
60
0
30
60
0
30
60
0
30
60
Time (minules)
Fig. 2. Heat stability of ribonuclease in extracts from larvae and adult females. Samples were diluted 3-fold (females) or 30-fold (larvae) in buffer (10 mM Tris-HC1, pH 8.0, 50 mM KC1, 0.25 M sucrose, 0.03 mM PMSF and 0.03% DMSO) and heated for 10min at 30 (A), 37 (11), 45 (A), 56 ([~), 65 (O) 80 (Q) and 100 (/X) °C. Control samples ( 0 ) were not heated. After heat treatment, the extract was divided into two portions and assayed at 30 or at 56°C as indicated. Values are averages of duplicate samples.
nuclease activity in larvae, as measured by the present assay at 30°C, was 64-fold higher than that in males (Table 1). Pupae and adult females, however, had only about 9 and 4-fold more ribonuclease activity, respectively, than adult males, In our earlier work on ribonucleases in cultured Ae. albopictus cells, we showed that there were at least two ribonuclease components in crude extracts: a heat labile component that was inactivated by brief treatment at 56°C, and a heat stable component that was more active at 56 than at 30°C. When extracts from the different stages of the mosquito life cycle were assayed at 56°C, (Table 1) activity in larvae and pupae was lower, by 63 and 51%, respectively, than that measured at 30°C. In contrast, activity in extracts from adult females doubled, and activity in males rose slightly, relative to activity at 30°C. These data suggest that larval extracts are enriched for heat-labile ribonuclease activity, whereas extracts from adult females are enriched for heat-stable activity.
Thermal stability To examine the lability of ribonucleases in larvae and in adult female mosquitoes in more detail, we pretreated extracts at various temperatures ranging from 30 to 100°C for 10rain, and then measured hydrolysis of labelled substrate at 56 and at 30°C (Fig. 2). Untreated larval extract was about twice as active at 30 as at 56°C. When assays were done at 30°C, pretreatment at 56°C resulted in a 74% loss of activity, and no activity remained after a 10min pretreatment at 80°C. Activity measured at 56°C,
however, was inhibited by only 44% after a 10 min pretreatment at the same temperature. In contrast to the larval extract, untreated extract from adult female mosquitoes contained ribonuclease activity that was more than twice as active at 56 than at 30°C. As much as 40% of the heat-stable activity still remained when samples that had been boiled for 10 rain were assayed at 56°C. These results suggest that the enzymes responsible for total ribonuclease activity change as larvae mature to adults. From these data alone, however, it cannot be determined whether these changes are quantitative or qualitative in nature.
pH optima Ribonucleases with optimum activity in acidic or alkaline pH ranges have been identified in extracts from dipteran species and from cultured mosquito cells. In extracts from female fat body (Fig. 3) and from adult female mosquitoes (data not shown), there was a sharp peak of activity at pH 5.0-5.5; at 56°C, this activity was 3-4 fold higher than that at pH 8.0. In contrast to these results with adult female fat body, larval extracts at 30°C were almost 2-fold more active at pH 8.0 than at pH 5.5.
Distribution of ribonuclease in adult female mosquitoes In blood fed mosquitoes, a cycle of accumulation and degradation of total R N A accompanies synthesis of vitellogenin by the fat body (Hagedorn et al., 1973). It was therefore of interest to determine whether changes in ribonuclease activity are associated with changes in fat body RNA content. To provide a framework for evaluating the magnitude of changes in fat body ribonuclease activity, we mea-
358
MARY ANN FRITZ et al. I
Table 2. Ribonuclease activity in tissues from adult female mosquitoes Tlme after feedln~:
Body
Unfed
6h
12h
18h
36h
Assa x , ~C
--
Counts per m i n u t e x tO -3
in
'Q
6
Head
30 ~ 56 °
5.3 9.6
4.8 ]2,6
6.8 10.6
5.3 |].4
5.1 8.6
Thorax
30 ° 56 =
8.8 14.9
8.6 14,2
8.2 14.3
8.4 15.6
7.7 13.4
Midgut
305 56 °
2.6 6,6
7.4 13.6
6.7 11.5
8.5 15.9
8.0 13.4
Ovary
30 ° 56 °
0.4 0.7
2.1 4.0
4.6 8.9
2.1 3.3
I.I 1.9
Fat body
30 ° 56 ~
8.7 14.9
11.0 15.1
9.5 12.4
8.8 13.1
8.2 16.6
Blood meal
30 ° 56 °
---
12.6 13.3
15.2 15.5
15.1 17.9
---
i
"d w(P
2
j G
0---4; 4.5
I'=1
'~ 5.5
I
E5
Ribonuclease activity in body parts from adult femalemosquitoes. Female mosquitoeswere fed on a guinea pig, dissectedat the indicated timesfollowingthe blood meal, and extracts of body parts were assayed for activity. Values represent substrate hydrolyzed (cpm) during 30 rain at the indicated temperature. Each value is averaged from at least two differentexperiments, representing body parts of separate batches of 5 mosquitoes. Values are expressedon a per individual basis.
I=1
7.5
85
pH Fig. 3, Ribonuclease activity in fat body as a function of pH. Crude fat body extract was diluted 3-fold in buffer to a final concentration of 18mM KC1, 88 mM sucrose, 0.035raM PMSF, 0.035% DMSO and 0.05M sodium acetate (pH 4.5-5.5), sodium phosphate (pH 6.0) or Tris-HCl (pH 6.8-8.3). Samples were incubated in the presence of [3H]rRNA at 30°C (©) or at 56"C (O) for 60 min. Blanks, without enzyme, were also assayed at 30°C (A) and at 56°C (A). Similar results were obtained in three independent experiments.
~_
I
I
I
sured hydrolysis o f labelled substrate by extracts from the head, thorax, midgut, ovaries, and fat body o f blood-fed mosquitoes (Table 2). Activity was found in all body parts as well as in the blood meal itself; activity in the blood meal may represent enzymes from guinea-pig serum (Lechner and D u q u e Magalh~es, 1973) or enzymes secreted by midgut cells. In all b o d y parts, but not in the b l o o d meal, activity was a b o u t 1.5-2-fold higher at 56 than at
l
1
I Adult
I I Females
i_
"
0
25
50
75
I00 0
2.5
50
75
I00
MnCl= (raM} Fig. 4. Effect of manganese chloride on ribonuclease activity in extracts from larvae and adult females. Samples were diluted 30-fold (larvae) or 3-fold (adult females) in buffer containing 10 mM Tris-HC1, pH 8.0, and 0.25 M sucrose; manganese chloride was added to the indicated concentrations. Each assay mixture was incubated with [3H]rRNA for 60 min at 30 (O) or 56°C (0). Blanks assayed in the absence of enzyme have been subtracted. Similar results were obtained in three independent experiments.
Ribonucleases in Aedes aegypti 30°C, consistent with the data shown in Table 1 and Fig. 2; no body part showed extremely high activity, relative to other parts. Activity in ovaries, although low relative to that in other tissues, increased severalfold during the period of rapid growth following the blood meal. In several different experiments, including assays at both pH 8.0 or 5.4, and at 30 or at 56°C, levels of activity in female fat body as a function of time (6 to 48 hr) after the blood meal varied within a 3-fold range; no outstanding pattern to this variation was detected and no major increases in ribonuclease activity occurred at 36-48 hr after the blood meal, when fat body RNA content is decreasing (Table 2 and other experiments not shown). Moreover, the magnitude of changes in ribonuclease activity in mosquito fat body was no greater than the fluctuations in activity in extracts from other body parts.
Effect of manganese and other agents In extracts from cultured mosquito cells, ribonuelease activity measured at 30°C was inhibited by manganese; at 56°C, a slight inhibition of ribonuclease activity was detected at manganese concentrations below 5 mM, but activity increased at higher concentrations (Fritz and Fallon, 1985). It was therefore of interest to compare hydrolysis of rRNA by extracts from larvae and adult females in the presence
1
BP
-
BP
-
2
3
4
359
of manganese (Fig. 4). At 30°C, activity in extracts from both larvae and adult females was markedly inhibited by manganese. Heat-stable activity assayed at 56°C was somewhat more resistant to manganese at both developmental stages. Activity in extracts from whole adult females, but not from larvae, exhibited a decrease at low manganese concentrations, followed by partial recovery of activity at higher concentrations when assayed at 56°C, similar to what we have observed with ribonuclease in extracts from cultured cells. However, unlike ribonuclease activity from cultured cells, heat-stable activity from females did not recover to initial levels at higher manganese concentrations. Extracts from fat bodies of unfed females showed essentially the same pattern of activity in the presence of manganese as shown for adult females in Fig. 4, and no changes in this pattern were detected in fat body extracts prepared at different times after the blood meal. P-chloromercuribenzoate (p CMB) has been shown to activate latent ribonucleases in extracts from other Diptera (Aoki and Natori, 1981). However, the addition of pCMB to extracts from larvae or from mosquito fat body did not result in increased activity. Aoki and Natori (1983) effected the release of latent ribonuclease activity from fat body membranes of Sarcophaga by the addition of calcium, and calcium-dependent ribonuclease has been described
5
6
7
-
BP
--,¢RNase
Fig. 5. Electrophoretic analysis of ribonuclease activities at different developmental stages ofAe. aegypti. A 12.5% polyacrylamide gel containing [32p]rRNA (4 x 105cpm) was prepared as described previously (Fritz and Fallon, 1985). After electrophoresis (3hr at 150V) the gel was washed with 25% isopropanol/10mM Tris HC1, pH 8.0, to remove SDS, and incubated overnight at 37°C in 0.1 M Tris-HCl, pH 8.0, to allow hydrolysis of the labelled substrate. The gel was exposed to Kodak X-Omat film to localize ribonuclease activity (clear bands), or RNA-binding proteins (dark bands). Lane 1, extract from cultured cells (80/tg total protein); 2, larval extract (7.05/~g protein); 3, adult female extract (3.45/~g protein); 4, pupal extract (5.10/~g protein); 5, larval extract (16.92 gg protein); 6, adult female extract (8.28 gg protein); 7, pupal extract (12.24/~g protein). The amount of extract added to the gel was chosen on the basis of activity in solution; activity from cultured cells, in solution, is low relative to that of insect extracts.
MARY ANN FRITZ et al.
360
in polysome preparations from cockroaches (Englemann, 1977) and locusts (Reid and Chen, 1981). However, the addition of calcium chloride to our fat body preparations (up to a concentration of 1 raM) had no effect on activity; concentrations of 10 mM or higher were inhibitory.
Electrophoretic analysis Assays involving hydrolysis of labelled R N A by crude extracts do not distinguish among the many enzymes which may contribute to the total activity. The data we have presented above, regarding heat stability, pH dependence, and the effects of manganese, as well as our previous work on ribonuclease activity in extracts from cultured Ae. albopictus cells, suggest that a multiplicity of ribonucleases is present in the mosquito. Electrophoretic detection of ribonucleases provides a method for identification, on the basis of size, of those enzymes that recover activity after removal of SDS from polyacrylamide gels (Huet et al., 1978). Extracts from larvae, pupae and adult mosquitoes were therefore compared with extracts from mosquito cells on 12.5% SDS-polyacrylamide gels containing [32p]rRNA substrate (Fig. 5). In contrast to results obtained with assays in solution, activity detected in gels was particularly low for the larval extract (lanes 2 and 5); only a weak band of activity was present (at a molecular mass of approximately 16,000 daltons). Extracts from pupae (lanes 4 and 7) and adult females (lanes 3 and 6) contained much stronger bands of activity at about 16,000 daltons. In addition, extracts from pupae and adult females contained diffuse ribonuclease activity of higher mass and at least one of the RNA-binding proteins (BP; molecular mass about 33,000 daltons) that also occur in extracts from cultured cells (lane 1). DISCUSSION
Our previous work with cultured mosquito (Ae. albopictus) cells suggested that a multiplicity of ribonuclease activities would be present in mosquito tissues. Activities that were detected in cultured cells differed from each other with respect to pH optima, heat stability, subcellular localization, and the effect of divalent cations (Fritz and Fallon, 1985). In the mosquito, total ribonuclease activity varied with developmental stage. During the larval to adult transition, the predominant activity shifted from a heat-labile component with a broad alkaline pH optimum, to a heat-stable component with optimal activity at pH 5.0-5.5. Activity in extracts from pupae reflected intermediate properties between these two extremes. On a per animal basis, overall ribonuclease activity in the larval extract was much higher than that at other developmental stages. The properties of the major component of the larval preparation were reminiscent of the heat-labile activity from cultured Ae. albopictus cells. Although some heat-stable activity could be detected in larvae, activity at 30°C was at least 2-fold greater than that at 56°C, and the absolute level of ribonuclease activity (corrected for endogenous RNA) was 17-fold greater than that in female extracts assayed at 30°C. Finally, the heat
labile component in larval extracts resembled that of cultured cells in its sensitivity to manganese. We have previously suggested that, in extracts from cultured cells, the low molecular mass (16 kDa) activity is heat-stable (Fritz and Fallon, 1985). Consistent with this hypothesis is the observation that electrophoretic analyses showed relatively little low molecular weight activity in larval extracts, relative to that in extracts from pupae or adults. Moreover, the inability to reproduce in gels the high activity of larval extracts in solution suggests that the heat-labile component does not recover activity after electrophoresis under the described conditions. In pupae, absolute ribonuclease activity per animal was about 7-fold less than that in larvae at 30°C, but the ratio of heat-stable to heat-labile activity increased slightly. Extracts from adult females contained approximately the same amount of total ribonuclease activity as extracts from pupae, but were markedly enriched for heat-stable activity. Extracts from females also contained a component that was active even after boiling. Activity stable to boiling was not detected in extracts from cultured cells. In Ae. aegypti, synthesis of vitellogenin by adult female fat body is accompanied by a cycle of RNA accumulation and degradation (Hagedorn et al., 1973). To evaluate a possible role for ribonuclease during the mosquito vitellogenic cycle, we compared activity in extracts from fat body at various times after a blood meal, with that in extracts from dissected parts from adult female mosquitoes and with that in fat bodies from unfed females. Activity in all the extracts examined exhibited the same general properties, and no remarkable changes were detected during the vitellogenic cycle. Only a slight decrease in activity at 12-24 hr post blood meal, and a slight increase in activity at 36-48 hr post feeding was detected. The extent and timing of this fluctuation varied slightly from experiment to experiment, and was more obvious at 56 than at 30°C. On the basis of the present data it seems unlikely that modulation of total cellular ribonuclease activity is primarily responsible for the accumulation and degradation of R N A in fat body of blood-fed mosquitoes. These experiments do not, however, eliminate the possibility that changes in activity of a specific ribonuclease, such as a ribosome-associated enzyme, might be obscured by higher activities of other enzymes. Extracts from cultured Aedes albopictus cells contain heat-stable and heat-labile activity in approximately equal proportions. In contrast, extracts from intact mosquitoes appear to be enriched for different activities at different developmental stages. Extracts from larval and adult female mosquitoes, respectively, may therefore facilitate further characterization of heat-labile and heat-stable ribonucleases. Acknowledgements--This work was supported by a grant from the National Institute of Allergy and Infectious Diseases (AI20385). We thank Eleanor Kells and Patti Vendula for typing the manuscript. REFERENCES
Aoki Y. and Natori S. (1981) Activation of latent ribonuclease in the fat-body of fleshfly (Sarcophaga peregrina) larvae on pupation. Biochem. J. 196, 699 703.
Ribonucleases in Aedes aegypti Aoki Y. and Natori S. (1983) Activation of an RNAse in a membrane fraction from fat body of Sarcophaga peregrina larvae by calcium, lnsect Biochem. 13, 403-406. Brogdon W. G. (1984) Mosquito protein microassay--1. Protein determinations from small portions of single mosquito homogenates. Comp. Biochem. Physiol. 79B, 457-459. Doenecke D., Marmaras V. J. and Sekeris C. E. (1972) Increased RNase H (hybridase) activity in the integument of blowfly larvae during development and under the influence of fl-ecdysone. FEBS Lett. 22, 261-264. Englemann F. (1977) Undegraded vitellogenin polysomes from female insect fat bodies. Biochem. Biophys. Res. Comm. 78, 641~47. Fritz M. A. and Fallon A. M. (1985) Evidence for multiple ribonucleases in crude extracts from cultured mosquito cells. Insect Biochem. 15, 817-825. Garcia-Segura J. M. and Gavilanes J. G. (1982) Study of the RNA-degrading activities during the development of the insect Ceratitis capitata. Comp. Biochem. Physiol. 73B, 835-838. Hagedorn H. H., Fallon A. M. and Laufer H. (1973)
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Vitellogenin synthesis by the fat body of the mosquito Aedes aegypti: evidence for transcriptional control. Devel. Biol. 31, 285-294. Hagedorn H. H., Turner S., Hagedorn E. A., Pontecorvo D., Greenbaum P., Pfeiffer D., Wheelock G. and Flanagan T. R. (1977) Postemergence growth of the ovarian follicle of Aedes aegypti. J. Insect Physiol. 23, 203-206. Hotchkin P. (1985) The duration of larval life of Aedes aegypti as affected by time of hatch. J. Am. Mosq. Cont. Assoc. 1, 489-492. Huet J., Sentenac A. and Fromageot P. (1978) Detection of nucleases degrading double helical RNA and of nucleic acid-binding proteins following SDS-gel electrophoresis. FEBS Lett. 94, 28-32. Lechner M. C. and Duque Magalh~es M. C. (1973) RNase activities in blood serum of several vertebrates. Experientia 29, 1479-1480. Reid P. C. and Chen T. T. (1981) Juvenile hormonecontrolled vitellogenin synthesis in the fat body of the locust (Locusta migratoria): isolation and characterization of vitellogenin polysomes and their induction in vivo. Insect Biochem. 11, 297-305.
0305-0491/86 $3.00+0.00 Pergamon Journals Ltd
CHANGES IN RIBONUCLEASE ACTIVITY DURING DEVELOPMENT OF THE MOSQUITO, AEDES AEG YPTI MARY ANN FRITZ, PHYLLIS G. HOTCHKIN and ANN MARIE FALLON Department of Microbiology, University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, P.O. Box 55, Piscataway, NJ 08854, USA
(Received 24 October 1985) Abstract--l. In the mosquito Aedes aegypti, quantitative and qualitative changes have been detected in
ribonuclease activity during development. 2. Ribonuclease activity is particularly high in extracts from larvae, relative to that in extracts from pupae or adults. 3. Larval extract is enriched for a ribonuclease that is heat-labile, has an alkaline pH optimum, and is extremely sensitive to the divalent cation, manganese. 4. Extract from adult females is enriched for a heat-stable component that has an acidic pH optimum and is more active at 56 than at 30°C. 5. Throughout the vitellogenic cycle, no major changes in ribonuclease activity were detected in fat body extracts.
INTRODUCTION Ribonucleases are essential to normal cell growth and metabolism. Their precise roles in the cell, and the mechanisms by which their activities are regulated, however, are poorly understood. We have recently shown that cultured Aedes albopictus (mosquito) cells contain multiple ribonucleases that degrade labelled r R N A from Escherichia coil (Fritz and Fallon, 1985). In mosquito cells, ribonuclease activities differed from each other with respect to heat stability, pH optima, sensitivity to cations, subcellular localization, and electrophoretic mobility. Studies using extracts from intact insects representing other dipteran species provide further evidence for a multiplicity of ribonucleases that function during development. Doenecke et al. (1972) suggested that the activity o f ribonuclease H (an enzyme that specifically degrades the R N A component of R N A / D N A hybrids) in the integument of Calliphora larvae may be influenced by 20-hydroxyecdysone. Although it is not clear whether ribonuclease activity is directly responsive to changes in the hormonal milieu, Garcia-Segura and Gavilanes (1982) have shown that in Ceratitis capitata, ribonuclease activity varied over a twelve-fold range during development. Total alkaline ribonuclease and phosphodiesterase activities were particularly high in homogenates from larvae; acid ribonuclease was prominent during the pharate adult stage. Aoki and Natori (1981, 1983) have described two distinct ribonucleases in Sarcophaga peregrina : a latent activity that could be activated by p-chloromercuribenzoate in crude extracts from larval fat body, and a membrane-associated, calciumdependent ribonuclease that appeared to be activated at pupation. We have assayed ribonuclease activity during development of the mosquito, Aedes aegypti, in extracts 355
from whole insects and from dissected tissues of adult females. The properties of ribonucleases from mosquito tissues were in general similar to those we have described from cultured cells. In this report, we describe evidence for several ribonucleases that differ from each other in biochemical properties and in activity during different stages of the mosquito life cycle. MATERIALS AND METHODS
Mosquitoes Aedes aegypti (Rockefeller strain) larvae were reared at 24°C and fed with pulverized rat chow as described by Hotchkin (1985). Adults were maintained at 27°C under a 14 hr light cycle. Guinea-pigs were used to provide the blood meal. Preparation of extracts Larvae (at the end of the last instar), pupae, adult mosquitoes, or dissected parts from adult female mosquitoes (see Table 2), were frozen on dry ice and stored at -70°C. Larvae and pupae were washed with distilled water and blotted dry before freezing. Females were dissected in Aedes saline (Hagedorn et al., 1977). Heads were removed at the neck; thoraces were separated from the abdomen at the junction of the thorax and the first abdominal segment; legs and wings were removed at thoracic junctures and discarded. The last two abdominal segments and cerci, and the attached hindgut (including Malpighian tubules), were removed and discarded. The abdomen was torn open along either pleural region; ovaries were removed; midgut from blood-fed females was torn, and allowed to contract away from the partially digested blood meal. Fat body was left attached to the abdominal wall as described by Hagedorn et al. (1973). Insects were disrupted using a motor-driven Teflon pestle and glass homogenizer (15-20 strokes) in cold extraction buffer (135-150 #1 per insect) containing 0.01 M Tris HCI, pH 8.0, 0.05 M KCI, 0.25 M sucrose (ribonuclease-free,
MARY ANN FRITZ et al.
356
Bethesda Research Laboratories, Inc.), 0.1mM phenylmethyl sulfonyl fluoride (PMSF) and 0.1% dimethylsulfoxide (DMSO). Homogenates were sonicated on ice (one or two 60 sec pulses) with a Kontes sonicator equipped with a microprobe. Particulate material was removed by centrifugation at approx. 12,000g for 10 min at 4°C. Supernatants (crude extracts) were stored at -70°C. Dissected tissues from adult female mosquitoes were sonicated directly, without prior homogenization, in 60 #1 of extraction buffer per body part, and processed as described above. Generally, samples represented pooled tissues from 5 insects.
,~
Electrophoretic analysis Polyacrylamide gels containing [nP]rRNA were used to detect ribonuclease activity after electrophoresis. Gels were prepared by a modification of the method of Huet et al. (1978) as described in the legend to Table 5. The crude whole-cell extract from cultured Ae. albopictus cells was prepared as previously described (Fritz and Fallon, 1985).
RESULTS
Changes in ribonudease activity during development T o establish the developmental profile of ribonuclease activity in Ae. aegypti, we m e a s u r e d hydrolysis o f [3H]rRNA substrate at 30°C by crude extracts p r e p a r e d from larvae, pupae, a n d adult male a n d female mosquitoes (Fig. 1). O n a per animal basis, ribonuclease activity in extracts from larvae after a one h o u r i n c u b a t i o n was more t h a n 4-fold higher t h a n t h a t in pupae, a b o u t 7-fold higher t h a n t h a t in adult females, a n d a b o u t 10-fold higher t h a n t h a t in adult males. The levels of ribonuclease activity at different
i
I
30
60
90
6
4 e ,-r
E
2
0
Time
RNA, l~g ~ r animal
Larva
19.0
]41
Pupa
10.2
115
6.9
1Ol
2.8
Adult
female
Adult male
2.8
Protein~ L~E per anima]
74
RNase 30*C c~/anlmal
(minutes)
developmental stages as they are represented in Fig. 1 are n o t corrected for the " e n d o g e n o u s " R N A c o n t r i b u t e d by the crude extracts a n d the c o n s e q u e n t variation in the specific activity of the labelled R N A substrate in the different assay mixtures. The R N A a n d protein c o n t e n t o f m o s q u i t o extracts are s h o w n in Table 1. The R N A c o n t e n t per larval m o s q u i t o was almost 7-fold higher t h a n t h a t per adult male, 3-fold higher t h a n t h a t per adult female, and 2-fold higher t h a n t h a t per pupa. In contrast, the total protein c o n t e n t in extracts from the various stages varied by less t h a n 2-fold. O n the basis of the relative R N A c o n t e n t in extracts from larvae, pupae, and adults, it can be estimated t h a t the absolute level of ribo-
~atlo, relative to adult male%
RNase, 56aC, cpm/an~mal
1,7 ~ 106
64.2
6.3 x 105
4.5 x 105
9.0
2.2 x 105
4.0
3.9
5.5
6.9
1.0
2,0 x i05
× 10 5
1.8 x 105
120
Fig. 1. Hydrolysis of [3H]rRNA by extracts from larvae (O), pupae (A), adult female (~') and adult male (11) Ae. aegypti. Crude extracts (see Materials and Methods) were diluted 30-fold in buffer (50 mM Tris-HCl, pH 8.0, 83 mM sucrose, 17 mM KC1, 0.03 mM PMSF and 0.03% DMSO) and incubated at 30°C for the indicated time. Blank values, obtained from samples assayed in the absence of extract (approx. 500cpm) have been subtracted. Similar results were obtained in 3 independent experiments.
Table 1. Changes in RNA and protein levels and in ribonuclease activity at different stages of the mosquito life cycle Sta~e
I
U
Ribonuclease assay Ribonuclease activity was assayed by the hydrolysis of [3H]rRNA from E. coli as described previously (Fritz and Fallon, 1985). Briefly, crude extract (22.5 #1) was incubated with 2.5 #1 (10,000 cpm; 4 × 104 cpm/#g) of the [3H]rRNA substrate at 30 or at 56°C, as indicated in the legends to figures and tables. The reaction was stopped by addition of an equal volume (25/~1) of cold 10% trichloroacetic acid (TCA). Samples were maintained on ice for at least 10 min, centrifuged at 10,000g for 10 min, and radioactivity in the supernatant was determined by scintillation counting. Total protein concentration was determined by the Coomassie blue method of Brogdon (1984), using bovine serum albumin (BSA) as the standard. Total RNA in the crude extracts was determined spectrophotometrically after precipitation with perchloric acid and hydrolysis in potassium hydroxide essentially as described previously (Hagedorn et al., 1973).
I
x 10 5
Ratio, relative to adult male% 21.4
I.N
RNA and protein in crude soluble extracts were determined as described in the Materials and Methods. Ribonuclease (RNase) activities are the average of duplicate experiments similar to the one as shown in Fig. 1; values at 60 min were used to calculate activity per animal, tRatios represent activity relative to that in adult males, corrected for the endogenous RNA contributed by the extract. Ratios therefore are the product of [RNase activity (cpm) at stage S/RNase activity in males (I .8 x 10~cpm)] × [RNA(/~g) at stage S/RNA in males (2.8 #g)].
Ribonucleases in Aedes aegypti
Larvae 8
Adult
30 °
56 °
357
Females
30 •
56 °
7
"
6
i $}
' 4
____t 0
30
60
0
30
60
0
30
60
0
30
60
Time (minules)
Fig. 2. Heat stability of ribonuclease in extracts from larvae and adult females. Samples were diluted 3-fold (females) or 30-fold (larvae) in buffer (10 mM Tris-HC1, pH 8.0, 50 mM KC1, 0.25 M sucrose, 0.03 mM PMSF and 0.03% DMSO) and heated for 10min at 30 (A), 37 (11), 45 (A), 56 ([~), 65 (O) 80 (Q) and 100 (/X) °C. Control samples ( 0 ) were not heated. After heat treatment, the extract was divided into two portions and assayed at 30 or at 56°C as indicated. Values are averages of duplicate samples.
nuclease activity in larvae, as measured by the present assay at 30°C, was 64-fold higher than that in males (Table 1). Pupae and adult females, however, had only about 9 and 4-fold more ribonuclease activity, respectively, than adult males, In our earlier work on ribonucleases in cultured Ae. albopictus cells, we showed that there were at least two ribonuclease components in crude extracts: a heat labile component that was inactivated by brief treatment at 56°C, and a heat stable component that was more active at 56 than at 30°C. When extracts from the different stages of the mosquito life cycle were assayed at 56°C, (Table 1) activity in larvae and pupae was lower, by 63 and 51%, respectively, than that measured at 30°C. In contrast, activity in extracts from adult females doubled, and activity in males rose slightly, relative to activity at 30°C. These data suggest that larval extracts are enriched for heat-labile ribonuclease activity, whereas extracts from adult females are enriched for heat-stable activity.
Thermal stability To examine the lability of ribonucleases in larvae and in adult female mosquitoes in more detail, we pretreated extracts at various temperatures ranging from 30 to 100°C for 10rain, and then measured hydrolysis of labelled substrate at 56 and at 30°C (Fig. 2). Untreated larval extract was about twice as active at 30 as at 56°C. When assays were done at 30°C, pretreatment at 56°C resulted in a 74% loss of activity, and no activity remained after a 10min pretreatment at 80°C. Activity measured at 56°C,
however, was inhibited by only 44% after a 10 min pretreatment at the same temperature. In contrast to the larval extract, untreated extract from adult female mosquitoes contained ribonuclease activity that was more than twice as active at 56 than at 30°C. As much as 40% of the heat-stable activity still remained when samples that had been boiled for 10 rain were assayed at 56°C. These results suggest that the enzymes responsible for total ribonuclease activity change as larvae mature to adults. From these data alone, however, it cannot be determined whether these changes are quantitative or qualitative in nature.
pH optima Ribonucleases with optimum activity in acidic or alkaline pH ranges have been identified in extracts from dipteran species and from cultured mosquito cells. In extracts from female fat body (Fig. 3) and from adult female mosquitoes (data not shown), there was a sharp peak of activity at pH 5.0-5.5; at 56°C, this activity was 3-4 fold higher than that at pH 8.0. In contrast to these results with adult female fat body, larval extracts at 30°C were almost 2-fold more active at pH 8.0 than at pH 5.5.
Distribution of ribonuclease in adult female mosquitoes In blood fed mosquitoes, a cycle of accumulation and degradation of total R N A accompanies synthesis of vitellogenin by the fat body (Hagedorn et al., 1973). It was therefore of interest to determine whether changes in ribonuclease activity are associated with changes in fat body RNA content. To provide a framework for evaluating the magnitude of changes in fat body ribonuclease activity, we mea-
358
MARY ANN FRITZ et al. I
Table 2. Ribonuclease activity in tissues from adult female mosquitoes Tlme after feedln~:
Body
Unfed
6h
12h
18h
36h
Assa x , ~C
--
Counts per m i n u t e x tO -3
in
'Q
6
Head
30 ~ 56 °
5.3 9.6
4.8 ]2,6
6.8 10.6
5.3 |].4
5.1 8.6
Thorax
30 ° 56 =
8.8 14.9
8.6 14,2
8.2 14.3
8.4 15.6
7.7 13.4
Midgut
305 56 °
2.6 6,6
7.4 13.6
6.7 11.5
8.5 15.9
8.0 13.4
Ovary
30 ° 56 °
0.4 0.7
2.1 4.0
4.6 8.9
2.1 3.3
I.I 1.9
Fat body
30 ° 56 ~
8.7 14.9
11.0 15.1
9.5 12.4
8.8 13.1
8.2 16.6
Blood meal
30 ° 56 °
---
12.6 13.3
15.2 15.5
15.1 17.9
---
i
"d w(P
2
j G
0---4; 4.5
I'=1
'~ 5.5
I
E5
Ribonuclease activity in body parts from adult femalemosquitoes. Female mosquitoeswere fed on a guinea pig, dissectedat the indicated timesfollowingthe blood meal, and extracts of body parts were assayed for activity. Values represent substrate hydrolyzed (cpm) during 30 rain at the indicated temperature. Each value is averaged from at least two differentexperiments, representing body parts of separate batches of 5 mosquitoes. Values are expressedon a per individual basis.
I=1
7.5
85
pH Fig. 3, Ribonuclease activity in fat body as a function of pH. Crude fat body extract was diluted 3-fold in buffer to a final concentration of 18mM KC1, 88 mM sucrose, 0.035raM PMSF, 0.035% DMSO and 0.05M sodium acetate (pH 4.5-5.5), sodium phosphate (pH 6.0) or Tris-HCl (pH 6.8-8.3). Samples were incubated in the presence of [3H]rRNA at 30°C (©) or at 56"C (O) for 60 min. Blanks, without enzyme, were also assayed at 30°C (A) and at 56°C (A). Similar results were obtained in three independent experiments.
~_
I
I
I
sured hydrolysis o f labelled substrate by extracts from the head, thorax, midgut, ovaries, and fat body o f blood-fed mosquitoes (Table 2). Activity was found in all body parts as well as in the blood meal itself; activity in the blood meal may represent enzymes from guinea-pig serum (Lechner and D u q u e Magalh~es, 1973) or enzymes secreted by midgut cells. In all b o d y parts, but not in the b l o o d meal, activity was a b o u t 1.5-2-fold higher at 56 than at
l
1
I Adult
I I Females
i_
"
0
25
50
75
I00 0
2.5
50
75
I00
MnCl= (raM} Fig. 4. Effect of manganese chloride on ribonuclease activity in extracts from larvae and adult females. Samples were diluted 30-fold (larvae) or 3-fold (adult females) in buffer containing 10 mM Tris-HC1, pH 8.0, and 0.25 M sucrose; manganese chloride was added to the indicated concentrations. Each assay mixture was incubated with [3H]rRNA for 60 min at 30 (O) or 56°C (0). Blanks assayed in the absence of enzyme have been subtracted. Similar results were obtained in three independent experiments.
Ribonucleases in Aedes aegypti 30°C, consistent with the data shown in Table 1 and Fig. 2; no body part showed extremely high activity, relative to other parts. Activity in ovaries, although low relative to that in other tissues, increased severalfold during the period of rapid growth following the blood meal. In several different experiments, including assays at both pH 8.0 or 5.4, and at 30 or at 56°C, levels of activity in female fat body as a function of time (6 to 48 hr) after the blood meal varied within a 3-fold range; no outstanding pattern to this variation was detected and no major increases in ribonuclease activity occurred at 36-48 hr after the blood meal, when fat body RNA content is decreasing (Table 2 and other experiments not shown). Moreover, the magnitude of changes in ribonuclease activity in mosquito fat body was no greater than the fluctuations in activity in extracts from other body parts.
Effect of manganese and other agents In extracts from cultured mosquito cells, ribonuelease activity measured at 30°C was inhibited by manganese; at 56°C, a slight inhibition of ribonuclease activity was detected at manganese concentrations below 5 mM, but activity increased at higher concentrations (Fritz and Fallon, 1985). It was therefore of interest to compare hydrolysis of rRNA by extracts from larvae and adult females in the presence
1
BP
-
BP
-
2
3
4
359
of manganese (Fig. 4). At 30°C, activity in extracts from both larvae and adult females was markedly inhibited by manganese. Heat-stable activity assayed at 56°C was somewhat more resistant to manganese at both developmental stages. Activity in extracts from whole adult females, but not from larvae, exhibited a decrease at low manganese concentrations, followed by partial recovery of activity at higher concentrations when assayed at 56°C, similar to what we have observed with ribonuclease in extracts from cultured cells. However, unlike ribonuclease activity from cultured cells, heat-stable activity from females did not recover to initial levels at higher manganese concentrations. Extracts from fat bodies of unfed females showed essentially the same pattern of activity in the presence of manganese as shown for adult females in Fig. 4, and no changes in this pattern were detected in fat body extracts prepared at different times after the blood meal. P-chloromercuribenzoate (p CMB) has been shown to activate latent ribonucleases in extracts from other Diptera (Aoki and Natori, 1981). However, the addition of pCMB to extracts from larvae or from mosquito fat body did not result in increased activity. Aoki and Natori (1983) effected the release of latent ribonuclease activity from fat body membranes of Sarcophaga by the addition of calcium, and calcium-dependent ribonuclease has been described
5
6
7
-
BP
--,¢RNase
Fig. 5. Electrophoretic analysis of ribonuclease activities at different developmental stages ofAe. aegypti. A 12.5% polyacrylamide gel containing [32p]rRNA (4 x 105cpm) was prepared as described previously (Fritz and Fallon, 1985). After electrophoresis (3hr at 150V) the gel was washed with 25% isopropanol/10mM Tris HC1, pH 8.0, to remove SDS, and incubated overnight at 37°C in 0.1 M Tris-HCl, pH 8.0, to allow hydrolysis of the labelled substrate. The gel was exposed to Kodak X-Omat film to localize ribonuclease activity (clear bands), or RNA-binding proteins (dark bands). Lane 1, extract from cultured cells (80/tg total protein); 2, larval extract (7.05/~g protein); 3, adult female extract (3.45/~g protein); 4, pupal extract (5.10/~g protein); 5, larval extract (16.92 gg protein); 6, adult female extract (8.28 gg protein); 7, pupal extract (12.24/~g protein). The amount of extract added to the gel was chosen on the basis of activity in solution; activity from cultured cells, in solution, is low relative to that of insect extracts.
MARY ANN FRITZ et al.
360
in polysome preparations from cockroaches (Englemann, 1977) and locusts (Reid and Chen, 1981). However, the addition of calcium chloride to our fat body preparations (up to a concentration of 1 raM) had no effect on activity; concentrations of 10 mM or higher were inhibitory.
Electrophoretic analysis Assays involving hydrolysis of labelled R N A by crude extracts do not distinguish among the many enzymes which may contribute to the total activity. The data we have presented above, regarding heat stability, pH dependence, and the effects of manganese, as well as our previous work on ribonuclease activity in extracts from cultured Ae. albopictus cells, suggest that a multiplicity of ribonucleases is present in the mosquito. Electrophoretic detection of ribonucleases provides a method for identification, on the basis of size, of those enzymes that recover activity after removal of SDS from polyacrylamide gels (Huet et al., 1978). Extracts from larvae, pupae and adult mosquitoes were therefore compared with extracts from mosquito cells on 12.5% SDS-polyacrylamide gels containing [32p]rRNA substrate (Fig. 5). In contrast to results obtained with assays in solution, activity detected in gels was particularly low for the larval extract (lanes 2 and 5); only a weak band of activity was present (at a molecular mass of approximately 16,000 daltons). Extracts from pupae (lanes 4 and 7) and adult females (lanes 3 and 6) contained much stronger bands of activity at about 16,000 daltons. In addition, extracts from pupae and adult females contained diffuse ribonuclease activity of higher mass and at least one of the RNA-binding proteins (BP; molecular mass about 33,000 daltons) that also occur in extracts from cultured cells (lane 1). DISCUSSION
Our previous work with cultured mosquito (Ae. albopictus) cells suggested that a multiplicity of ribonuclease activities would be present in mosquito tissues. Activities that were detected in cultured cells differed from each other with respect to pH optima, heat stability, subcellular localization, and the effect of divalent cations (Fritz and Fallon, 1985). In the mosquito, total ribonuclease activity varied with developmental stage. During the larval to adult transition, the predominant activity shifted from a heat-labile component with a broad alkaline pH optimum, to a heat-stable component with optimal activity at pH 5.0-5.5. Activity in extracts from pupae reflected intermediate properties between these two extremes. On a per animal basis, overall ribonuclease activity in the larval extract was much higher than that at other developmental stages. The properties of the major component of the larval preparation were reminiscent of the heat-labile activity from cultured Ae. albopictus cells. Although some heat-stable activity could be detected in larvae, activity at 30°C was at least 2-fold greater than that at 56°C, and the absolute level of ribonuclease activity (corrected for endogenous RNA) was 17-fold greater than that in female extracts assayed at 30°C. Finally, the heat
labile component in larval extracts resembled that of cultured cells in its sensitivity to manganese. We have previously suggested that, in extracts from cultured cells, the low molecular mass (16 kDa) activity is heat-stable (Fritz and Fallon, 1985). Consistent with this hypothesis is the observation that electrophoretic analyses showed relatively little low molecular weight activity in larval extracts, relative to that in extracts from pupae or adults. Moreover, the inability to reproduce in gels the high activity of larval extracts in solution suggests that the heat-labile component does not recover activity after electrophoresis under the described conditions. In pupae, absolute ribonuclease activity per animal was about 7-fold less than that in larvae at 30°C, but the ratio of heat-stable to heat-labile activity increased slightly. Extracts from adult females contained approximately the same amount of total ribonuclease activity as extracts from pupae, but were markedly enriched for heat-stable activity. Extracts from females also contained a component that was active even after boiling. Activity stable to boiling was not detected in extracts from cultured cells. In Ae. aegypti, synthesis of vitellogenin by adult female fat body is accompanied by a cycle of RNA accumulation and degradation (Hagedorn et al., 1973). To evaluate a possible role for ribonuclease during the mosquito vitellogenic cycle, we compared activity in extracts from fat body at various times after a blood meal, with that in extracts from dissected parts from adult female mosquitoes and with that in fat bodies from unfed females. Activity in all the extracts examined exhibited the same general properties, and no remarkable changes were detected during the vitellogenic cycle. Only a slight decrease in activity at 12-24 hr post blood meal, and a slight increase in activity at 36-48 hr post feeding was detected. The extent and timing of this fluctuation varied slightly from experiment to experiment, and was more obvious at 56 than at 30°C. On the basis of the present data it seems unlikely that modulation of total cellular ribonuclease activity is primarily responsible for the accumulation and degradation of R N A in fat body of blood-fed mosquitoes. These experiments do not, however, eliminate the possibility that changes in activity of a specific ribonuclease, such as a ribosome-associated enzyme, might be obscured by higher activities of other enzymes. Extracts from cultured Aedes albopictus cells contain heat-stable and heat-labile activity in approximately equal proportions. In contrast, extracts from intact mosquitoes appear to be enriched for different activities at different developmental stages. Extracts from larval and adult female mosquitoes, respectively, may therefore facilitate further characterization of heat-labile and heat-stable ribonucleases. Acknowledgements--This work was supported by a grant from the National Institute of Allergy and Infectious Diseases (AI20385). We thank Eleanor Kells and Patti Vendula for typing the manuscript. REFERENCES
Aoki Y. and Natori S. (1981) Activation of latent ribonuclease in the fat-body of fleshfly (Sarcophaga peregrina) larvae on pupation. Biochem. J. 196, 699 703.
Ribonucleases in Aedes aegypti Aoki Y. and Natori S. (1983) Activation of an RNAse in a membrane fraction from fat body of Sarcophaga peregrina larvae by calcium, lnsect Biochem. 13, 403-406. Brogdon W. G. (1984) Mosquito protein microassay--1. Protein determinations from small portions of single mosquito homogenates. Comp. Biochem. Physiol. 79B, 457-459. Doenecke D., Marmaras V. J. and Sekeris C. E. (1972) Increased RNase H (hybridase) activity in the integument of blowfly larvae during development and under the influence of fl-ecdysone. FEBS Lett. 22, 261-264. Englemann F. (1977) Undegraded vitellogenin polysomes from female insect fat bodies. Biochem. Biophys. Res. Comm. 78, 641~47. Fritz M. A. and Fallon A. M. (1985) Evidence for multiple ribonucleases in crude extracts from cultured mosquito cells. Insect Biochem. 15, 817-825. Garcia-Segura J. M. and Gavilanes J. G. (1982) Study of the RNA-degrading activities during the development of the insect Ceratitis capitata. Comp. Biochem. Physiol. 73B, 835-838. Hagedorn H. H., Fallon A. M. and Laufer H. (1973)
361
Vitellogenin synthesis by the fat body of the mosquito Aedes aegypti: evidence for transcriptional control. Devel. Biol. 31, 285-294. Hagedorn H. H., Turner S., Hagedorn E. A., Pontecorvo D., Greenbaum P., Pfeiffer D., Wheelock G. and Flanagan T. R. (1977) Postemergence growth of the ovarian follicle of Aedes aegypti. J. Insect Physiol. 23, 203-206. Hotchkin P. (1985) The duration of larval life of Aedes aegypti as affected by time of hatch. J. Am. Mosq. Cont. Assoc. 1, 489-492. Huet J., Sentenac A. and Fromageot P. (1978) Detection of nucleases degrading double helical RNA and of nucleic acid-binding proteins following SDS-gel electrophoresis. FEBS Lett. 94, 28-32. Lechner M. C. and Duque Magalh~es M. C. (1973) RNase activities in blood serum of several vertebrates. Experientia 29, 1479-1480. Reid P. C. and Chen T. T. (1981) Juvenile hormonecontrolled vitellogenin synthesis in the fat body of the locust (Locusta migratoria): isolation and characterization of vitellogenin polysomes and their induction in vivo. Insect Biochem. 11, 297-305.